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WO2010023340A2 - Novel biomarker as therapeutic target in lung cancer - Google Patents

Novel biomarker as therapeutic target in lung cancer Download PDF

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Publication number
WO2010023340A2
WO2010023340A2 PCT/ES2009/070347 ES2009070347W WO2010023340A2 WO 2010023340 A2 WO2010023340 A2 WO 2010023340A2 ES 2009070347 W ES2009070347 W ES 2009070347W WO 2010023340 A2 WO2010023340 A2 WO 2010023340A2
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Prior art keywords
rhob
gene
expression
subject
lung adenocarcinoma
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PCT/ES2009/070347
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Spanish (es)
French (fr)
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WO2010023340A3 (en
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Fernando Lecanda Cordero
Diego Luis-Ravelo Salazar
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Proyecto De Biomedicina Cima, S.L.
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Publication of WO2010023340A2 publication Critical patent/WO2010023340A2/en
Publication of WO2010023340A3 publication Critical patent/WO2010023340A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the area of cancer diagnosis and therapy.
  • it refers to RhoB gene overexpression and its clinical impact on non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • Lung cancer is the leading cause of cancer-related deaths worldwide. Since it tends to spread, or produce metastases, very early in its evolution, it is a cancer with high lethality and one of the most difficult treatment cancers. Lung cancer can spread to certain organs - particularly the adrenal glands, the liver, the brain, and the bones - the most common organs for metastasis. Despite advances in surgery, chemotherapy and radiotherapy, the survival rate has improved little in the last decade, and long-term survival is very low. It has been established that lung cancer arises as a result of the accumulation of multiple genetic changes in critical genes that control motility, proliferation, differentiation and cell apoptosis. The identification and characterization of these genetic changes that cause the development and progression of lung cancer is of great interest to improve early diagnosis and the development of a novel targeted therapy (Mazieres et al. 2004).
  • Lung cancers also known as bronchogenic carcinomas, are broadly classified into two types: small or small cell lung cancers (SCLC) and non-small or non-small cell lung cancers microcytic (NSCLC). This classification is based on the microscopic appearance of the tumor cells themselves. NSCLCs are the most common lung cancers, representing approximately 80% of all lung cancers. There are three main types of NSCLC that are called based on the type of cells found in the tumor: adenocarcinomas, squamous cell carcinomas, large cell carcinomas, and mixtures of different types of NSCLC are also observed. Among lung cancers and particularly NSCLC, lung adenocarcinoma is the most frequent, accounting for 30-35% of all cases of lung cancer.
  • SCLC small or small cell lung cancers
  • NSCLC non-small or non-small cell lung cancers microcytic
  • Lung adenocarcinoma is defined by the World Health Organization (WHO) as "a malignant epithelial tumor with patterns of tubular, acinar, or papillary growth, and / or mucosal production by tumor cells.”
  • WHO World Health Organization
  • the WHO currently recognizes four categories of adenocarcinomas: acinar, papillary, bronchioloalveolar, and solid carcinoma with mucus formation.
  • Most adenocarcinomas appear in the periphery of the lung, and, as a result, they are frequently asymptomatic in much of their evolution. They are often located at the subpleural level and cause pleural retraction and thickening by radiographs.
  • Ras proteins which regulate important cellular functions that vary from differentiation to cell proliferation. They function as molecular switches, between inactive GDP binding states and active GTP binding states.
  • the ras oncogene plays a fundamental role in the malignant transformation Ras is mutated resulting in a deficient form of GTPase that leads to its constitutive activation resulting in uncontrolled proliferation in approximately 50% of lung adenocarcinoma.
  • Rho small guanosine triphosphatases
  • GTPases small guanosine triphosphatases
  • RhoB While most Rho proteins have been shown to have a positive role in the proliferation and transformation of malignant tumors, the specific role of RhoB in these processes seems to be more divergent.
  • RhoB protein was expressed in normal lung and decreased sharply throughout the progression of early stage lung cancer, concluding that loss of RhoB expression occurs very frequently in lung carcinogenesis, reinforcing its potential activity as a tumor suppressor gene.
  • Sato et al. (2007) analyzed the changes in RhoB NSCLC, demonstrating that RhoB expression is frequently regulated negatively in NSCLCs by multiple mechanisms, and suggesting that RhoB is a tumor suppressor gene in NSCLC.
  • US7135463 describes the use of RhoB protein and its variants to inhibit growth, migration, invasion, metastasis of cancer cells, the transformation of tumor cells, and / or to modulate oncogenic signaling, in which the introduction of RhoB directly or indirectly through a nucleic acid sequence encoding RhoB, in a transformed tumor cell or a cancer cell decreases the phosphorylation of Erk and Akt proteins that inhibit the mechanism of cellular survival Pl3-kinase / Akt and boost programmed cell death or apoptosis .
  • RhoB variant polypeptides are introduced directly or indirectly through a nucleic acid sequence encoding the RhoB variant polypeptide, into a transformed tumor cell or a cancer cell, in which the RhoB variant polypeptide suppresses tumor growth and drives apoptosis
  • WO2008031910 describes several non-small cell lung cancer cell lines that can experience bone metastasis very effectively.
  • it describes specific cell lines derived from a carcinoma cell line bronchioalveolar (A459) that share a common gene expression pattern characterized in the overexpression of at least 3, 4, 5, or 6 genes selected from ILIl, PITXl, HDAC4, RHOB, ROBOl, and SLC26A2.
  • RhoB plays a critical role in bone metastasis, by increasing cell motility and boosting bone colonization.
  • RhoB overexpression in lung adenocarcinoma represents a new and potent prognostic biomarker potentially useful in the clinical treatment of cancer of lung
  • PR stable disease
  • TTP time to progression
  • PFS progression free survival
  • GS overall survival
  • Bisphosphonates are indicated for a wide variety of diseases related to bone metabolism, including osteoporosis, Paget's disease, multiple myeloma, and bone metastases secondary to advanced solid tumors.
  • bone metastases can lead to skeletal-related events (SREs), including pathological fractures, compression of the spine, hypercalcemia, bone pain that requires palliative radiotherapy, and orthopedic surgery.
  • SREs skeletal-related events
  • These skeletal complications frequently lead to the loss of functional independence and the decrease in the quality of life and, thus, the goal of therapy is to prevent or delay the onset of SREs to preserve independence.
  • Bisphosphonates are the medication of choice for the treatment of osteolytic and osteoblastic lesions associated with bone metastases and have been found to significantly reduce the risk of SREs. (Costa et al. 2007)
  • lovastatin which belongs to the statin group, an HMG-CoA reductase inhibitor on expression, Rho isoform activity, and association with guanine nucleotide dissociation inhibitors, thus providing a molecular mechanism in which statins cause an accumulation of non-pre-piled Rho-GTP.
  • WO2004024165 describes a pharmaceutical composition for the treatment of malignant tumors, in particular multiple myeloma (MM), said composition comprising in combination with a bisphosphonate, for example zoledronic acid or a salt or ester, a 3-hydroxy-3-methylglutaryl inhibitor -coenzyme A (HMG-CoA) reductase, for simultaneous, sequential or separate use.
  • MM multiple myeloma
  • WO2000016809 describes antisense compounds, compositions and procedures for modulating RhoB expression.
  • the compositions comprise antisense compounds, particularly antisense oligonucleotides, directed to nucleic acids encoding RhoB. Procedures for using these compounds are provided for the modulation of RhoB expression and for the treatment of diseases associated with RhoB expression.
  • RhoB in NSCLC, such as lung adenocarcinoma
  • the inventors have surprisingly observed that a combination comprising lovastatin and zoledronic acid significantly improves overall survival when It is compared with the administration of zoledronic acid alone.
  • the inventors have also observed that the administration of lentivirally transduced lung cells with several rRNAs against RhoB (shRhoB) to immunocompromised mice resulted in a decrease in their promtatatic activity, including a very marked reduction of osteolytic lesions and the burden of tumor, compared to control cells.
  • the combination comprising lovastatin and zoledronic acid, as well as shRhoB-based RNAi agents, have proven useful in the treatment of NSCLC, such as lung adenocarcinoma, for example in slowing the progression of bone metastasis. .
  • the present invention relates to a method of prognosis of the evolution of the disease in a subject with lung adenocarcinoma comprising: • the measurement of RhoB gene expression; or • measurement of the number of copies of the RhoB gene; in a sample of said subject with lung adenocarcinoma, characterized in that the detection of overexpression or increase in the number of copies of the RhoB gene in relation to the expression or number of copies of the same gene in a control sample is indicative of the risk of metastasis, or recurrence or recurrence of neoplastic activity.
  • the present invention relates to a screening procedure for active drugs against lung adenocarcinoma, said method comprising: a) measuring the expression of the RhoB gene, or measuring the copy number of the RhoB gene , in a sample with lung adenocarcinoma cells in the presence of a drug, or the measurement of binding affinity between a drug and a RhoB polypeptide; b) comparing the expression or measured copy numbers of the RhoB gene, or the binding affinity of step a) to a control sample; characterized in that a reduced expression or copy number of the RhoB gene, or a higher binding affinity in step a), in relation to the expression or the number of copies of the same gene or the binding affinity of the same polypeptide in a sample Control is indicative of the activity of the drug against lung adenocarcinoma.
  • the present invention relates to a method for determining the efficacy of a therapeutic treatment regimen in a subject with lung adenocarcinoma, said method comprising a) measuring RhoB gene expression, or measuring number of copies of the RhoB gene, in a sample with lung adenocarcinoma cells of said subject, thereby generating an initial level; b) measurement of RhoB gene expression, or measurement of RhoB gene copy number, in a second sample with lung adenocarcinoma cells of the same subject at a time after the administration of the treatment regimen, thereby obtaining a level of evidence; and c) comparison of initial and test levels; characterized in that a reduced expression or number of copies of the RhoB gene at the test level in relation to the initial level is indicative that the treatment pattern is effective in the subject.
  • RhoB gene overexpression inhibitors are provided for use as a medicine or for use in the treatment of NSCLC.
  • these inhibitors are selected from:
  • a Rhob gene inhibitor nucleic acid selected from an antisense oligonucleotide, a ribozyme, and an RNAi agent selected from RNAds and shRNA; or
  • RNAsh molecule that has at least 70% homology with SEQ ID No. 3, preferably in the form of a retroviral vector, for use in lung adenocarcinoma gene therapy.
  • the combination comprising lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof is provided for use in the treatment of lung adenocarcinoma.
  • A549 adenocarcinoma cells were inoculated intracardiacly in immunosuppressed mice. After radiographic detection of bone metastasis, these subpopulations were isolated, expanded in a culture, and inoculated again. This procedure was repeated until obtaining highly metastatic subpopulations MlMl, M1M4, and M1M3. These were inoculated again in other groups of mice, as well as the parental population. Its metastatic ability was evaluated by analyzing microradiograph images of the long bones, and Kaplan-Meier metastasis free survival curves.
  • RhoB was identified as one of the overexpressed genes in all metastatic subpopulations. Overexpression was validated by RT-PCR and by Western blot analysis.
  • FIG. 3 Preparation of RhoB inhibitor in lung cancer cells. to. Preparation of RhoB inhibition by a lentiviral vector with rRNA specific for RhoB. For the overexpressed gene a retroviral vector was used. b. Effect on in vitro proliferation of the inhibitor of
  • RhoB and the negative control vector ("scramble"). No changes in proliferation were observed in vitro by
  • RhoB inhibition decreases metastasis in vivo Cells were inoculated with rRNA against RhoB and with a control vector ("Scramble"). After 35 days after inoculation, a significant decrease in the radiographic area was observed by image analysis. In addition, the decrease in tumor burden was verified by bioluminescence.
  • Figure 6 Pharmacological effect of a combination of lovastatin and zoledronic acid in patients with adenocarcinoma of the lung and cells treated in vitro.
  • A549 TM MlMl Treatment with A549 TM MlMl in culture.
  • FIG. 7 TM5 MlMl metastatic adenocarcinoma A549 cells were treated for 4 days with a vehicle (Control); Lovastatin (Lov) 1 ⁇ M and 0.5 ⁇ M; Zoledronic acid (ZA) 10 ⁇ M and 5 ⁇ M; and the 1 ⁇ M + 10 ⁇ M ZA combinations; and 0.5 ⁇ M Lov + 5 ⁇ M ZA diluted in culture medium.
  • the inoculation lasted 2 days and the density was 2500 cells / well.
  • the photographs taken of the cultures that followed the different treatments mentioned show the efficacy of the combinations 1 ⁇ M + 10 ⁇ M ZA and 0.5 ⁇ M Lov + 5 ⁇ M ZA.
  • Figure 8 After 24 hours the cell viability of metastatic TM MlMl cells of adenocarcinoma A549 was measured in the presence of different pharmacological treatments. The treatments were with vehicle (Control), 1 ⁇ M lovastatin (Lov), 10 ⁇ M zoledronic acid (ZA), and the combination 1 ⁇ M + 10 ⁇ M ZA (Lov + ZA) diluted in culture medium, and applied for 72 h after sowing in 96-well culture plates. Cell viability was determined by measuring the concentration of MTT, a water-soluble yellow tetrazolium dye that is transformed into water-soluble violet formazan only in living cell mitochondria.
  • MTT a water-soluble yellow tetrazolium dye that is transformed into water-soluble violet formazan only in living cell mitochondria.
  • FIG. 9 TM5 MlMl metastatic adenocarcinoma A549 cells were treated for 24 hours with: vehicle (Control); Lovastatin 1 ⁇ M (Lov); Zoledronic acid 10 ⁇ M (ZA); and the combination Lov l ⁇ M + 10 ⁇ M ZA (Lov + ZA) diluted in culture medium.
  • the test was performed on a 96-well plate; The cells were seeded (1000 cells / well) and maintained for 72 h after treatment. Caspase-3 activity was determined by testing with luminescent caspase-Glo 3/7. The tests were performed on 12 replicates for each treatment. ** p ⁇ 0.001 vs. control, Lov or ZA (one-way ANOVA followed subsequently by Tamhane test and contrast). Values, means +/- SEM. The results show that the combination of lovastatin and zoledronic acid increases caspase activity 3/7.
  • Figure 10 The H727 non-small cell lung cancer human cell line derived from carcinoid tumor was treated for 24 h with a vehicle (Control); 1 ⁇ M lovastatin (Lov); Zoledronic acid (ZA) 10 ⁇ M; and the combination 1 ⁇ M Lov + 10 ⁇ M ZA diluted in culture medium. The incubation lasted 6 days and the density was 5000 cells / well. The photographs taken of the cultures undergoing the different previous treatments demonstrate the effectiveness of the 1 ⁇ M Lov + 10 ⁇ M ZA combination.
  • a vehicle Control
  • 1 lovastatin Lov
  • ZA Zoledronic acid
  • the cell viability with MTT of the same human H727 cell line was measured in the presence of the same treatments.
  • the cells were seeded and treated for 24 hours with vehicle (Control); Lovastatin (Lov) 1 ⁇ M; Zoledronic acid (ZA) 10 ⁇ M or the combination Lov l ⁇ M + ZA 10 ⁇ M (Lov + ZA) diluted in culture medium.
  • FIG. 11 The H727 cell line was treated for 24 hours with a vehicle (Control); 5 ⁇ M lovastatin (Lov); Zoledronic acid (ZA) 5 ⁇ M; and the combination 5 ⁇ M Lov + 5 ⁇ M ZA diluted in culture medium. The incubation lasted 6 days and the density was 5000 cells / well. The photographs taken of the cultures undergoing the different previous treatments demonstrate the effectiveness of the 5 ⁇ M Lov + 5 ⁇ M ZA combination.
  • the cell viability with MTT of the same human cell line of non-small cell lung cancer H727 derived from carcinoid tumor was measured in the presence of the same treatments.
  • the cells were seeded and treated for 24 hours with vehicle (Control); Lovastatin (Lov) 5 ⁇ M; Zoledronic acid (ZA) 5 ⁇ M or the combination 5 ⁇ M Lov + 5 ⁇ M ZA (Lov + ZA) diluted in culture medium. Planting density 1000 cells / well; ** p ⁇ 0.001 vs. control; t p ⁇ 0.001 vs. Lov or ZA (one-way ANOVA followed subsequently by Tamhane test and contrast). Values, means +/- SEM.
  • FIG. 12 The H727 cell line was treated for 72 hours with a vehicle (Control); 5 ⁇ M lovastatin (Lov); Zoledronic acid (ZA) 5 ⁇ M; and the combination 5 ⁇ M Lov + 5 ⁇ M ZA diluted in culture medium. The incubation lasted 4 days and the density was 5000 cells / well. The photographs taken of the cultures undergoing the different previous treatments demonstrate the effectiveness of the 5 ⁇ M Lov + 5 ⁇ M ZA combination.
  • the cell viability with MTT of the same H727 cell line was measured in the presence of the same treatments.
  • Cells were seeded and treated for 72 hours with vehicle (Control); Lovastatin (Lov) 5 ⁇ M; Zoledronic acid (ZA) 5 ⁇ M or the combination 5 ⁇ M Lov + 5 ⁇ M ZA (Lov + ZA) diluted in culture medium.
  • Vehicle Control
  • Lovastatin Lov
  • ZA Zoledronic acid
  • Figure 13 The H460 non-small cell lung cancer human cell line derived from a large cell carcinoma was treated for 24 h with a vehicle (Control); 1 ⁇ M lovastatin (Lov); Zoledronic acid (ZA) 10 ⁇ M; and the combination 1 ⁇ M Lov + 10 ⁇ M ZA diluted in culture medium. The incubation lasted 5 days and the density was 1500 cells / well. The photographs taken of the cultures undergoing the different previous treatments demonstrate the effectiveness of the 1 ⁇ M Lov + 10 ⁇ M ZA combination.
  • the cell viability with MTT of the same H460 cell line was measured in the presence of the same treatments. Cells were seeded and treated for 24 hours with vehicle
  • FIG. 14 The H460 cell line was treated for 24 hours with a vehicle (Control); 5 ⁇ M lovastatin (Lov); Zoledronic acid (ZA) 5 ⁇ M; and the combination 5 ⁇ M Lov + 5 ⁇ M ZA diluted in culture medium. The incubation lasted 5 days and the density was 1500 cells / well. The photographs taken of the crops experienced by the different Previous treatments demonstrate the effectiveness of the 5 ⁇ M + 5 ⁇ M ZA combination.
  • a vehicle Control
  • 5 ⁇ M lovastatin Lov
  • ZA Zoledronic acid
  • the cell viability with MTT of the same H460 cell line was measured in the presence of the same treatments. Cells were seeded and treated for 24 hours with vehicle
  • FIG. 15 The H460 cell line was treated for 72 hours with a vehicle (Control); 5 ⁇ M lovastatin (Lov); Zoledronic acid (ZA) 5 ⁇ M; and the combination 5 ⁇ M Lov + 5 ⁇ M ZA diluted in culture medium. The incubation lasted 3 days and the density was 1500 cells / well. The photographs taken of the cultures undergoing the different previous treatments demonstrate the effectiveness of the 5 ⁇ M Lov + 5 ⁇ M ZA combination.
  • the cell viability with MTT of the same H460 cell line was measured in the presence of the same treatments.
  • Cells were seeded and treated for 72 hours with vehicle (Control); Lovastatin (Lov) 5 ⁇ M; Zoledronic acid (ZA) 5 ⁇ M or the combination 5 ⁇ M Lov + 5 ⁇ M ZA (Lov + ZA) diluted in culture medium.
  • Vehicle Control
  • Lovastatin Lov
  • ZA Zoledronic acid
  • the present invention relates to a method of prognosis of the evolution of the disease in a subject with lung adenocarcinoma comprising: a) the measurement of RhoB gene expression; b) measurement of the number of copies of the RhoB gene; in a sample of said subject with lung adenocarcinoma, characterized in that the detection of overexpression or the increase in the number of copies of the RhoB gene in relation to the expression or the number of copies of the same gene in a control sample is indicative of risk for metastasis, or recurrence or recurrence of neoplastic activity.
  • prognosis of disease progression refers to providing information regarding the impact of the presence of lung adenocarcinoma, for example, as determined by the methods of the present invention, in the future health of a subject, or in other words, the prognosis of the evolution of the disease provides a prediction of how the disease will progress in a subject and whether there is a possibility of recovery.
  • the methods of the present invention can be used to determine the progression of cancer; for example, from lung carcinoma in situ to metastasis, in particular to bone, or recurrence or recurrence of neoplastic activity.
  • the prognosis of disease progression comprises the determination of a clinical outcome selected from the group consisting of response speed (RR), complete response (CR), partial response (PR), stable disease (SD), time to progression (TTP), overall survival (OS), and clinical benefit, which includes complete response (CR), partial response (PR), and stable disease (SD).
  • response speed RR
  • CR complete response
  • PR partial response
  • SD stable disease
  • TTP time to progression
  • OS overall survival
  • clinical benefit which includes complete response (CR), partial response (PR), and stable disease (SD).
  • RR response speed
  • RR refers to the percentage of patients whose cancer decreases or disappears after treatment.
  • CR complete response
  • partial response refers to a decrease in the size of a tumor, or in the extent of cancer in the body, in response to treatment. It is also called partial remission.
  • stable disease refers to a cancer that does not grow or decrease in extent or severity.
  • time to progression is a synonym for "progression-free survival (PFS)” and refers to a measurement of time after a disease has been diagnosed or treated, until the disease begins to worsen .
  • PFS progression-free survival
  • the measurement for each patient is equal to the time elapsed from the start of treatment in a patient in a test - as defined in the test protocol - until the detection of a tumor progression - as defined in the test protocol - or the appearance of any fatality, whichever comes first. If patient observation was stopped (for example, at the end of the study) after a period and no event was observed, then this observation time is called “censored.”
  • OS global survival
  • TTD time to death
  • rate of survival refers to the percentage of subjects in a study who have survived for a defined period of time. This percentage is usually indicated along with the period of time from the date of diagnosis or treatment.
  • overall survival is measured from the day of the surgery until the death related to the tumor
  • the measurement for each patient is equal to the time elapsed from the beginning of the treatment of a patient in a test -as defined in the protocol- until the appearance of any fatality If the observation of the patient was stopped, for example, at the end of the study, after a period t and the patient survived this time, then this observation time t is called “censored”.
  • subject refers to an organism affected by lung cancer, particularly non-small cell lung cancer, more particularly lung adenocarcinoma.
  • This subject may be a mammal, preferably a human, rat, mouse, or a non-human primate. Usually the subject is a patient.
  • metastatic cancer refers to the movement or expansion of cancer cells from one organ or tissue to another. Cancer cells normally expand through the bloodstream or lymphatic system.
  • metalastasis also refers to the formation of foci of secondary tumors in progressive growth at discontinuous points to the primary lesion.
  • the metastatic process is a multi-stage mechanism in which a metastatic cancer cell escapes from the primary tumor, enters the circulation, invades a distant tissue point and develops a macroscopic tumor at the target site.
  • neoplastic activity refers to an unregulated or abnormally regulated cell proliferation and the process of angiogenesis or the formation of new vasculature that supports a neoplasm in the endothelial microenvironment around the neoplasm.
  • tumor refers to tumor cells, cells that have an unregulated or abnormally regulated proliferation as a result of instability or genetic mutation, and an endothelium in which endothelial cells have an unregulated or abnormally regulated proliferation as a result of a pathogenic state.
  • sample refers to a composition that is obtained or derived from a subject of interest that contains a cellular entity and / or another molecular entity to be characterized and / or identified in based on, for example, physical, biochemical, chemical and / or physiological characteristics. Any suitable sample in which the overexpression of the RhoB gene or the number of copies thereof can be determined is included within the scope of the present invention.
  • the sample is a biological material obtained from a subject, and it can be any organic sample, tissue sample, cell sample, body fluid, body fluid precipitate, or an isolated wash sample from a subject having a lung cancer or with the risk of lung cancer (for example, based on family history or personal history, such as chronic smoker).
  • a sample may be obtained from a subject in any of the ways used in clinical devices to obtain a sample comprising the required cell or nucleic acid, i.e. DNA or RNA
  • samples may be obtained from recent, frozen or paraffin surgical samples or biopsies of an organ or tissue comprising the nucleic acid or cell suitable for testing.
  • Suitable samples used in procedures for the detection or prognosis of cancer can be obtained from a breast sample, ducts or lobes of the breast tissues, sample from the gastrointestinal tract (for example, a biopsy of a polyp), a sample of liver, a sample of pancreatic tissue, a bone marrow sample, a skin sample, a lymph node sample, a kidney sample, a lung sample, a muscle sample, a bone sample, a brain sample, or similar.
  • the sample may be derived from a biological fluid, for example, blood, bone marrow, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymphatic fluid, serum, plasma, pancreatic juice, urine, kilo, feces, ejaculate, sputum, nipple suction , duct fluid, saliva, samples obtained with organic duct swabs, colon wash samples, and brush samples.
  • a biological fluid for example, blood, bone marrow, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymphatic fluid, serum, plasma, pancreatic juice, urine, kilo, feces, ejaculate, sputum, nipple suction , duct fluid, saliva, samples obtained with organic duct swabs, colon wash samples, and brush samples.
  • Tissues and body fluids can be collected using any of the procedures known in the art. If appropriate, the sample can be obtained by using an aspiration procedure.
  • Typical samples for the detection or prognosis of lung adenocarcinoma include, without limitation, cells or tissue (for example, from a biopsy or autopsy), solid lung tumors, sputum, cough, bronchoalveolar lavage, bronchial brushes, mucosa buccal, peripheral blood, whole blood, red blood cell concentrates, platelet concentrates, leukocyte concentrates, red blood cell proteins, blood plasma, platelet rich plasma, a plasma concentrate, a precipitate of any fraction of the plasma, a supernatant of any fraction of the plasma, fractions of blood plasma proteins, proteins or other purified or partially purified blood components, serum, tissue or biopsy samples with fine needle and pleural fluid, etc. isolated from a subject with lung cancer, or any other tumor, or any other extract thereof, obtained from a test subject, healthy volunteer or experimental animal.
  • a sample may be substantially free of tumor cells, for example, it contains less than 1, 5, 10, 20, 30, 40, or 50% of tumor cells compared to non-tumor cells in the sample.
  • the non-tumor cells may be epithelial cells or lymphocytes.
  • a sample containing the lung cancer cell can be purified cells from a tissue.
  • biological samples can be obtained from a subject or a subject at different time points, including before, during and / or after a treatment.
  • a sample may also include, without limitation, products produced in cell cultures by normal or transformed cells, for example, through recombinant DNA technology or monoclonal antibodies.
  • a sample can also be a cell or a cell line created under experimental conditions that are not isolate directly from a subject.
  • a sample can also be cell free, derived or artificially synthesized.
  • a sample may be from a cell or tissue known to be cancerous, suspected of being cancerous, or believed to be non-cancerous (for example, normal or control).
  • RhoB gene refers to the process of converting genetic information encoded in the RhoB gene into RNA, for example, mRNA, rRNA, tRNA, or mRNA, through gene transcription, that is, through the enzymatic action of an RNA polymerase, and for genes that encode proteins, in the protein through the translation of mature mRNA.
  • Gene expression can be regulated in many stages of the process.
  • “Positive regulation” or “activation” refers to regulation that increases the production of gene expression products, that is, RNA or protein
  • negative regulation or “repression” refers to regulation that decreases the production.
  • Molecules for example, transcription factors, which are involved in positive regulation or negative regulation are often referred to as “activators” and “repressors,” respectively.
  • RhoB gene expression can be measured at the protein level, at the mRNA level, or at the cDNA level.
  • Protein level measurement RhoB-derived protein expression can be measured using any of the immunoassays available in the art.
  • immunoassays useful in determining the level of protein expression include, but are not limited to, immunoprecipitation, radioimmunoassay, assay of Western blotting, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay, and enzyme-linked immunoabsorbent assay (ELISA).
  • the above immunoassays can be used in combination, such as immunoprecipitation followed by Western blotting. The above procedures are described in Principies and Practice of Immunoassay, Price and Newman, eds. , Stochton Press, last ed.
  • Such assays may be direct, indirect, competitive, or non-competitive immunoassays as described by the technique (Oelbrick, N. (1984) J. Clin. Chem. Clin. Biochem., 22: 895-904).
  • the protein to be analyzed by such procedures can be obtained directly from the sample.
  • the protein can be obtained as a crude lysate or can be further purified by methods known to those skilled in the art, including immunoaffinity chromatography using antibodies to the RhoB protein (Sambrook, J. et al (2001) in "Molecular Cloning: A Laboratory Manual ", CoId Spring Harbor Press, Plainview, NY).
  • RhoB protein levels can be detected by immunohistochemistry of fixed or frozen tumor sections.
  • polyclonal or monoclonal anti-RhoB antibodies can be used. If polyclonal antibodies are desired, serum containing polyclonal antibodies to the RhoB protein can be used or the polyclonal antibodies can be purified from antigens present in the serum by immunoaffinity chromatography. Alternatively, monoclonal antibodies directed against RhoB can be easily produced by one skilled in the art. The methods of producing monoclonal or polyclonal antibodies are known to a person skilled in the art. (Goding, JW (1996) Monoclonal antibodies: Principies and Practice, Plodermic Press, Inc., NY, NY, pp. 56-97; Hurn, BAL et al. (1980) Meth. Enzymol., 70: 104-141) .
  • Suitable immunogens that can be used to produce polyclonal or monoclonal antibodies include Cellular used prepared from cells transcribed with a recombinant RhoB protein, a partially or substantially purified recombinant or native RhoB protein, or peptides derived from the amino acid sequence of RhoB. Vectors and their use in the production of recombinant proteins are known to those skilled in the art (Sambrook, J. et al. (2001) in "Molecular Cloning, A Laboratory Manual", CoId Spring Harbor Press, Plainview, N. Y.).
  • Antibodies or antigen binding fragments can also be produced by genetic engineering.
  • the technology for the expression of both heavy chain and light chain genes in E. coli is the subject of PCT patent applications; Publication number WO 901443, WO 901443, and WO 9014424 and in Huse et al. (1989) Science, 246: 1275-1281.
  • anti-RhoB antibodies can be induced by administration of anti-idiotype antibodies as immunogens. Practically, a purified anti-RhoB antibody is used to induce an anti-idiotype antibody in a host animal.
  • antibodies can be used in situ to detect RhoB protein in the sample.
  • the antibodies can be used in direct or indirect immunofluorescence.
  • RhoB protein can be detected in in situ by the use of radiolabeled anti-RhoB antibody or by the use of an unlabeled anti-RhoB antibody followed by a second radiolabeled antibody reactive to the anti-RhoB antibody.
  • Antibodies can also be used to immunoprecipitate RhoB protein from a mixture of proteins.
  • the use of immunoprecipitation as a sensitive and specific technique to detect and quantify target antigens in protein mixtures is well known to those skilled in the art (see, Molecular Cloning, A Laboratory Manual, 2d Edition, (2001) CoId Spring Harbor Press) .
  • the antibodies can also be fixed to solid supports for use in the isolation of Rhob protein by immunoaffinity chromatography.
  • Techniques for immunoaffinity chromatography are well known in the art (Harlow, E. and La ⁇ e, D. (1888) "Antibodies: A Laboratory Manual", CoId Spring Harbor Laboratory, CoId Spring Harbor, NY) and include techniques for fixing antibodies to solid supports, so that they maintain their immunoselective activity;
  • the techniques used may be those in which the antibodies are adsorbed to the support, as well as those in which the antibodies are covalently bound to the support.
  • Rho B (119) sc-180 An antibody suitable for the measurement of Rho B gene expression at the protein level is Rho B (119) sc-180 commercially available from Santa Cruz Biotechnology, Inc.
  • Rho B (119) is an affinity purified rabbit polyclonal antibody developed against a peptide that is mapped into an internal Rho B region of human origin.
  • the antibodies described above and the fragments Binding to them can be supplied in a diagnostic kit useful for the detection of alterations in Rhob protein expression.
  • RhoB gene expression can be controlled at the level of messenger RNA (mRNA) according to methods known in the art. Procedures that utilize hybridization of RhoB transcript nucleic acid probes, including microarray technology, gel visualization, and Northern blotting, can be used to measure the presence and / or level of RhoB mRNA. The mRNA can also be evaluated using amplification techniques, such as real-time quantitative RT-PCR or RT-PCR.
  • mRNA messenger RNA
  • Hybridization analysis which is based on the specificity of nucleotide interactions, uses oligonucleotides or cDNAs to selectively identify or capture DNA or mRNA from the specific sequence composition.
  • the amount of mRNA or cDNA hybridized to a known capture sequence can be determined quantitatively or qualitatively, thereby providing information on the relative representation of the RhoB protein.
  • Hybridization can also be performed to arrays or chips of both DNA, cDNA, oligonucleotides or RNA, where these can be produced according to any suitable procedure known in the art. For example, the methods of producing large oligonucleotide arrays are described in US5,134,854, and US5,445,934 using light-directed synthesis techniques.
  • a heterogeneous array of monomers is converted, through simultaneous coupling at a number of reaction sites, into a heterogeneous array of polymers.
  • the microarrays are generated by the deposition of presynthesized oligonucleotides on a solid substrate, for example as described in WO95 / 35505.
  • Sequencing-based procedures are an alternative. These procedures began with the use of expressed sequence markers (ESTs), and now include procedures based on short markers, such as serial analysis of gene expression (SAGE) and MPSS (Massively Parallel Signature Sequencing). As such, mRNA expression levels in a test sample can be determined by generating a library of ESTs from said test sample. The enumeration of the relative representation of ESTs in the library can be used to approximate the relative representation of a gene transcript in the starting sample. The results of the EST analyzes of a test sample can then be compared with the EST analysis of a reference sample comprising control cells to determine the relative expression levels of a polynucleotide encoding RhoB.
  • ESTs expressed sequence markers
  • SAGE serial analysis of gene expression
  • MPSS Massively Parallel Signature Sequencing
  • SAGE (Velculescu et al., Science (1995) 270: 484) involves the isolation of unique short sequence markers from a specific location in each transcript. Sequence markers are concatenated, cloned and sequenced. The frequency of particular transcripts in the starting test sample is reflected by the number of times the associated sequence marker is found in the sequence population.
  • MPSS described by Brenner et al., Nature Biotechnology 18: 630-634 (2000), is a sequencing strategy that combines non-gel sequencing with in vitro cloning of millions of templates in separate microspheres of 5 ⁇ m in diameter.
  • a DNA template microsphere library is constructed by in vitro cloning. This is followed by the assembly of a flat array of microspheres containing templates in a flow cell at a high density (usually greater than 3 ⁇ O 6 microspheres / cm 2 ). The free ends of the cloned templates in each microsphere are analyzed simultaneously, using a fluorescence-based sequencing procedure that does not require separation of DNA fragments.
  • This technique allows the efficient identification and isolation of many hundreds of thousands of individual sequences, the generation of a "library" of small RNAs.
  • the abundance or frequency of occurrence of each different sequence of a "library” of small RNAs is indicative of the amount in the original sample from which the RNA was obtained.
  • sequences which are usually 17-20 nucleotides in length
  • genomic DNA database it is possible to determine the points in the DNA that act as the origin for small RNAs. Comparisons with genome annotations, cDNA databases, and other data can often be used to identify the largest RNA precursors of small RNAs. More significantly, MPSS provides the ability to direct the biology of small RNA to a broad genome scale.
  • RhoB gene expression can also be controlled at the cDNA level. RhoB gene expression levels are determined using the reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR is a well known technique in the sector that is based on the fact that the enzyme reverse transcriptase reverse transcribes the mRNA to form cDNA, which can then be amplified in a standard PCR reaction. The protocols and kits for carrying out the RT-PCR are well known to those skilled in the art and are commercially available.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • RT-PCR can be carried out in a non-quantitative manner.
  • Said end-point RT-PCR measures changes in expression levels using three different procedures: relative, competitive and comparative. These traditional procedures are well known in the art, for example, visualization of the amplicon in a gel electrophoresis.
  • the RT-PCR is carried out in real time and quantitatively.
  • Real-time PCR is a technique used to monitor the progress of a real-time PCR reaction.
  • a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified.
  • Real-time PCR is based on the detection of fluorescence produced by a reporter molecule that increases as the reaction progresses. This occurs due to the accumulation of the PCR product with each amplification cycle.
  • These fluorescent reporter molecules include fluorochromes that bind to double stranded DNA or specific sequence probes.
  • the present invention provides primers with SEQ ID NO 4-6 for performing real-time RT-PCR or PCR.
  • Primers with SEQ ID NO 4 and 5 are particularly suitable for use in the expression profile of the RhoB gene using RT-PCR
  • the recognition of multiple DNA alterations can increase the effective identification of cancer.
  • the probes allow multiple types of DNA to be measured in the same sample (multiplex PCR), since fluorescent dyes with different emission spectra can bind to different probes. Multiplex PCR allows internal controls to be co-amplified. These hybridization probes allow a level of discrimination impossible to obtain with SYBR Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or other non-specific products.
  • Variants of the basic PCR technique such as nested PCR, and the like are also included within the scope of the present invention.
  • Examples include isothermal amplification techniques such as NASBA, 3SR, TMA, and triamplification, all known in the industry and commercially available.
  • Other suitable amplification procedures include ligase chain reaction (CSF) (Barringer et al, 1990), Methylation Specific Multiple Ligand-dependent Probe Amplification assay (MS-MLPA) (Nygrenet al., Nucleic Acids Res.
  • CSF ligase chain reaction
  • MS-MLPA Methylation Specific Multiple Ligand-dependent Probe Amplification assay
  • the relative representation of a gene expressed in a sample can then be estimated based on the relative representation of the fragment associated with the gene that is in the group of all possible fragments.
  • Methods and compositions for carrying out DD are known in the art, see, for example, US 5,776,683; and US5,807,680.
  • the genomic DNA, protein, mRNA, and cDNA sequences of the RhoB gene, or a region thereof, are provided below.
  • the primers and probes described in the present invention are provided as SEQ ID NO: 4-6.
  • the nucleotide sequence of the human RhoB genomic DNA (NC_000002.10) (5 ' ⁇ 3', 2367 bp) is provided in the following sequence SEQ ID NO: 1:
  • the amino acid sequence of the human RhoB protein (NP_004031.1) (196 aa) is provided in the following sequence SEQ ID NO: 2: MAAIRKKLVVVGDGACGKTCLLIVFSKDEFPEVYVPTVFENYVADIEVDGKQVELALW DTAGQEDYDRLRPLSYPDTDVILMCFSVDSPDSLENIPEKWVPEVKHFCPNVPIILVA NKKDLRSKTQRKVKVKVKVKVKVKVKVKVKTKVKVKVKVKVKVKTKRKVKVKVKVKVKVKTKRKVKVKVKVKVKTKRKVKVKVKVKTKRKVKVKVKVKTKRKVKVKVKVKTKRKVKVKVKVKTKRKVKVKVKVKTKRKVKVKVKVKTKRKVKVKVKVKTKRKVKVKV
  • the nucleotide sequence of human RhoB mRNA (NM_004040.2) (5 ' ⁇ 3', 2384 bp) is provided in the following sequence SEQ ID No. 7: ATCTGCCACCGCAGTCTGGTTGGAGCTGTTGTCTTGTATGCTCAGCGAGGCCCGGAGA GACCCGGGAGAGCTAGGCCGAGTCCACCGCCCGAGTCTGCTGCCCGAGCCCGCGTT ACGCACAAAGCCGCCGATCCCCGGCCTGGGGTGAGCAGAGCGACCACCGCCCGGGAGC AGCGCGGCGAGACGCACGGTGCCCTATGCCCCCGCGCCCCCACCGCCCCCGCCGCGCG GCAGCCGAAGCGCAGCGAGAGAACGCGCCACCGCGGGGCCCGGGTGCAGCTAGCGACC CTCTCGCCACCTGCGCAGCCCGAGGTGAGCAGTGAGCGGCGAGCGGGAGGGCAGCG AGGCGTTCGCGGGCCCCCTCCTGCTGCCCGGGCCCGGCCCGCTCATGGCGGCCATCCG CAAGAAGC
  • the nucleotide sequence of the human RhoB cDNA (CCDS1699.1) (5 ' ⁇ 3', 591 bp) is provided in the following sequence SEQ ID NO: 8:
  • sequences of genomic DNA, protein, mRNA, cDNA, primers, and probes presented in the present invention also comprise those sequences that have minus 70% homology with respect to the sequences in SEQ ID NO. 1-8, preferably at least 80%, and more preferably at least 90%, 95%, 96%, 97%, 98%, 99% homology.
  • the forms generated by alternative splicing are also within the scope of the present invention.
  • homology refers to the level of similarity between the nucleic acid or amino acid sequences in terms of percentage of positional identity of nucleotides or amino acids, respectively, that is, the similarity or identity of the sequences.
  • the percentage of homology between two sequences can be calculated according to the FASTA or BLAST algorithms (Altschul et al. "Basic local alignment search tool” J. Mol. Biol. 1990, 215, 403-410, http: //www.ncbi. nlm.nih.gov/blast; Lipman et al. "Rapid and sensitive protein similarity searches”. Science 1985, 227, 1435-1441. http://www.ebi.ac.uk), which are incorporated into the present invention by reference
  • Rhob gene expression at the level of protein, mRNA, or cDNA is also within the scope of the present invention.
  • the method of prognosis of disease progression in a subject with lung adenocarcinoma comprises measuring the copy number of the RhoB gene.
  • in situ hybridization for example, fluorescence in situ hybridization (FISH) (see, Angerer, 1987 Meth. Enzymol 152: 649).
  • FISH fluorescence in situ hybridization
  • in situ hybridization comprises the following main stages: (1) fixation of the tissue or biological structure to be analyzed; (2) Prehybridization treatment of the biological structure to increase accessibility to the target DNA, and to reduce non-specific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove fragments of unbound nucleic acids in hybridization, and (5) detection of hybridized nucleic acid fragments.
  • FISH fluorescence in situ hybridization
  • the probes used in such applications are usually labeled, for example, with radioisotopes or fluorescent reporters.
  • Preferred probes are long enough, for example, from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to allow specific hybridization with the target acid or nucleic acids under stringent conditions.
  • CGH comparative genomic hybridization
  • Hybridization protocols suitable for use with the methods of the present invention are described, for example, in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Nati Acad. Sci. USA 85: 9138-9142; 430402EPAE PO Pub. No. 430: 402; Methods in Molecular Biology, VoI. 33: In Situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, NJ (1994).
  • Amplification-based assays can also be used to measure the copy number of the RhoB gene.
  • the corresponding RhoB nucleic acid sequence acts as a template in an amplification reaction (for example, Polymerase Chain Reaction or PCR).
  • an amplification reaction for example, Polymerase Chain Reaction or PCR
  • the amount of amplification product will be proportional to the amount of template in the original sample.
  • Comparison with appropriate controls provides a measure of the copy number of the RhoB gene, corresponding to the specific probe used, according to the principle described above.
  • Real-time quantitative PCR procedures using Taqman probes are known in the art. Detailed protocols for real-time quantitative PCR are provided, for example, for RNA in: Gibson et al., 1996, A novel method for real time quantitative RT-PCR. Genome Res. 10: 995-1001; and for DNA in: Heid et al., 1996, Real time quantitative PCR. Genome Res. 10: 986-994.
  • Southern blotting can also be used to measure the copy number of the RhoB gene.
  • the DNA is analyzed in agarose or acrylamide gels to fractionate the DNA according to the size followed by the transfer of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane.
  • the immobilized DNA is hybridized with a labeled probe to detect DNA samples complementary to the probe used.
  • the DNA can be divided with restriction enzymes before electrophoresis. After electrophoresis, the DNA can be partially stripped and denatured before or during the transfer to the solid support.
  • Southern blots are a common tool for molecular biologists (J. Sambrook et al., Molecular Cloning: A Laboratory Manual, CoId Spring Harbor Press, NY, p. 9.31-9.58 [1989]).
  • Microarray technology can be used because it offers high resolution.
  • traditional CGH generally has a mapping resolution limited to 20 Mb; whereas in CGH based on arrays, the fluorescence ratios of the differentially labeled test DNAs and genomic reference DNA provide a "locus per locus" measurement of the variation in the number of copies of DNA, thus achieving greater mapping resolution
  • Details of a microarray procedure can be found in the literature. See, for example, 6232068USB US 6,232,068; Pollack et al., Nat Genet, 1999, 23 (l): 41-6.
  • overexpression or increase in Number of copies of the RhoB gene in relation to the expression or number of copies of the same gene in a control sample are useful markers for the prognosis of lung adenocarcinoma.
  • said overexpression or increase in the number of copies of the RhoB gene is indicative of the risk of metastasis, or recurrence or recurrence of neoplastic activity.
  • RhoB gene or a "higher” level or “high” level of a RhoB polynucleotide or protein refers to a level of RhoB polynucleotide or protein which, compared to the RhoB control level, is in a higher detectable way.
  • increase in the number of copies of the RhoB gene refers to a level of the number of copies of RhoB which, in comparison to a level of RhoB control, is detectably higher.
  • Overexpression or increase in the number of copies of the RhoB gene are relative terms that mean that a detectable difference (beyond the contribution of noise in the system used to measure it) is found in the amount of expression or number of copies of the gene of RhoB relative to a certain baseline.
  • the baseline is the measured gene expression or the number of copies of a control sample, the term of which is defined below.
  • the comparison can be carried out by statistical analysis on the numerical measurements of the expression or number of copies of the RhoB gene; or, it can be done through visual examination of experimental results by qualified researchers.
  • the gene expression intensities of a Sick tissue can be compared with the expression intensities generated from normal tissue of the same type (for example, sample of diseased lung tissue vs. sample of normal lung tissue). A relationship of these expression intensities indicates the times that gene expression changes between the test and control samples.
  • overexpression of the RhoB gene is determined when there is at least a 1.5-fold difference in its expression relative to the expression of the same gene in a control sample.
  • an increase in the number of copies of the RhoB gene is determined when there is at least a 1.5-fold difference in its number of copies in relation to the number of copies of the same gene in a control sample.
  • the Student t test is an example of a robust statistical test that can be used to find significant differences between two groups.
  • a p value of less than 0.05 by means of the t test shows that the gene is significantly different.
  • a more conclusive evidence is a value of p less than
  • control sample refers to a sample of biological material representative of healthy subjects without cancer.
  • the expression level of the RhoB gene or the number Copies of the RhoB gene in a control sample are desirably customary for the general population of normal subjects without cancer of the same species.
  • This sample can be collected from a subject for the purpose of being used in the procedures described in the present invention or, it can be any biological material representative of normal subjects without cancer obtained for other reasons, but nevertheless, suitable for use in Procedures of the present invention.
  • a control sample can also be obtained from normal tissue of the subject who has cancer or is suspected of having cancer.
  • a control sample can also refer to a certain level of RhoB gene expression or the number of RhoB gene copies representative of the non-cancer population, which have been previously established based on measurements of normal subjects without cancer.
  • a control sample does not need to be a material, but can be data or information related to a control sample. This data or information is usually stored in electronic form or paper.
  • a biological control sample may refer to a sample that is obtained from a different individual or it may be a normalized value based on baseline values found in a population.
  • a control sample can be defined by age, sex, ethnicity or other specific demographic parameters. In some situations, control is implicit in the concrete measurement.
  • it is considered a detection procedure that can only detect RhoB or the number of copies of the RhoB gene when a higher level than usual is present in a normal subject without cancer, for example, an immunohistochemical test, to evaluate the RhoB level or the number of copies of the RhoB gene compared to the level of control or the number of copies of the RhoB gene, since the level of control or the number of copies is natural and known in the assay.
  • the present invention further relates to the use of a RhoB gene, or a region thereof, as a prognostic marker of metastasis, or the recurrence or recurrence of neoplastic activity in a subject with lung adenocarcinoma.
  • the present invention further provides a method for screening active drugs against lung adenocarcinoma, said method comprising: a) measuring the expression of the RhoB gene, or measuring the number of copies of the RhoB gene, in a sample with lung adenocarcinoma cells in the presence of a drug, or the measurement of binding affinity between a drug and a RhoB polypeptide; b) comparing the expression or measured copy numbers of the RhoB gene, or the binding affinity of step a) to a control sample; characterized in that a reduced expression or copy number of the RhoB gene, or a greater enzymatic activity in step a), in relation to the expression or the number of copies of the same gene or the binding affinity of the same polypeptide in a sample of Control is indicative of the activity of the drug against lung adenocarcinoma.
  • the present invention provides methods for identifying compounds or agents that can be used to treat disorders characterized by, or associated with, a reduced expression or number of copies of the RhoB gene.
  • These procedures usually include the screening stage of a candidate, test compound or agent for identifying compounds that are an agonist or antagonist of a RhoB polypeptide, and specifically for the ability to interact with (for example, bind to) a RhoB polypeptide, to modulate the interaction of a RhoB polypeptide and a target molecule , and / or to modulate RhoB nucleic acid expression and / or RhoB polypeptide activity.
  • Candidates, test compounds or agents that have one or more of these capabilities can be used as drugs to treat disorders characterized by a reduced expression or number of copies of the RhoB gene.
  • RhoB gene marker in a lung adenocarcinoma can also help predict that cancer's response to specific therapies.
  • This molecular marker can be used to select the most appropriate therapy for the subject.
  • the present invention further provides a method for determining the efficacy of a therapeutic treatment regimen in a subject with lung adenocarcinoma, said method comprising a) measuring RhoB gene expression, or measuring copy number of the RhoB gene, in a sample with lung adenocarcinoma cells of said subject, thereby generating an initial level; b) the measurement of the RhoB gene expression, or the measurement of the copy number of the RhoB gene, in a second sample with lung adenocarcinoma cells of the same subject at a time after the administration of the treatment regimen, thus obtaining a test level; and b) comparison of initial and test levels; characterized in that a reduced expression or number of copies of the RhoB gene at the level of test in relation to With the initial level it is indicative that the treatment regimen is effective in the subject.
  • the present invention further relates to the use of a nucleotide molecule or antibody that interacts with DNA, RNA, RhoB protein, or a fragment thereof, for the prognosis of disease progression in a subject with lung adenocarcinoma, for screening active drugs against lung adenocarcinoma, or to determine the efficacy of a therapeutic treatment regimen in a subject with lung adenocarcinoma.
  • Said nucleotide molecule is a nucleotide that has at least 70% homology with respect to SEQ ID NO. 4-6, preferably at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% homology with respect to SEQ ID NO. 4-6.
  • the antibody molecule that interacts with DNA, RNA, Rhob protein, or a fragment thereof can be monoclonal, polyclonal, chimeric, humanized or bispecific, or is conjugated to a second antibody, or a fragment thereof.
  • the antibody molecule is the polyclonal Rho B (119) antibody, or an antibody molecule that preferably has at least 80% and more preferably at least 85%, 90%, 95%, 96%, 97 %, 98%, 99% or 100% homology with the polyclonal antibody Rho B (119).
  • a “fragment” is a part of a full length natural, native or endogenous mature Rhob sequence that has one or more amino acid residues removed.
  • the amino acid residue or residues removed may be anywhere in the polypeptide, including at the N-terminal or C-terminal end, or internally.
  • Said fragment will have at least one biological property in common with RhoB.
  • the fragments of RhoB will usually have a consecutive sequence of at least 10, 15, 20, 25, 30, 40, 50 or 60 amino acid residues that are identical to the isolated RhoB sequences of a mammal, including RhoB of SEQ ID NO: 2 .
  • antibody is interchangeable with “immunoglobulin” and refers to immunoglobulins in the general form found in vertebrate species, including mammals, such as humans, primates, rodents, rabbits and many other species. in which said immunoglobulins have been identified.
  • Antibody means molecules that correspond to complete immunoglobulins, as well as fragments thereof, such as Fab, F (ab ') 2, and Fv, which are capable of binding to the epitopic determinant.
  • the term "monoclonal antibody” refers to a population of antibody molecules that contain only a species from an antigen binding site capable of immunoreacting with a particular epitope of an antigen, while the term “polyclonal antibody” refers to a population of antibody molecules that contain multiple species of antigen binding sites capable of interacting with a particular antigen.
  • a composition with monoclonal antibodies usually shows a unique binding affinity for a particular antigen with which it immunoreacts.
  • mice can be immunized with a polypeptide of the present invention or a cell expressing said peptide.
  • an immune response for example, antibodies specific to the antigen in the mouse serum
  • the mouse spleen is collected and the splenocytes are isolated.
  • the splenocytes are then fused by known techniques to any suitable myeloma cell.
  • the hybridomas are selected and cloned by limited dilution.
  • hybridoma clones are assayed by methods known in the art for cells that secrete antibodies capable of binding to a polypeptide of the present invention.
  • Ascetic fluids which generally contain high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • Polyclonal antibodies are usually produced by immunization of a suitable mammal, such as a mouse, rabbit or goat. Frequently larger mammals are preferred, since the amount of serum that can be collected is greater. An antigen is injected into the mammal. This induces B lymphocytes to produce specific IgG immunoglobulins for the antigen. This polyclonal IgG is purified from mammalian serum. Instead, monoclonal antibodies are derived from a single cell line.
  • chimeric antibody refers to an antibody in which the constant region comes from an antibody of one species (usually human) and the variable region comes from an antibody of another species (usually rodent). Methods for producing chimeric antibodies are known in the art.
  • humanized antibody refers to antibody molecules in which amino acids have been substituted in the regions that are not antigen-binding in order that it resembles as much as possible a human antibody, while still maintaining the original binding capacity.
  • this expression may also include "primate” antibodies, in which a first antibody obtained from an organism that is not primate has been modified to closely resemble a primate immunoglobulin.
  • bispecific or bifunctional antibody is an artificial hybrid antibody that has two different heavy / light chain partners and two different binding sites.
  • Bispecific antibodies can be produced by a series of procedures including hybridoma fusion or Fab 'fragment binding. See, for example, Songsivilai & Lachmann, Clin Exp.
  • a further embodiment of the present invention provides a kit for the prognosis of disease progression in a subject with lung adenocarcinoma comprising reagents for measuring expression or copy number of the RhoB gene.
  • Said kit may additionally comprise suitable reagents for performing a microarray analysis.
  • the kit according to the present invention comprises Rho B (119): sc-180 Santa Cruz Biotechnology, Inc.
  • Rho B (119) is an affinity purified rabbit polyclonal antibody developed against a peptide that maps to an internal region of RhoB of human origin.
  • the kit according to the present invention comprises primers with SEQ ID No. 4-6 for performing real-time quantitative RT-PCR or PCR.
  • the present invention further provides an amplicon of Isolated RhoB gene, where the amplicon comprises more than one copy of a polynucleotide selected from: a) a polynucleotide encoding the polypeptide set forth in SEQ ID NO. 2 ; b) a polynucleotide established in SEQ ID NO. one ; c) a polynucleotide having at least 70% homology with the polynucleotide of a) or b); and d) a polynucleotide that is overexpressed in lung adenocarcinoma cells that has at least 70% homology with the polynucleotide of a) or b).
  • a polynucleotide selected from: a) a polynucleotide encoding the polypeptide set forth in SEQ ID NO. 2 ; b) a polynucleotide established in SEQ ID NO. one ; c) a polyn
  • the present invention further relates to inhibitors of RhoB gene overexpression for use as a medicament.
  • Such use as a medicament or method of treatment comprises administration to subjects with NSCLC or to subjects susceptible to developing NSCLC including lung adenocarcinoma, squamous cell carcinoma, or large cell carcinoma of an effective amount of an RhoB gene overexpression inhibitor. to combat the conditions associated with NSCLC, in particular to prevent the development after bone metastasis, or the recurrence or recurrence of neoplastic activity.
  • the present invention also relates to the use of RhoB gene overexpression inhibitors or any subgroup thereof in the manufacture of a medicament for the treatment of NSCLC including lung adenocarcinoma, squamous cell carcinoma, or large cell carcinoma. , in particular to prevent further development in bone metastases, or the recurrence or recurrence of neoplastic activity.
  • the present invention provides inhibitors of RhoB gene overexpression for use in the treatment of NSCLC including adenocarcinoma. of lung, squamous cell carcinoma, or large cell carcinoma, in particular to prevent the development after bone metastasis, or the recurrence or recurrence of neoplastic activity.
  • treat refers to reversing, alleviating or inhibiting the progress of the disorder or condition to which said term applies, or one or more symptoms of said disorders or condition.
  • treatment refers to the act of treating, which has been defined above.
  • terapéuticaally effective amount or “effective amount” as used in the present invention means the amount of compound or component or active pharmaceutical agent that obtains the biological or medical response in a tissue, system, animal or human that is It is seeking, in the light of the present invention, a researcher, veterinarian, doctor or other clinical professional, which includes the improvement of the symptoms of the disease being treated.
  • the "therapeutically effective amount” is that amount of the agents taken together, so that the combined effect provides the desired biological or medical response.
  • inhibitor is intended to include antagonists and, refers to a molecule that directly or indirectly reduces the overexpression of the RhoB gene, or the biological activity of a RhoB polypeptide or protein.
  • RhoB gene overexpression inhibitors may include antibodies, nucleic acids, or other molecules.
  • RhoB gene overexpression inhibitors may be selected from: a) an antibody or fragment thereof that specifically binds to a RhoB protein, or a polypeptide comprising a RhoB binding region; b) a Rhob polypeptide mimetic peptide; c) a RhoB gene inhibitor nucleic acid selected from an antisense oligonucleotide, a ribozyme, and an RNAi agent selected from RNAds and shsh; od) a combination comprising lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof.
  • the antibody molecule that specifically binds to a RhoB protein can be monoclonal, polyclonal, chimeric, humanized or bispecific, as described above in the present invention.
  • antibody fragment refers more specifically to these immunoglobulin-like fragments and / or polypeptides that do not comprise a complete immunoglobulin.
  • a "polypeptide comprising a RhoB specific binding region” means a polypeptide that incorporates one or more binding regions that specifically bind to the RhoB protein of SEQ ID NO. 2.
  • a classic type of antigen specific binding region is a complementarity determining region (CDR) found in an immunoglobulin.
  • CDRs are short amino acid sequences that specifically bind to the antigen in question and provide the basis for the binding selectivity of the polypeptide in which it resides.
  • CDRs are usually identified from immunoglobulins, but can be generate by other means.
  • Antigen specific binding regions also include relatively short amino acid sequences that bind to an antigen, even if these sequences are not derived from CDRs.
  • WO2004 / 044011 incorporated in the present invention by reference, provides an example of how said antigen specific binding regions (which are not CDRs) can be identified and developed. There are other procedures known to those skilled in the art. Once developed, the RhoB-specific binding region can be used in a wide variety of known and futuristic structures and frameworks, including any immunoglobulin isotype or fragment thereof, and other structure or framework polypeptides that are not immunoglobulins to provide the specificity of antigen binding. All these polypeptides are considered in the present invention as "polypeptides comprising a specific RhoB binding region".
  • a "mimetic peptide” is a synthetically derived non-peptide agent created based on knowledge of the critical residues of the polypeptide of a subject that can mimic the normal function of the polypeptide. Peptide mimetics can break the binding of a polypeptide to its receptor or other proteins and, thus, interfere with the normal function of a polypeptide. For example, a RhoB mimetic would interfere with normal RhoB function.
  • Nucleic acid sequence refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or parts thereof, whose polymeric components may be DNA, RNA, modified nucleotides, nucleotide mimetics, or combinations thereof; and they can be of genomic or synthetic origin, and they can be single or double strands, and represent the sense or antisense chain.
  • antisense refers to nucleotide sequences that are complementary to a specific DNA or RNA sequence, usually a target mRNA. They bind to mRNA by pairing Watson-Crick bases, and inhibit their target message through degradation by ribonuclease (RNase) H or by interference with the translation machinery.
  • RNase ribonuclease
  • the antisense oligonucleotides can optionally be stabilized with phosphorothionates, methylphosphonates, polyalkylcyanoacrylate nanoparticles, and 2 'modifications of the sugars.
  • the term “antisense chain” is used in reference to a nucleic acid chain that is complementary to the "sense chain”.
  • Antisense molecules can be produced by any method, including synthesis by binding the gene of interest in reverse orientation to a viral promoter that allows the synthesis of a complementary chain.
  • the designation "negative” is sometimes used in reference to the antisense chain, and “positive” is sometimes used in reference to the sense chain.
  • ribozyme means an RNA molecule that has an enzymatic activity that is capable of dividing or splicing other separate RNA molecules into a nucleotide base sequence in a specific manner.
  • a catalytic or enzymatic RNA molecule is meant an RNA molecule that has complementarity in a substrate binding region with RhoB mRNA, and also has enzymatic activity that is active to divide and / or splice that mRNA, thereby altering it
  • RNAi agent refers to compounds and compositions that can act through an RNA interference mechanism (see, for general reference, He and Hannon, (2004) Nat. Genet. 5: 522-532).
  • RNAi agents are used such as short interference RNA (siRNA), double stranded RNA (RNAs), short hairpin RNA (shRNA), also sometimes referred to as small hairpin RNA, others are in development.
  • siRNA short interference RNA
  • RNAs double stranded RNA
  • shRNA short hairpin RNA
  • RNAi agents are incorporated into a macromolecular complex that uses chains of the RNAi agent to label and divide RNA chains that contain the complementary (or substantially complementary) sequence.
  • antisense oligonucleotides, triple helix DNA, RNA aptamers, RNAi agents such as siRNA, RNAds, and shRNAs, ribozymes and single stranded RNA are designed to inhibit RhoB expression, so that the nucleotide sequence chosen from the inhibitor molecule is designed to cause the inhibition of endogenous RhoB protein synthesis.
  • RhoB nucleotide sequence can be used to design an siRNA molecule that inhibits RhoB expression without too much experimentation.
  • ribozymes can be synthesized to recognize specific nucleotide sequences of a protein from interest and divide it (Cech. J. Amer. Med Assn. 260: 3030 (1988)). Techniques for the design of such molecules for use in the directed inhibition of gene expression are well known to those skilled in the art.
  • the RhoB gene overexpression inhibitor is an RNAsh molecule that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99%, or 100% homology with SEQ ID NO. 3, as provided below.
  • Antisense oligonucleotides, ribozymes, and RNAi agents can be prepared by any method known in the art for the synthesis of nucleic acid molecules. Techniques for chemical synthesis, such as chemical synthesis of solid phase phosphoramidite, are included. Alternatively, antisense oligonucleotides, ribozymes, and RNAi agents can be generated by in vitro transcription of DNA sequences encoding the relevant molecule. Such DNA sequences can be incorporated into a wide variety of vectors with suitable RNA polymerase promoters.
  • Antisense oligonucleotides, ribozymes, and RNAi agents can be released to cells by direct application, such as transfection or formulated in liposomes.
  • Conventional transfection techniques include a variety of techniques recognized in the sector for introducing exogenous nucleic acid (eg, DNA or RNA) into a host cell, including coprecipitation with calcium phosphate or calcium chloride, DEAE-dextran-mediated transfection, lipofection, (micro) injection, or electroporation
  • Liposomes can be prepared using conventional techniques including sonication, chelation dialysis, homogenization, solvent infusion coupled with extrusion, freeze-thaw extrusion, microemulsification, and others.
  • Reagents used to crosslink a liposome or other agent containing lipids with antisense oligonucleotides, ribozymes, and RNAi agents comprise a phospholipid derivative to anchor at one end of the cross-linking in the lipid layer and a reactive group at the other end to provide a point binding to the target biomolecule.
  • Polymerized liposomes or polymer coated liposomes can also be used. Such polymers can stabilize the liposome, reduce its clearance from the body, and / or reduce its immunogenicity.
  • the liposome can be loaded with a functional residue such as a diagnostic or therapeutic agent during or after its formation.
  • the agent may be contained in the aqueous nucleus of the liposome or it may be incorporated or bound to its surrounding
  • antisense oligonucleotides, ribozymes, and RNAi agents are performed through a gene therapy strategy in which vectors, including plasmids, cosmids, bacteriophages, or viral vectors, particularly retroviral, lentiviral, or adenoviral vectors, are they release a cell or subject; or cells that direct the expression of the molecules, for example, cells in which a vector that directs the expression of the molecule has been introduced, are administered to the subject.
  • vectors that direct the in vivo synthesis of antisense oligonucleotides, ribozymes, and RNAi agents can be introduce into cell lines, cells or tissues constitutively or inducibly.
  • a "vector" molecule is a nucleic acid molecule into which heterologous nucleic acid can be inserted that can then be introduced into an appropriate host cell.
  • the vectors preferably have one or more origins of replication, and one or more sites where the recombinant DNA can be inserted.
  • Vectors often have suitable means by which cells with vectors can be selected from those without vectors, for example, which encode drug resistance genes.
  • a "host cell,” as used in the present invention, refers to a prokaryotic or eukaryotic cell that contains heterologous DNA that has been introduced into the cell by any means, for example, electroporation, calcium phosphate precipitation, microinjection. , transformation, viral infection, and the like.
  • Heterologous as used in the present invention means “of different natural origin” or represents an unnatural state. For example, if a host cell is transformed with a DNA or gene derived from another organism, particularly from another species, that gene is heterologous with respect to the host cell and also with respect to the descendants of the host cell carrying that gene. Similarly, heterologous refers to a nucleotide sequence derived from and inserted into the same original natural cell type, but which is present in an unnatural state, for example a number of different copies, or under the control of different elements.
  • Additional gene therapy protocols may involve isolating a population of cells, for example, stem cells or cells of a subject's immune system, optionally expanding the cells in tissue culture, and administering a gene therapy vector to the cells in vitro. Next, the cells can be returned to the subject.
  • cells expressing the desired antisense oligonucleotide, ribozyme, or RNAi agent may be selected in vitro before being introduced into the subject.
  • a population of cells can be used, which can be cells of a cell line or an individual that is not the subject.
  • the isolation procedures of stem cells, immune system cells, etc., of a subject are well known in the art. Such procedures are used, for example, for bone marrow transplantation, peripheral blood stem cell transplantation, etc., in individuals undergoing chemotherapy.
  • the RhoB gene overexpression inhibitor is a combination comprising lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof.
  • lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof refers to the following combinations: a) lovastatin and zoledronic acid; b) a pharmaceutically acceptable salt of lovastatin and zoledronic acid; c) lovastatin and a pharmaceutically acceptable salt of zoledronic acid; Y d) a pharmaceutically acceptable salt of lovastatin and a pharmaceutically acceptable salt of zoledronic acid.
  • Lovastatin is a commercial product that can be provided by the companies TCI Europe NV, Sigma Aldrich, among others.
  • the CAS registry number of lovastatin is 75330-75-5, and its structural formula is:
  • Zoledronic acid which is also referred to as zoledronate, is a commercial product that can be provided by Interchim Ambinter, ACIC
  • pharmaceutically acceptable means that a compound or combination of compounds must be compatible with the other ingredients of a formulation, and not be prejudicial for the patient.
  • the salts of lovastatin or zoledronic acid are those in which the counterion is pharmaceutically acceptable.
  • Pharmaceutically acceptable acid addition salts as mentioned above are intended to comprise the forms of therapeutically active non-toxic acid addition salts that lovastatin and zoledronic acid are each capable of forming.
  • Pharmaceutically acceptable acid addition salts can be suitably obtained by treating the basic form of lovastatin and zoledronic acid with said appropriate acid.
  • Suitable acids comprise, for example, inorganic acids such as hydracids, for example hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and similar acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e.
  • butanedioic acid maleic, fumaric, malic (i.e. hydroxybutanedioic acid) , tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and similar acids.
  • said salt forms can be converted by treatment with an appropriate base in the free base form.
  • the combination comprising lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof can be administered simultaneously, separately or sequentially.
  • kits may comprise two separate pharmaceutical formulations comprising: 1) lovastatin or pharmaceutically acceptable salts thereof; and 2) zoledronic acid or pharmaceutically acceptable salts thereof.
  • the kit may also comprise a container for separate formulations, such as a compartmentalized bottle or a compartmentalized aluminum foil container. Additional examples of containers include syringes, boxes, bags and the like.
  • a kit comprises instructions for the administration of the separate components.
  • the kit form is particularly advantageous when the separated components are preferably administered in different dosage forms (eg, oral and parenteral), are administered at different dosage intervals, or when adjustment of the individual components of the combination is desired by professional. clinical.
  • an effective daily amount of zoledronic acid of about 0.5 to about 2000 mg can be used.
  • An effective daily amount of lovastatin can vary from about 5 mg to about 400 mg.
  • RhoB gene overexpression inhibitors depend on the particular compound or combination used, the specific condition being treated, the severity of the condition being treated, age, weight, sex, the extent of the disorder and the general physical condition of the specific patient, as well as other medication that the individual may be taking, as is well known by those skilled in the art. Furthermore, it is evident that said effective daily amount may decrease or increase depending on the response of the treated subject and / or depending on the evaluation of the prescribing physician of the compounds of the present invention. The variations of the effective daily amount mentioned above therefore serve only as guides.
  • Atomic nude mice four weeks old from Har ⁇ an (Barcelona, Spain) were acquired and kept in specific pathogen-free conditions.
  • the A549 adenocarcinoma cell line was obtained from Dr. Gazdar. All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum.
  • RNA 6000 nanochips (Agilent Technologies, Germany).
  • DNA was used to synthesize complementary probes (cDNA) using the one-cycle cDNA synthesis kit (Affimetrix).
  • cRNA was synthesized and labeled with cDNA as a template using the IVT labeling kit (Affimetrix), and purified by GeneChip Sample Cleanup Module (Affimetrix).
  • the labeled cRNAs were fragmented and hybridized to high density oligonucleotide microarrays of Affimetrix Human Genome U133 2.0 GeneChip according to the protocols described in Gene Expression Analysis Technical Manual (http://www.affimetrix.com). 3 microarrays were hybridized with 3 independent biological replicates per cell line of 3 different lines (called MlMl, M1M3, and M1M4). For each cell line, a group of three independent mRNA extractions was used to hybridize each GeneChip human high density oligonucleotide microarray. As controls, 6 lines of parental tumor cells were used: 3 transcribed with the luciferase vector and 3 untranslated. In total, a set of 15 samples was hybridized: 6 controls and 9 metastatic lines.
  • RNA from 70-80% of confluent cultures was isolated using Trizol RNA (2 ⁇ g) and DNase I, treated and transcribed in reverse using Superscript II reverse transcriptase and oligo (dT) primers.
  • An Assay-on-Demand TM (Applied Biosystems) was performed using a Gene Amp 7300 (Applied Biosystems) sequence detection system for RHOB according to the manufacturer's recommendations.
  • the average threshold cycle value (Ct) of the triplicate samples was used to calculate gene expression.
  • the PCR products were normalized to ⁇ -actin levels. The experiment was repeated three times with identical results.
  • microarray data was performed using the strategy and procedures described in the literature (Castellano et al. 2007).
  • the RMA algorithm was used for base correction, intra- and intermicroarray normalization and expression signal calculation (2-4).
  • the main test was performed by contrasting 4 control samples against 9 highly metastatic samples. However, with 3 different metastatic lines (MlMl, M1M3, MM1M4) performed in triplicate, multiple SAM analyzes were performed to find the genes that showed a significant differential expression that were also stable in different SAM tests. A cut of FDR ⁇ 0.20 was used for all differential expression calculations. All these procedures were applied using the statistical tools "R” and Bioconductor. After identifying the set of probes for differentially expressed genes, the corresponding array of expression values for all microarray hybridizations was analyzed using the "hclust" clustering algorithm implemented in "R".
  • This algorithm performs a hierarchical clustering analysis with complete binding to look for similarities between the sets of probes based on their expression values in the different microarray of chips analyzed.
  • the algorithm classifies probe sets into correlated groups that have similar expression profiles or expression signatures. This global strategy allowed the selection of significant key genes with similar expression profiles.
  • 10 ⁇ m 2D CT images were reconstructed using a standard retro-convolution projection procedure with a Shepp-Logan filter.
  • the images were stored in 3D groups with a voxel size of 19 ⁇ m x 19 ⁇ m x 23 ⁇ m.
  • Knock-down constructions Lentiviral constructions were obtained for human RhoB shRNA from Sigma (St. Louis, MO, USA). To obtain viral particles, packaging cells were transiected with 8 ⁇ g of DNA for each type of fusion using the classic phosphate procedure according to the manufacturer's recommendations. The pLKO "scramble" vector was used as a control (containing a shRHOB sequence in which the nucleotide bases have been randomly changed). Two days after the transection, the supernatants were centrifuged for 10 minutes at 600 g and filtered through a 0.45 ⁇ m pore cellulose acetate filter.
  • A549 was seeded into IxIO 5 cells and incubated overnight with viral supernatants in the presence of 4 ⁇ g / ml polybrene (Sigma). Forty-eight hours after infection, cell populations were incubated in medium containing the appropriate antibiotic for two additional weeks. Antibiotic resistant groups expanded and frozen in each cell passage. Isolation of colonies derived from a single cell (SCC) from long bones
  • Metastatic cells from the bone marrow were isolated from the lower extremities. Mice were sacrificed according to the approved protocols of the Local Animal Committee (University of Navarra, protocol 003-04). Long bones were removed, and washed from all soft tissues. The marrow cells were released by flushing, introducing 5-10 ml of CC-MEM medium containing 2 x penicillin / streptomycin with a 27 gauge needle into the distal epiphysis through the bone marrow compartment. The cell clusters were disaggregated by passing the medium containing the cells through the 27G needle syringe. The cells were seeded in expanded 10 or 15 cm plates for 5 days in a medium containing 0.4 mg / ml of G-418. This procedure was performed separately for each femur and tibia of 7 mice per group. SCCs were counted under the light of a microscope after staining with crystal violet.
  • the hind legs were removed, soft tissues of the femur were carefully removed, fixed for 24 h in 10% neutral buffered formalin, dehydrated and decalcified in Osteosoft® solution.
  • PD Progressive Disease
  • DF No Disease
  • the PD group included patients with a history of a completely resected primary lung cancer that relapsed (recurrence or metastasis) after a disease-free period of 6 months after surgery.
  • DF patients were the rest of individuals with completely resected primary lung cancer with no signs of the disease.
  • Patients who performed any treatment, whether radiotherapy or chemotherapy, after surgery were excluded from the analysis.
  • the study protocol was approved by the Local Ethical Committee. Written consent and questionnaires (background and follow-up) were obtained for each patient.
  • Inclusion criteria were: histology (NSCLC: adenocarcinoma), and absence of cancer in the 5 years prior to the lung cancer surgery, except melanoma or skin cancer.
  • the histological diagnosis was determined according to the WHO classification.
  • the pathological staging of the tumors was performed according to the international system for lung cancer. The follow-up period continued until October 31, 2007.
  • the recidivism time was calculated from the surgical intervention until the recidivism date.
  • Sections fixed to formalin and embedded in paraffin were used for immunohistochemical procedures.
  • the paraffin was extracted from the tissues and the sections were hydrated through a graduated series of ethanol. Endogenous peroxidase activity was stopped with 3% hydrogen peroxidase for 10 minutes.
  • Microwave antigen extraction was carried out with EDTA buffer (mM, pH 8) for 2 x 15 min. Non-specific binding sites were blocked with normal serum of 5% goat in TBS-Tween (wash buffer, Dako) for 30 minutes. Sections were incubated with rabbit anti-RhoB polyclonal antibody (sc-180, Santa Cruz Biotechnology, Inc) overnight at 4 0 C. The working dilution was: 1:75.
  • RhoB RhoB extension was scored as the percentage of positive cells (0-100%) and the intensity of staining was evaluated compared to a known external positive control (tidal 1+, light; 2+, moderate and 3+, intense ). Discordant independent readings were resolved by simultaneous review by both observers.
  • RhoB is frequently downregulated in non-small-cell lung cancer and resides in the 2p24 homozygous deletion region of a lung cancer cell line. Int J Cancer. 120 (3): 543-51.
  • Tusher VG Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Nati Acad Sci U S A 2001; 98: 5116-21.
  • TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. J Clin Invest 1999; 103: 197-206

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Abstract

The present invention relates to a method for prognosis of the progression of the disease of a subject suffering from adenocarcinoma of the lung, which comprises either measuring expression of the Rho B gene or measuring the number of copies of the Rho B gene in a sample taken from said subject suffering from adenocarcinoma of the lung, characterized in that detection of the overexpression or increase in the number of copies of the Rho B gene as compared with expression or the number of copies of same gene in a control sample is indicative of the risk of metastasis or reappearance or recurrence of neoplastic activity. The present invention further provides screening methods for drugs that are active against adenocarcinoma of the lung with a view to determining the efficacy of a therapeutic treatment model in a subject suffering from adenocarcinoma of the lung and also drugs that inhibit overexpression of the Rho B gene, for use as a drug or for use in the treatment of non-small-cell lung cancers (NSCLC).

Description

NUEVO BIOMARCADOR COMO DIANA TERAPÉUTICA EN CÁNCER DE PULMÓN NEW BIOMARCATOR AS A THERAPEUTIC DIANA IN CANCER DE PULMÓN
Campo de la invención La presente invención se refiere al área del diagnóstico y terapia del cáncer. En particular, se refiere a la sobreexpresión del gen RhoB y su impacto clínico en el cáncer de pulmón no microcítico (NSCLC) .Field of the Invention The present invention relates to the area of cancer diagnosis and therapy. In particular, it refers to RhoB gene overexpression and its clinical impact on non-small cell lung cancer (NSCLC).
Antecedentes de la invenciónBackground of the invention
El cáncer de pulmón es la causa principal de las muertes relacionadas con el cáncer a nivel mundial. Dado que tiende a extenderse, o producir metástasis, de manera muy temprana en su evolución, es un cáncer con alta letalidad y uno de los cánceres de más difícil tratamiento. El cáncer de pulmón se puede extender a ciertos órganos - particularmente las glándulas adrenales, el hígado, el cerebro, y los huesos - los órganos más habituales para la metástasis. A pesar de los avances en la cirugía, la quimioterapia y la radioterapia, la tasa de supervivencia ha mejorado poco en la última década, y la supervivencia a largo plazo es muy baja. Se ha establecido que el cáncer de pulmón surge como consecuencia de la acumulación de múltiples cambios genéticos en genes críticos que controlan la motilidad, proliferación, diferenciación y apoptosis celular. La identificación y caracterización de estos cambios genéticos que ocasionan el desarrollo y progresión del cáncer de pulmón es de gran interés para mejorar el diagnóstico precoz y el desarrollo de una terapia dirigida novedosa (Mazieres et al. 2004) .Lung cancer is the leading cause of cancer-related deaths worldwide. Since it tends to spread, or produce metastases, very early in its evolution, it is a cancer with high lethality and one of the most difficult treatment cancers. Lung cancer can spread to certain organs - particularly the adrenal glands, the liver, the brain, and the bones - the most common organs for metastasis. Despite advances in surgery, chemotherapy and radiotherapy, the survival rate has improved little in the last decade, and long-term survival is very low. It has been established that lung cancer arises as a result of the accumulation of multiple genetic changes in critical genes that control motility, proliferation, differentiation and cell apoptosis. The identification and characterization of these genetic changes that cause the development and progression of lung cancer is of great interest to improve early diagnosis and the development of a novel targeted therapy (Mazieres et al. 2004).
Los cánceres de pulmón, también conocidos como carcinomas broncogénicos, se clasifican ampliamente en dos tipos: cánceres de pulmón de célula pequeña o microcíticos (SCLC) y cánceres de pulmón de célula no pequeña o no microcíticos (NSCLC) . Esta clasificación se basa en la apariencia microscópica de las propias células tumorales. Los NSCLC son los cánceres de pulmón más habituales, representando aproximadamente el 80% de todos los cánceres de pulmón. Existen tres tipos principales de NSCLC que se denominan en base al tipo de células que se encuentran en el tumor: adenocarcinomas, carcinomas de células escamosas, carcinomas de células grandes y también se observan mezclas de diferentes tipos de NSCLC. Entre los cánceres de pulmón y particularmente NSCLC, el adenocarcinoma de pulmón es el más frecuente, representando un 30-35% de todos los casos de cáncer de pulmón. El adenocarcinoma de pulmón se define por la Organización Mundial de la Salud (OMS) como "un tumor epitelial maligno con patrones de crecimiento tubular, acinar, o papilar, y/o la producción de mucosidad por las células tumorales". Actualmente la OMS reconoce cuatro categorías de adenocarcinomas: acinar, papilar, bronquioloalveolar , y carcinoma sólido con formación de mucosidad. La mayoría de los adenocarcinomas aparecen en la periferia del pulmón, y, como resultado son frecuentemente asintomáticos en gran parte de su evolución. Frecuentemente se localizan a nivel subpleural y provocan una retracción pleural y un engrosamiento mediante radiografías.Lung cancers, also known as bronchogenic carcinomas, are broadly classified into two types: small or small cell lung cancers (SCLC) and non-small or non-small cell lung cancers microcytic (NSCLC). This classification is based on the microscopic appearance of the tumor cells themselves. NSCLCs are the most common lung cancers, representing approximately 80% of all lung cancers. There are three main types of NSCLC that are called based on the type of cells found in the tumor: adenocarcinomas, squamous cell carcinomas, large cell carcinomas, and mixtures of different types of NSCLC are also observed. Among lung cancers and particularly NSCLC, lung adenocarcinoma is the most frequent, accounting for 30-35% of all cases of lung cancer. Lung adenocarcinoma is defined by the World Health Organization (WHO) as "a malignant epithelial tumor with patterns of tubular, acinar, or papillary growth, and / or mucosal production by tumor cells." The WHO currently recognizes four categories of adenocarcinomas: acinar, papillary, bronchioloalveolar, and solid carcinoma with mucus formation. Most adenocarcinomas appear in the periphery of the lung, and, as a result, they are frequently asymptomatic in much of their evolution. They are often located at the subpleural level and cause pleural retraction and thickening by radiographs.
Muchos estudios han examinado la potencial utilidad de marcadores moleculares en el diagnóstico precoz y la terapia dirigida. Una familia particularmente interesante de marcadores son las proteínas Ras, las cuales regulan importantes funciones celulares que varían desde la diferenciación hasta la proliferación celular. Funcionan como interruptores moleculares, entre los estados de unión a GDP inactivo y los estados de unión a GTP activo. El oncogén ras juega un papel fundamental en la transformación maligna. Ras se encuentra mutado dando lugar a una forma deficiente de GTPasa que conduce a su activación constitutiva dando lugar a la proliferación descontrolada en aproximadamente el 50% del adenocarcinoma de pulmón. Los miembros estrechamente relacionados de la familia de Ras, tales como guanosina trifosfatasas pequeñas (GTPasas) homologas de Ras (Rho) , también están implicados en la regulación de una serie de procesos celulares, tales como la organización del citoesqueleto de actina, señalización inducida por estrés genotóxico, y la transformación celulares. Estudios recientes han confirmado adicionalmente el papel de las proteínas Rho en el cáncer al demostrar su implicación en la transformación, supervivencia, invasión, metástasis, y angiogénesis celulares. Además, los análisis en tumores humanos demuestran la sobreexpresión de varias proteínas Rho en cánceres de mama, de RhoA en tumores de células germinales testiculares, y de RhoC en adenocarcinoma pancreático, en melanoma, y en cáncer de mama (Mazieres et al. 2004) .Many studies have examined the potential utility of molecular markers in early diagnosis and targeted therapy. A particularly interesting family of markers are Ras proteins, which regulate important cellular functions that vary from differentiation to cell proliferation. They function as molecular switches, between inactive GDP binding states and active GTP binding states. The ras oncogene plays a fundamental role in the malignant transformation Ras is mutated resulting in a deficient form of GTPase that leads to its constitutive activation resulting in uncontrolled proliferation in approximately 50% of lung adenocarcinoma. The closely related members of the Ras family, such as small guanosine triphosphatases (GTPases) homologs of Ras (Rho), are also involved in the regulation of a series of cellular processes, such as the organization of the actin cytoskeleton, signaling induced by genotoxic stress, and cell transformation. Recent studies have further confirmed the role of Rho proteins in cancer by demonstrating their involvement in cell transformation, survival, invasion, metastasis, and angiogenesis. In addition, analyzes in human tumors demonstrate the overexpression of several Rho proteins in breast cancers, RhoA in testicular germ cell tumors, and RhoC in pancreatic adenocarcinoma, in melanoma, and in breast cancer (Mazieres et al. 2004) .
Mientras que la mayoría de las proteínas Rho han demostrado tener una función positiva en la proliferación y la transformación de tumores malignos, la función específica de RhoB en estos procesos parece ser más divergente .While most Rho proteins have been shown to have a positive role in the proliferation and transformation of malignant tumors, the specific role of RhoB in these processes seems to be more divergent.
Por un lado, Mazieres et al. (2004) mostraron que la proteína RhoB se expresaba en pulmón normal y disminuyó bruscamente a lo largo de la progresión del cáncer de pulmón en estadios iniciales, concluyendo que la pérdida de expresión de RhoB ocurre muy frecuentemente en carcinogénesis de pulmón, reforzando su actividad potencial como un gen supresor de tumores. Sato et al. (2007) analizaron las alteraciones en NSCLC de RhoB, demostrando que la expresión de RhoB está frecuentemente regulada de manera negativa en NSCLCs mediante múltiples mecanismos, y sugiriendo que RhoB es un gen supresor de tumores en NSCLC.On the one hand, Mazieres et al. (2004) showed that RhoB protein was expressed in normal lung and decreased sharply throughout the progression of early stage lung cancer, concluding that loss of RhoB expression occurs very frequently in lung carcinogenesis, reinforcing its potential activity as a tumor suppressor gene. Sato et al. (2007) analyzed the changes in RhoB NSCLC, demonstrating that RhoB expression is frequently regulated negatively in NSCLCs by multiple mechanisms, and suggesting that RhoB is a tumor suppressor gene in NSCLC.
US7135463 describe el uso de la proteina RhoB y sus variantes para inhibir el crecimiento, migración, invasión, metástasis de células cancerosas, la transformación de células tumorales, y/o para modular la señalización oncogénica, en el que la introducción de RhoB directa o indirectamente a través de una secuencia de ácidos nucleicos que codifica RhoB, en una célula tumoral transformada o una célula cancerosa disminuye la fosforilación de las proteínas Erk y Akt que inhiben el mecanismo de supervivencia celular Pl3-quinasa/Akt e impulsan la muerte celular programada o apoptosis.US7135463 describes the use of RhoB protein and its variants to inhibit growth, migration, invasion, metastasis of cancer cells, the transformation of tumor cells, and / or to modulate oncogenic signaling, in which the introduction of RhoB directly or indirectly through a nucleic acid sequence encoding RhoB, in a transformed tumor cell or a cancer cell decreases the phosphorylation of Erk and Akt proteins that inhibit the mechanism of cellular survival Pl3-kinase / Akt and boost programmed cell death or apoptosis .
De manera similar, WO2007011415 describe el uso de variantes de la proteína RhoB para inhibir el crecimiento, metástasis, invasión, migración de células cancerosas, la transformación de células tumorales, y/o para modular la señalización oncogénica. Los polipéptidos variantes de RhoB se introducen directa o indirectamente a través de una secuencia de ácidos nucleicos que codifica el polipéptido variante de RhoB, en una célula tumoral transformada o una célula cancerosa, en el que el polipéptido variante de RhoB suprime el crecimiento tumoral e impulsa la apoptosis.Similarly, WO2007011415 describes the use of RhoB protein variants to inhibit growth, metastasis, invasion, cancer cell migration, tumor cell transformation, and / or to modulate oncogenic signaling. RhoB variant polypeptides are introduced directly or indirectly through a nucleic acid sequence encoding the RhoB variant polypeptide, into a transformed tumor cell or a cancer cell, in which the RhoB variant polypeptide suppresses tumor growth and drives apoptosis
Por otro lado, el documento WO2008031910 describe varias líneas celulares de cáncer de pulmón no microcítico que pueden experimentar metástasis hasta el hueso con gran eficacia. Particularmente, describe líneas celulares específicas derivadas de una línea celular de carcinoma bronquioalveolar (A459) que comparten un patrón de expresión génica común caracterizado en la sobreexpresión de por lo menos 3, 4, 5, ó 6 genes seleccionados entre ILIl, PITXl, HDAC4, RHOB, ROBOl, y SLC26A2.On the other hand, WO2008031910 describes several non-small cell lung cancer cell lines that can experience bone metastasis very effectively. In particular, it describes specific cell lines derived from a carcinoma cell line bronchioalveolar (A459) that share a common gene expression pattern characterized in the overexpression of at least 3, 4, 5, or 6 genes selected from ILIl, PITXl, HDAC4, RHOB, ROBOl, and SLC26A2.
La detección precoz del cáncer de pulmón ha sido desde hace tiempo un objetivo perseguido por los profesionales sanitarios y los patólogos. Actualmente, la etapa de la enfermedad, en base al sistema TNM (tumor primario / nodulo linfático / metástasis distante) , es el factor individual más importante en la determinación de la supervivencia de los pacientes con cáncer de pulmón. La identificación de pacientes con una mayor probabilidad de metástasis, reaparición o reincidencia del cáncer es un foco principal en la investigación molecular del cáncer. Como tal, y el conocimiento de cambios moleculares iniciales puede tener fines predictivos, ya que reflejan frecuentemente la duración de la supervivencia del paciente o la sensibilidad a la terapia. En este contexto, existe la necesidad de marcadores adicionales que pueden servir para el pronóstico en pacientes con NSCLC, particularmente pacientes con adenocarcinoma de pulmón. Además, existe la necesidad de biomarcadores validados que se puedan aplicar directamente y utilizar en oncología clínica.Early detection of lung cancer has long been a goal pursued by health professionals and pathologists. Currently, the stage of the disease, based on the TNM system (primary tumor / lymph node / distant metastasis), is the most important individual factor in determining the survival of patients with lung cancer. The identification of patients with a higher probability of metastasis, recurrence or recurrence of cancer is a main focus in molecular cancer research. As such, and the knowledge of initial molecular changes can have predictive purposes, since they often reflect the duration of patient survival or sensitivity to therapy. In this context, there is a need for additional markers that can be used for prognosis in patients with NSCLC, particularly patients with lung adenocarcinoma. In addition, there is a need for validated biomarkers that can be applied directly and used in clinical oncology.
En un esfuerzo por identificar y caracterizar los genes diana claves implicados en la evolución del NSCLC, particularmente del adenocarcinoma de pulmón, los inventores han hallado sorprendentemente que RhoB juega un papel crítico en la metástasis ósea, al aumentar la motilidad celular e impulsar la colonización ósea. La sobreexpresión de RhoB en el adenocarcinoma de pulmón representa un biomarcador de pronóstico nuevo y potente potencialmente útil en el tratamiento clínico del cáncer de pulmón .In an effort to identify and characterize the key target genes involved in the evolution of NSCLC, particularly lung adenocarcinoma, the inventors have surprisingly found that RhoB plays a critical role in bone metastasis, by increasing cell motility and boosting bone colonization. . RhoB overexpression in lung adenocarcinoma represents a new and potent prognostic biomarker potentially useful in the clinical treatment of cancer of lung
Dado que actualmente los resultados del tratamiento estándar del adenocarcinoma de pulmón son generalmente escasos, existe la necesidad de agentes terapéuticos nuevos que puedan prolongar o mejorar la tasa de respuestaSince the results of standard treatment of lung adenocarcinoma are currently scarce, there is a need for new therapeutic agents that can prolong or improve the response rate
(RR) , la respuesta completa (CR) , la respuesta parcial(RR), the complete answer (CR), the partial answer
(PR), la enfermedad estable (SD), el tiempo hasta progresión (TTP), la supervivencia libre de progresión (PFS), la supervivencia global (GS), o el beneficio clínico, en pacientes afectados.(PR), stable disease (SD), time to progression (TTP), progression free survival (PFS), overall survival (GS), or clinical benefit, in affected patients.
Los bisfosfonatos están indicados para una amplia variedad de enfermedades relacionadas con el metabolismo óseo, incluyendo la osteoporosis, la enfermedad de Paget, mieloma múltiple, y metástasis óseas secundarias a tumores sólidos avanzados. En pacientes con tumores, las metástasis óseas pueden dar lugar a eventos relacionados con el esqueleto (SREs), incluyendo fracturas patológicas, compresión de la columna vertebral, hipercalcemia, dolor óseo que requiere radioterapia paliativa, y cirugía ortopédica. Estas complicaciones esqueléticas conducen frecuentemente a la pérdida de independencia funcional y a la disminución de la calidad de vida y, de este modo, el objetivo de la terapia es prevenir o retrasar la aparición de SREs para preservar la independencia. Los bisfosfonatos son el medicamento de elección para el tratamiento de lesiones osteolíticas y osteoblásticas asociadas con la metástasis ósea y se ha observado que reducen significativamente el riesgo de SREs. (Costa et al. 2007)Bisphosphonates are indicated for a wide variety of diseases related to bone metabolism, including osteoporosis, Paget's disease, multiple myeloma, and bone metastases secondary to advanced solid tumors. In patients with tumors, bone metastases can lead to skeletal-related events (SREs), including pathological fractures, compression of the spine, hypercalcemia, bone pain that requires palliative radiotherapy, and orthopedic surgery. These skeletal complications frequently lead to the loss of functional independence and the decrease in the quality of life and, thus, the goal of therapy is to prevent or delay the onset of SREs to preserve independence. Bisphosphonates are the medication of choice for the treatment of osteolytic and osteoblastic lesions associated with bone metastases and have been found to significantly reduce the risk of SREs. (Costa et al. 2007)
En particular, Li et al. (2008) han demostrado que el ácido zoledrónico, un bifosfonato nitrogenado de tercera generación, es incapaz de inducir la apoptosis, pero ralentiza el crecimiento tumoral y prolonga la supervivencia para los cánceres de pulmón de célula no pequeña .In particular, Li et al. (2008) have shown that zoledronic acid, a third-generation nitrogen bisphosphonate, is unable to induce apoptosis, but slows tumor growth and prolongs Survival for non-small cell lung cancers.
Turner et al. (2007) discuten los efectos de la lovastatina, que pertenece al grupo de las estatinas, un inhibidor de la HMG-CoA reductasa sobre la expresión, actividad de la isoforma de Rho, y la asociación con inhibidores de la disociación de nucleótidos de guanina, proporcionando de esta manera un mecanismo molecular en el que las estatinas provocan una acumulación de Rho-GTP no prenilado .Turner et al. (2007) discuss the effects of lovastatin, which belongs to the statin group, an HMG-CoA reductase inhibitor on expression, Rho isoform activity, and association with guanine nucleotide dissociation inhibitors, thus providing a molecular mechanism in which statins cause an accumulation of non-pre-piled Rho-GTP.
Hawk et al. (1996) sugirieron que la lovastatina podía suprimir la formación de tumores de pulmón inducidos por el carcinógeno químico NNK, posiblemente en una fase promocional inicial, aunque esta supresión no parecía estar relacionada con la presencia de K-ras mutadas o a cambios en la expresión de K-ras.Hawk et al. (1996) suggested that lovastatin could suppress the formation of lung tumors induced by the chemical carcinogen NNK, possibly in an initial promotional phase, although this suppression did not appear to be related to the presence of mutated K-ras or changes in the expression of K-ras.
WO2004024165 describe una composición farmacéutica para el tratamiento de tumores malignos, en particular el mieloma múltiple (MM) , comprendiendo dicha composición en combinación con un bisfosfonato, por ejemplo ácido zoledrónico o una sal o éster, un inhibidor de 3-hidroxi- 3-metilglutaril-coenzima A (HMG-CoA) reductasa, para el uso simultáneo, secuencial o separado.WO2004024165 describes a pharmaceutical composition for the treatment of malignant tumors, in particular multiple myeloma (MM), said composition comprising in combination with a bisphosphonate, for example zoledronic acid or a salt or ester, a 3-hydroxy-3-methylglutaryl inhibitor -coenzyme A (HMG-CoA) reductase, for simultaneous, sequential or separate use.
WO2000016809 describe compuestos antisentido, composiciones y procedimientos para modular la expresión de RhoB. Las composiciones comprenden compuestos antisentido, particularmente oligonucleótidos antisentido, dirigidos a ácidos nucleicos que codifican RhoB. Se proporcionan procedimientos de utilización de estos compuestos para la modulación de la expresión de RhoB y para el tratamiento de enfermedades asociadas con la expresión de RhoB.WO2000016809 describes antisense compounds, compositions and procedures for modulating RhoB expression. The compositions comprise antisense compounds, particularly antisense oligonucleotides, directed to nucleic acids encoding RhoB. Procedures for using these compounds are provided for the modulation of RhoB expression and for the treatment of diseases associated with RhoB expression.
Como parte del estudio exhaustivo sobre la diana terapéutica de la presente invención, es decir la sobreexpresión de RhoB en NSCLC, tal como el adenocarcinoma de pulmón, los inventores han observado sorprendentemente que una combinación que comprende lovastatina y ácido zoledrónico mejora significativamente la supervivencia global cuando se compara con la administración de ácido zoledrónico solo.As part of the comprehensive study on the therapeutic target of the present invention, that is to say overexpression of RhoB in NSCLC, such as lung adenocarcinoma, the inventors have surprisingly observed that a combination comprising lovastatin and zoledronic acid significantly improves overall survival when It is compared with the administration of zoledronic acid alone.
Además, los inventores también han observado que la administración de células de pulmón transducidas lentiviralmente con varios ARNsh contra RhoB (shRhoB) a ratones inmunodeprimidos daba lugar a un descenso de su actividad prometastática, incluyendo una reducción muy acusada de las lesiones osteoliticas y la carga del tumor, en comparación con las células control.In addition, the inventors have also observed that the administration of lentivirally transduced lung cells with several rRNAs against RhoB (shRhoB) to immunocompromised mice resulted in a decrease in their promtatatic activity, including a very marked reduction of osteolytic lesions and the burden of tumor, compared to control cells.
Como tal, la combinación que comprende lovastatina y ácido zoledrónico, asi como agentes de ARNi basados en shRhoB, han demostrado ser útiles en el tratamiento de NSCLC, tal como el adenocarcinoma de pulmón, por ejemplo en la ralentización de la progresión de la metástasis ósea.As such, the combination comprising lovastatin and zoledronic acid, as well as shRhoB-based RNAi agents, have proven useful in the treatment of NSCLC, such as lung adenocarcinoma, for example in slowing the progression of bone metastasis. .
Descripción resumida de la invenciónSummary Description of the Invention
La presente invención se refiere a un procedimiento de pronóstico de la evolución de la enfermedad en un sujeto con adenocarcinoma de pulmón que comprende: • la medición de la expresión del gen de RhoB; o • la medición del número de copias del gen de RhoB; en una muestra de dicho sujeto con adenocarcinoma de pulmón, caracterizado porque la detección de la sobreexpresión o aumento en el número de copias del gen de RhoB en relación con la expresión o el número de copias del mismo gen en una muestra de control es indicativa del riesgo de metástasis, o reaparición o reincidencia de actividad neoplásica.The present invention relates to a method of prognosis of the evolution of the disease in a subject with lung adenocarcinoma comprising: • the measurement of RhoB gene expression; or • measurement of the number of copies of the RhoB gene; in a sample of said subject with lung adenocarcinoma, characterized in that the detection of overexpression or increase in the number of copies of the RhoB gene in relation to the expression or number of copies of the same gene in a control sample is indicative of the risk of metastasis, or recurrence or recurrence of neoplastic activity.
En una realización, la presente invención se refiere a un procedimiento de screening para fármacos activos contra el adenocarcinoma de pulmón, comprendiendo dicho procedimiento : a) la medición de la expresión del gen de RhoB, o la medición del número de copias del gen de RhoB, en una muestra con células de adenocarcinoma de pulmón en presencia de un fármaco, o la medición de la afinidad de unión ente un fármaco y un polipéptido RhoB; b) la comparación de la expresión o números de copias medidas del gen de RhoB, o la afinidad de unión de la etapa a) a una muestra de control; caracterizada porque una expresión o número de copias reducido del gen de RhoB, o una mayor afinidad de unión en la etapa a) , en relación con la expresión o el número de copias del mismo gen o la afinidad de unión del mismo polipéptido en una muestra de control es indicativa de la actividad del fármaco contra el adenocarcinoma de pulmón.In one embodiment, the present invention relates to a screening procedure for active drugs against lung adenocarcinoma, said method comprising: a) measuring the expression of the RhoB gene, or measuring the copy number of the RhoB gene , in a sample with lung adenocarcinoma cells in the presence of a drug, or the measurement of binding affinity between a drug and a RhoB polypeptide; b) comparing the expression or measured copy numbers of the RhoB gene, or the binding affinity of step a) to a control sample; characterized in that a reduced expression or copy number of the RhoB gene, or a higher binding affinity in step a), in relation to the expression or the number of copies of the same gene or the binding affinity of the same polypeptide in a sample Control is indicative of the activity of the drug against lung adenocarcinoma.
En una realización adicional, la presente invención se refiere a un procedimiento para determinar la eficacia de una pauta de tratamiento terapéutico en un sujeto con adenocarcinoma de pulmón, comprendiendo dicho procedimiento a) la medición de la expresión del gen de RhoB, o la medición del número de copias del gen de RhoB, en una muestra con células de adenocarcinoma de pulmón de dicho sujeto, generando de este modo un nivel inicial; b) la medición de la expresión del gen de RhoB, o la medición del número de copias del gen de RhoB, en una segunda muestra con células de adenocarcinoma de pulmón del mismo sujeto en un momento posterior a la administración de la pauta de tratamiento, obteniendo de este modo un nivel de prueba; y c) la comparación de los niveles inicial y de prueba; caracterizada porque una expresión o número de copias reducido del gen de RhoB en el nivel de prueba en relación con el nivel inicial es indicativa de que la pauta de tratamiento es eficaz en el sujeto.In a further embodiment, the present invention relates to a method for determining the efficacy of a therapeutic treatment regimen in a subject with lung adenocarcinoma, said method comprising a) measuring RhoB gene expression, or measuring number of copies of the RhoB gene, in a sample with lung adenocarcinoma cells of said subject, thereby generating an initial level; b) measurement of RhoB gene expression, or measurement of RhoB gene copy number, in a second sample with lung adenocarcinoma cells of the same subject at a time after the administration of the treatment regimen, thereby obtaining a level of evidence; and c) comparison of initial and test levels; characterized in that a reduced expression or number of copies of the RhoB gene at the test level in relation to the initial level is indicative that the treatment pattern is effective in the subject.
Se proporcionan además inhibidores de la sobreexpresión del gen de RhoB para su uso como medicamento o para su uso en el tratamiento de NSCLC. Particularmente, estos inhibidores se seleccionan entre:In addition, RhoB gene overexpression inhibitors are provided for use as a medicine or for use in the treatment of NSCLC. In particular, these inhibitors are selected from:
• un anticuerpo o fragmento del mismo que se une específicamente a una proteína RhoB, o un polipéptido que comprende una región de unión a RhoB;• an antibody or fragment thereof that specifically binds to a RhoB protein, or a polypeptide comprising a RhoB binding region;
• un péptido mimético del polipéptido Rhob;• a Rhob polypeptide mimetic peptide;
• un ácido nucleico inhibidor del gen de Rhob seleccionado entre un oligonucleótido antisentido, un ribozima, y un agente ARNi seleccionado entre ARNds y ARNsh; o• a Rhob gene inhibitor nucleic acid selected from an antisense oligonucleotide, a ribozyme, and an RNAi agent selected from RNAds and shRNA; or
• una combinación que comprende lovastatina y ácido zoledrónico, y/o sales farmacéuticamente aceptables de la misma.• a combination comprising lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof.
Más particularmente, se proporciona una molécula de ARNsh que tiene por lo menos un 70% de homología con la SEC ID No. 3, preferiblemente en forma de un vector retroviral, para su uso en la terapia génica de adenocarcinoma de pulmón. También particularmente, se proporciona la combinación que comprende lovastatina y ácido zoledrónico, y/o sales farmacéuticamente aceptables de los mismos para su uso en el tratamiento del adenocarcinoma de pulmón . Descripción de los dibujosMore particularly, an RNAsh molecule is provided that has at least 70% homology with SEQ ID No. 3, preferably in the form of a retroviral vector, for use in lung adenocarcinoma gene therapy. Also particularly, the combination comprising lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof is provided for use in the treatment of lung adenocarcinoma. Description of the drawings
Figura 1: Aislamiento de poblaciones metastáticas hacia el huesoFigure 1: Isolation of metastatic populations towards the bone
Se inocularon células de adenocarcinoma A549 por vía intracardiaca en ratones inmunodeprimidos . Después de la detección radiográfica de la metástasis ósea, se aislaron esas subpoblaciones, se expandieron en un cultivo, y se volvieron a inocular. Este procedimiento se repitió hasta obtener las subpoblaciones altamente metastáticas MlMl, M1M4, y M1M3. Éstas se volvieron a inocular en otros grupos de ratones, asi como la población parental. Su capacidad metastática se evaluó mediante el análisis de imágenes de microrradiograflas de los huesos largos, y curvas de supervivencia libre metástasis de Kaplan-Meier .A549 adenocarcinoma cells were inoculated intracardiacly in immunosuppressed mice. After radiographic detection of bone metastasis, these subpopulations were isolated, expanded in a culture, and inoculated again. This procedure was repeated until obtaining highly metastatic subpopulations MlMl, M1M4, and M1M3. These were inoculated again in other groups of mice, as well as the parental population. Its metastatic ability was evaluated by analyzing microradiograph images of the long bones, and Kaplan-Meier metastasis free survival curves.
Figura 2: Identificación de RhoB en subpoblaciones metastáticasFigure 2: Identification of RhoB in metastatic subpopulations
Mediante microarrays de expresión, se identificó RhoB como uno de los genes sobreexpresados en todas las subpoblaciones metastáticas. La sobreexpresión se validó mediante RT-PCR y mediante análisis Western blot.Through expression microarrays, RhoB was identified as one of the overexpressed genes in all metastatic subpopulations. Overexpression was validated by RT-PCR and by Western blot analysis.
Figura 3 : Preparación de inhibidor de RhoB en células de cáncer de pulmón. a. Preparación de la inhibición de RhoB mediante un vector lentiviral con ARNsh especifico para RhoB. Para el gen sobreexpresado se utilizó un vector retroviral . b. Efecto sobre la proliferación in vitro del inhibidor deFigure 3: Preparation of RhoB inhibitor in lung cancer cells. to. Preparation of RhoB inhibition by a lentiviral vector with rRNA specific for RhoB. For the overexpressed gene a retroviral vector was used. b. Effect on in vitro proliferation of the inhibitor of
RhoB y el vector de control negativo ("scramble") . No se observaron cambios en la proliferación in vitro medianteRhoB and the negative control vector ("scramble"). No changes in proliferation were observed in vitro by
MTT entre el inhibidor de RhoB y el vector "scramble".MTT between the RhoB inhibitor and the scramble vector.
Figura 4: La inhibición de RhoB disminuye la metástasis in vivo Se inocularon células con ARNsh contra RhoB y con un vector de control ("Scramble") . Después de 35 días tras la inoculación, se observó un descenso significativo del área radiográfica mediante el análisis de imágenes. Además, se verificó la disminución de la carga del tumor mediante bioluminiscencia .Figure 4: RhoB inhibition decreases metastasis in vivo Cells were inoculated with rRNA against RhoB and with a control vector ("Scramble"). After 35 days after inoculation, a significant decrease in the radiographic area was observed by image analysis. In addition, the decrease in tumor burden was verified by bioluminescence.
Figura 5: Curvas de la tasa de supervivencia para pacientes con niveles altos y bajos de RhoBFigure 5: Survival rate curves for patients with high and low RhoB levels
Se realizó una inmunohistoquímica de biopsias de pacientes diagnosticados con adenocarcinoma con un anticuerpo específico contra RhoB.An immunohistochemistry of biopsies of patients diagnosed with adenocarcinoma with a specific antibody against RhoB was performed.
Figura 6 : Efecto farmacológico de una combinación de lovastatina y ácido zoledrónico en pacientes con adenocarcinoma de pulmón y células tratadas in vitro.Figure 6: Pharmacological effect of a combination of lovastatin and zoledronic acid in patients with adenocarcinoma of the lung and cells treated in vitro.
A. Pauta de administración de vehículo, lovastatina (Lov) , ácido zoledrónico (ZA), y una combinación de lovastatina y ácido zoledrónico (Lov+ZA) .A. Vehicle administration schedule, lovastatin (Lov), zoledronic acid (ZA), and a combination of lovastatin and zoledronic acid (Lov + ZA).
B. Curvas de supervivencia global a los 40 días. Curvas de Kaplan-Meier que muestran la supervivencia global 40 días después de la inoculación celular de los diferentes grupos tratados: Vehículo (control), Lovastatina (Lov) (10 mg/Kg/d) , Ácido zoledrónico (ZA) (3 μg/Kg/d) , o una combinación (Lov+ZA) . * p< 0,05, *** p< 0,001 vs control; t p< 0,01 vs ZA (Test Mantel-Cox) . No hubo diferencias entre los grupos de Control y Lovastatina.B. Global survival curves at 40 days. Kaplan-Meier curves showing overall survival 40 days after cell inoculation of the different treated groups: Vehicle (control), Lovastatin (Lov) (10 mg / Kg / d), Zoledronic acid (ZA) (3 μg / Kg / d), or a combination (Lov + ZA). * p <0.05, *** p <0.001 vs control; t p <0.01 vs ZA (Mantel-Cox Test). There were no differences between the Control and Lovastatin groups.
C. Tratamiento con A549 TM MlMl en cultivo. Células MTT de A549 TM MlMl 24 h después de los diferentes tratamientos farmacológicos: vehículo (Control); Lovastatina (Lov) 1 μM; Ácido zoledrónico (ZA) 10 μM o combinación (Lov+ZA) (Lov lμM + ZA 10 μM) diluidos en medio de cultivo. La densidad de siembra 10.000 cls/ml; tratamiento 72 h después de la siembra en placas de cultivo de 96 pocilios. ** p< 0,001 vs . control; t p< 0,001 vs. Lov o ZA (ANOVA de una vía ANOVA seguido de test de Sheffé) . Valores, medias +/- SEM.C. Treatment with A549 TM MlMl in culture. MT5 cells of A549 TM MlMl 24 h after the different pharmacological treatments: vehicle (Control); Lovastatin (Lov) 1 μM; Zoledronic acid (ZA) 10 μM or combination (Lov + ZA) (Lov lμM + ZA 10 μM) diluted in culture medium. Planting density 10,000 cls / ml; 72 h treatment after planting in 96-well culture plates. ** p <0.001 vs. control; t p <0.001 vs. Lov or ZA (ANOVA one-way ANOVA followed by test of Sheffé). Values, means +/- SEM.
Figura 7: Se trataron células TM MlMl metastáticas de adenocarcinoma A549 durante 4 días con un vehículo (Control); lovastatina (Lov) 1 μM y 0,5 μM; ácido zoledrónico (ZA) 10 μM y 5 μM; y las combinaciones Lov 1 μM + ZA 10 μM; y Lov 0,5 μM + ZA 5 μM diluidos en medio de cultivo. La inoculación duró 2 días y la densidad fue 2500 células/pocilio. Las fotografías tomadas de los cultivos que siguieron los diferentes tratamientos mencionados muestran la eficacia de las combinaciones Lov 1 μM + ZA 10 μM y Lov 0,5 μM + ZA 5 μM.Figure 7: TM5 MlMl metastatic adenocarcinoma A549 cells were treated for 4 days with a vehicle (Control); Lovastatin (Lov) 1 μM and 0.5 μM; Zoledronic acid (ZA) 10 μM and 5 μM; and the 1 μM + 10 μM ZA combinations; and 0.5 μM Lov + 5 μM ZA diluted in culture medium. The inoculation lasted 2 days and the density was 2500 cells / well. The photographs taken of the cultures that followed the different treatments mentioned show the efficacy of the combinations 1 μM + 10 μM ZA and 0.5 μM Lov + 5 μM ZA.
Figura 8: Después de 24 horas se midió la viabilidad celular de células TM MlMl metastáticas de adenocarcinoma A549 en presencia de diferentes tratamientos farmacológicos. Los tratamientos fueron con vehículo (Control), lovastatina (Lov) 1 μM, ácido zoledrónico (ZA) 10 μM, y la combinación Lov 1 μM + ZA 10 μM (Lov+ZA) diluidos en medio de cultivo, y se aplicaron durante 72 h después de la siembra en placas de cultivo de 96 pocilios. La viabilidad celular se determinó midiendo la concentración de MTT, un colorante tetrazolio amarillo soluble en agua que se transforma en formazán violeta insoluble en agua únicamente en las mitocondrias de células vivas. Después de la solubilización de los cristales de formazán resultantes con isopropanol, la absorbancia de la solución se controló a 540 nm. Los valores se correlacionan directamente con el número de células vivas que permanecen en el cultivo al completar la incubación. Densidad de inoculación 1000 células/pocilio; ** p< 0,001 vs. control; t p< 0,001 vs . Lov o ZA (ANOVA de una vía seguido posteriormente de test Tamhane y contraste) . Valores, medias +/- SEM. Figura 9: Se trataron células TM MlMl metastáticas de adenocarcinoma A549 durante 24h con: vehículo (Control); Lovastatina 1 μM (Lov) ; Ácido zoledrónico 10 μM (ZA); y la combinación Lov lμM + ZA 10 μM (Lov+ZA) diluidos en medio de cultivo. El test se realizó en una placa de 96 pocilios; las células se sembraron (1000 células/pocilio) y se mantuvieron durante 72 h después del tratamiento. Se determinó la actividad de caspasa-3 mediante el ensayo con caspasa-Glo 3/7 luminiscente. Los ensayos se realizaron sobre 12 réplicas para cada tratamiento. ** p< 0,001 vs . control, Lov o ZA (ANOVA de una vía seguido posteriormente de test Tamhane y contraste) . Valores, medias +/- SEM. Los resultados demuestran que la combinación de lovastatina y ácido zoledrónico aumenta la actividad de caspasa 3/7.Figure 8: After 24 hours the cell viability of metastatic TM MlMl cells of adenocarcinoma A549 was measured in the presence of different pharmacological treatments. The treatments were with vehicle (Control), 1 μM lovastatin (Lov), 10 μM zoledronic acid (ZA), and the combination 1 μM + 10 μM ZA (Lov + ZA) diluted in culture medium, and applied for 72 h after sowing in 96-well culture plates. Cell viability was determined by measuring the concentration of MTT, a water-soluble yellow tetrazolium dye that is transformed into water-soluble violet formazan only in living cell mitochondria. After solubilization of the resulting formazan crystals with isopropanol, the absorbance of the solution was monitored at 540 nm. The values correlate directly with the number of living cells that remain in the culture at the completion of the incubation. Inoculation density 1000 cells / well; ** p <0.001 vs. control; t p <0.001 vs. Lov or ZA (one-way ANOVA followed subsequently by Tamhane test and contrast). Values, means +/- SEM. Figure 9: TM5 MlMl metastatic adenocarcinoma A549 cells were treated for 24 hours with: vehicle (Control); Lovastatin 1 μM (Lov); Zoledronic acid 10 μM (ZA); and the combination Lov lμM + 10 µM ZA (Lov + ZA) diluted in culture medium. The test was performed on a 96-well plate; The cells were seeded (1000 cells / well) and maintained for 72 h after treatment. Caspase-3 activity was determined by testing with luminescent caspase-Glo 3/7. The tests were performed on 12 replicates for each treatment. ** p <0.001 vs. control, Lov or ZA (one-way ANOVA followed subsequently by Tamhane test and contrast). Values, means +/- SEM. The results show that the combination of lovastatin and zoledronic acid increases caspase activity 3/7.
Figura 10: Se trató la línea celular humana de cáncer de pulmón no microcítico H727 derivada de tumor carcinoide durante 24 h con un vehículo (Control); lovastatina (Lov) 1 μM; ácido zoledrónico (ZA) 10 μM; y la combinación Lov 1 μM + ZA 10 μM diluidos en medio de cultivo. La incubación duró 6 días y la densidad fue de 5000 células/pocilio. Las fotografías tomadas de los cultivos que experimentan los diferentes tratamientos anteriores demuestran la eficacia de la combinación Lov 1 μM + ZA 10 μM.Figure 10: The H727 non-small cell lung cancer human cell line derived from carcinoid tumor was treated for 24 h with a vehicle (Control); 1 μM lovastatin (Lov); Zoledronic acid (ZA) 10 μM; and the combination 1 μM Lov + 10 μM ZA diluted in culture medium. The incubation lasted 6 days and the density was 5000 cells / well. The photographs taken of the cultures undergoing the different previous treatments demonstrate the effectiveness of the 1 μM Lov + 10 μM ZA combination.
Se midió la viabilidad celular con MTT de la misma línea celular humana H727 en presencia de los mismos tratamientos. Las células se sembraron y se trataron durante 24 h con vehículo (Control); Lovastatina (Lov) 1 μM; Ácido zoledrónico (ZA) 10 μM o la combinación Lov lμM + ZA 10 μM (Lov+ZA) diluidos en medio de cultivo. Densidad de siembra 1000 células/pocilio; ** p< 0,001 vs . control; t p< 0,001 vs. Lov o ZA (ANOVA de una vía seguido posteriormente de test Tamhane y contraste) . Valores, medias +/- SEM.The cell viability with MTT of the same human H727 cell line was measured in the presence of the same treatments. The cells were seeded and treated for 24 hours with vehicle (Control); Lovastatin (Lov) 1 μM; Zoledronic acid (ZA) 10 μM or the combination Lov lμM + ZA 10 μM (Lov + ZA) diluted in culture medium. Planting density 1000 cells / well; ** p <0.001 vs. control; t p <0.001 vs. Lov or ZA (one-way ANOVA followed subsequently Tamhane test and contrast). Values, means +/- SEM.
Figura 11: Se trató la linea celular H727 durante 24 h con un vehiculo (Control); lovastatina (Lov) 5 μM; ácido zoledrónico (ZA) 5 μM; y la combinación Lov 5 μM + ZA 5 μM diluidos en medio de cultivo. La incubación duró 6 dias y la densidad fue de 5000 células/pocilio. Las fotografías tomadas de los cultivos que experimentan los diferentes tratamientos anteriores demuestran la eficacia de la combinación Lov 5 μM + ZA 5 μM.Figure 11: The H727 cell line was treated for 24 hours with a vehicle (Control); 5 μM lovastatin (Lov); Zoledronic acid (ZA) 5 μM; and the combination 5 μM Lov + 5 μM ZA diluted in culture medium. The incubation lasted 6 days and the density was 5000 cells / well. The photographs taken of the cultures undergoing the different previous treatments demonstrate the effectiveness of the 5 μM Lov + 5 μM ZA combination.
Se midió la viabilidad celular con MTT de la misma linea celular humana de cáncer de pulmón no microcitico H727 derivada de tumor carcinoide en presencia de los mismos tratamientos. Las células se sembraron y trataron durante 24 h con vehiculo (Control); Lovastatina (Lov) 5 μM; Ácido zoledrónico (ZA) 5 μM o la combinación Lov 5μM + ZA 5 μM (Lov+ZA) diluidos en medio de cultivo. Densidad de siembra 1000 células/pocilio; ** p< 0,001 vs . control; t p< 0,001 vs. Lov o ZA (ANOVA de una via seguido posteriormente de test Tamhane y contraste) . Valores, medias +/- SEM.The cell viability with MTT of the same human cell line of non-small cell lung cancer H727 derived from carcinoid tumor was measured in the presence of the same treatments. The cells were seeded and treated for 24 hours with vehicle (Control); Lovastatin (Lov) 5 μM; Zoledronic acid (ZA) 5 μM or the combination 5 μM Lov + 5 μM ZA (Lov + ZA) diluted in culture medium. Planting density 1000 cells / well; ** p <0.001 vs. control; t p <0.001 vs. Lov or ZA (one-way ANOVA followed subsequently by Tamhane test and contrast). Values, means +/- SEM.
Figura 12: Se trató la linea celular H727 durante 72 h con un vehiculo (Control); lovastatina (Lov) 5 μM; ácido zoledrónico (ZA) 5 μM; y la combinación Lov 5 μM + ZA 5 μM diluidos en medio de cultivo. La incubación duró 4 dias y la densidad fue de 5000 células/pocilio. Las fotografías tomadas de los cultivos que experimentan los diferentes tratamientos anteriores demuestran la eficacia de la combinación Lov 5 μM + ZA 5 μM.Figure 12: The H727 cell line was treated for 72 hours with a vehicle (Control); 5 μM lovastatin (Lov); Zoledronic acid (ZA) 5 μM; and the combination 5 μM Lov + 5 μM ZA diluted in culture medium. The incubation lasted 4 days and the density was 5000 cells / well. The photographs taken of the cultures undergoing the different previous treatments demonstrate the effectiveness of the 5 μM Lov + 5 μM ZA combination.
Se midió la viabilidad celular con MTT de la misma linea celular H727 en presencia de los mismos tratamientos. Las células se sembraron y trataron durante 72 h con vehiculo (Control); Lovastatina (Lov) 5 μM; Ácido zoledrónico (ZA) 5 μM o la combinación Lov 5μM + ZA 5 μM (Lov+ZA) diluidos en medio de cultivo. Densidad de siembra 1000 células/pocilio; ** p< 0,001 vs . control; t P < 0,001 vs . Lov o ZA (ANOVA de una vía seguido posteriormente de test Tamhane y contraste) . Valores, medias +/- SEM.The cell viability with MTT of the same H727 cell line was measured in the presence of the same treatments. Cells were seeded and treated for 72 hours with vehicle (Control); Lovastatin (Lov) 5 μM; Zoledronic acid (ZA) 5 μM or the combination 5 μM Lov + 5 μM ZA (Lov + ZA) diluted in culture medium. Planting density 1000 cells / well; ** p <0.001 vs. control; t P <0.001 vs. Lov or ZA (one-way ANOVA followed subsequently by Tamhane test and contrast). Values, means +/- SEM.
Figura 13: Se trató la linea celular humana de cáncer de pulmón no microcitico H460 derivada de un carcinoma de células grandes durante 24 h con un vehículo (Control); lovastatina (Lov) 1 μM; ácido zoledrónico (ZA) 10 μM; y la combinación Lov 1 μM + ZA 10 μM diluidos en medio de cultivo. La incubación duró 5 dias y la densidad fue de 1500 células/pocilio. Las fotografías tomadas de los cultivos que experimentan los diferentes tratamientos anteriores demuestran la eficacia de la combinación Lov 1 μM + ZA 10 μM.Figure 13: The H460 non-small cell lung cancer human cell line derived from a large cell carcinoma was treated for 24 h with a vehicle (Control); 1 μM lovastatin (Lov); Zoledronic acid (ZA) 10 μM; and the combination 1 μM Lov + 10 μM ZA diluted in culture medium. The incubation lasted 5 days and the density was 1500 cells / well. The photographs taken of the cultures undergoing the different previous treatments demonstrate the effectiveness of the 1 μM Lov + 10 μM ZA combination.
Se midió la viabilidad celular con MTT de la misma linea celular H460 en presencia de los mismos tratamientos. Las células se sembraron y trataron durante 24 h con vehículoThe cell viability with MTT of the same H460 cell line was measured in the presence of the same treatments. Cells were seeded and treated for 24 hours with vehicle
(Control); Lovastatina (Lov) 1 μM; Ácido zoledrónico (ZA)(Control); Lovastatin (Lov) 1 μM; Zoledronic acid (ZA)
10 μM o la combinación Lov lμM + ZA 10 μM (Lov+ZA) diluidos en medio de cultivo. Densidad de siembra 1000 células/pocilio; ** p< 0,001 vs . control; t P< 0,001 vs .10 μM or the combination Lov lμM + ZA 10 μM (Lov + ZA) diluted in culture medium. Planting density 1000 cells / well; ** p <0.001 vs. control; t P <0.001 vs.
Lov o ZA (ANOVA de una via seguido posteriormente de testLov or ZA (one way ANOVA followed subsequently by test
Tamhane y contraste) . Valores, medias +/- SEM.Tamhane and contrast). Values, means +/- SEM.
Figura 14: Se trató la linea celular H460 durante 24 h con un vehículo (Control); lovastatina (Lov) 5 μM; ácido zoledrónico (ZA) 5 μM; y la combinación Lov 5 μM + ZA 5 μM diluidos en medio de cultivo. La incubación duró 5 dias y la densidad fue de 1500 células/pocilio. Las fotografías tomadas de los cultivos que experimentan los diferentes tratamientos anteriores demuestran la eficacia de la combinación Lov 5 μM + ZA 5 μM.Figure 14: The H460 cell line was treated for 24 hours with a vehicle (Control); 5 μM lovastatin (Lov); Zoledronic acid (ZA) 5 μM; and the combination 5 μM Lov + 5 μM ZA diluted in culture medium. The incubation lasted 5 days and the density was 1500 cells / well. The photographs taken of the crops experienced by the different Previous treatments demonstrate the effectiveness of the 5 μM + 5 μM ZA combination.
Se midió la viabilidad celular con MTT de la misma linea celular H460 en presencia de los mismos tratamientos. Las células se sembraron y trataron durante 24 h con vehículoThe cell viability with MTT of the same H460 cell line was measured in the presence of the same treatments. Cells were seeded and treated for 24 hours with vehicle
(Control); Lovastatina (Lov) 5 μM; Ácido zoledrónico (ZA)(Control); Lovastatin (Lov) 5 μM; Zoledronic acid (ZA)
5 μM o la combinación Lov 5μM + ZA 5 μM (Lov+ZA) diluidos en medio de cultivo. Densidad de siembra 1000 células/pocilio; ** p< 0,001 vs . control; t P< 0,001 vs .5 μM or the combination 5 μM Lov + 5 μM ZA (Lov + ZA) diluted in culture medium. Planting density 1000 cells / well; ** p <0.001 vs. control; t P <0.001 vs.
Lov o ZA (ANOVA de una via seguido posteriormente de testLov or ZA (one way ANOVA followed subsequently by test
Tamhane y contraste) . Valores, medias +/- SEM.Tamhane and contrast). Values, means +/- SEM.
Figura 15: Se trató la linea celular H460 durante 72 h con un vehículo (Control); lovastatina (Lov) 5 μM; ácido zoledrónico (ZA) 5 μM; y la combinación Lov 5 μM + ZA 5 μM diluidos en medio de cultivo. La incubación duró 3 dias y la densidad fue de 1500 células/pocilio. Las fotografías tomadas de los cultivos que experimentan los diferentes tratamientos anteriores demuestran la eficacia de la combinación Lov 5 μM + ZA 5 μM.Figure 15: The H460 cell line was treated for 72 hours with a vehicle (Control); 5 μM lovastatin (Lov); Zoledronic acid (ZA) 5 μM; and the combination 5 μM Lov + 5 μM ZA diluted in culture medium. The incubation lasted 3 days and the density was 1500 cells / well. The photographs taken of the cultures undergoing the different previous treatments demonstrate the effectiveness of the 5 μM Lov + 5 μM ZA combination.
Se midió la viabilidad celular con MTT de la misma linea celular H460 en presencia de los mismos tratamientos. Las células se sembraron y trataron durante 72 h con vehículo (Control); Lovastatina (Lov) 5 μM; Ácido zoledrónico (ZA) 5 μM o la combinación Lov 5μM + ZA 5 μM (Lov+ZA) diluidos en medio de cultivo. Densidad de siembra 1000 células/pocilio; ** p< 0,001 vs . control; t P < 0,001 vs . Lov o ZA (ANOVA de una via seguido posteriormente de test Tamhane y contraste) . Valores, medias +/- SEM.The cell viability with MTT of the same H460 cell line was measured in the presence of the same treatments. Cells were seeded and treated for 72 hours with vehicle (Control); Lovastatin (Lov) 5 μM; Zoledronic acid (ZA) 5 μM or the combination 5 μM Lov + 5 μM ZA (Lov + ZA) diluted in culture medium. Planting density 1000 cells / well; ** p <0.001 vs. control; t P <0.001 vs. Lov or ZA (one-way ANOVA followed subsequently by Tamhane test and contrast). Values, means +/- SEM.
Descripción de la invenciónDescription of the invention
La presente invención se refiere a un procedimiento de pronóstico de la evolución de la enfermedad en un sujeto con adenocarcinoma de pulmón que comprende: a) la medición de la expresión del gen de RhoB; o b) la medición del número de copias del gen de RhoB; en una muestra de dicho sujeto con adenocarcinoma de pulmón, caracterizado porque la detección de la sobreexpresión o el aumento en el número de copias del gen de RhoB en relación con la expresión o el número de copias del mismo gen en una muestra control es indicativa de riesgo para la metástasis, o reaparición o reincidencia de actividad neoplásica.The present invention relates to a method of prognosis of the evolution of the disease in a subject with lung adenocarcinoma comprising: a) the measurement of RhoB gene expression; b) measurement of the number of copies of the RhoB gene; in a sample of said subject with lung adenocarcinoma, characterized in that the detection of overexpression or the increase in the number of copies of the RhoB gene in relation to the expression or the number of copies of the same gene in a control sample is indicative of risk for metastasis, or recurrence or recurrence of neoplastic activity.
El término "pronóstico de la evolución de la enfermedad" se refiere a proporcionar información con respecto al impacto de la presencia de adenocarcinoma de pulmón, por ejemplo, según se determina mediante los procedimientos de la presente invención, en la futura salud de un sujeto, o en otras palabras, el pronóstico de la evolución de la enfermedad proporciona una predicción de cómo progresará la enfermedad en un sujeto y si existe la posibilidad de recuperación. Como tal, los procedimientos de la presente invención, tal como se describirá a continuación, se pueden utilizar para determinar la progresión del cáncer; por ejemplo, de carcinoma de pulmón in situ a metástasis, en particular al hueso, o reaparición o reincidencia de la actividad neoplásica.The term "prognosis of disease progression" refers to providing information regarding the impact of the presence of lung adenocarcinoma, for example, as determined by the methods of the present invention, in the future health of a subject, or in other words, the prognosis of the evolution of the disease provides a prediction of how the disease will progress in a subject and whether there is a possibility of recovery. As such, the methods of the present invention, as will be described below, can be used to determine the progression of cancer; for example, from lung carcinoma in situ to metastasis, in particular to bone, or recurrence or recurrence of neoplastic activity.
En una realización de la presente invención, el pronóstico de la evolución de la enfermedad comprende la determinación de un resultado clínico seleccionado del grupo que consiste en velocidad de respuesta (RR) , respuesta completa (CR) , respuesta parcial (PR) , enfermedad estable (SD), tiempo hasta la progresión (TTP), supervivencia global (OS), y beneficio clínico, que comprende respuesta completa (CR) , respuesta parcial (PR) , y enfermedad estable (SD) . El término "velocidad de respuesta (RR) " se refiere al porcentaje de pacientes cuyo cáncer disminuye o desaparece después del tratamiento.In one embodiment of the present invention, the prognosis of disease progression comprises the determination of a clinical outcome selected from the group consisting of response speed (RR), complete response (CR), partial response (PR), stable disease (SD), time to progression (TTP), overall survival (OS), and clinical benefit, which includes complete response (CR), partial response (PR), and stable disease (SD). The term "response speed (RR)" refers to the percentage of patients whose cancer decreases or disappears after treatment.
El término "respuesta completa (CR) " se refiere a la desaparición de todos los signos del cáncer en respuesta al tratamiento. Esto no siempre significa que el cáncer se haya curado. También se denomina remisión completa.The term "complete response (CR)" refers to the disappearance of all signs of cancer in response to treatment. This does not always mean that the cancer has been cured. It is also called complete remission.
El término "respuesta parcial (PR) " se refiere a un descenso en el tamaño de un tumor, o en la extensión del cáncer en el cuerpo, en respuesta al tratamiento. También se denomina remisión parcial.The term "partial response (PR)" refers to a decrease in the size of a tumor, or in the extent of cancer in the body, in response to treatment. It is also called partial remission.
El término "enfermedad estable (SD)" se refiere a un cáncer que no crece ni decrece en extensión o gravedad.The term "stable disease (SD)" refers to a cancer that does not grow or decrease in extent or severity.
El término "tiempo hasta la progresión (TTP)" es un sinónimo para "supervivencia libre de progresión (PFS)" y se refiere a una medición del tiempo después de que se haya diagnosticado o tratado una enfermedad, hasta que la enfermedad empieza a empeorar. Aquí la medición para cada paciente es igual al tiempo transcurrido desde el inicio del tratamiento en un paciente en una prueba -tal como se define en el protocolo de pruebas- hasta la detección de una progresión tumoral -tal como se define en el protocolo de pruebas - o la aparición de cualquier fatalidad, lo que sea primero. Si se detuvo la observación del paciente (por ejemplo, al final del estudio) después de un periodo y no se observó ningún evento, entonces este tiempo de observación se denomina "censurado".The term "time to progression (TTP)" is a synonym for "progression-free survival (PFS)" and refers to a measurement of time after a disease has been diagnosed or treated, until the disease begins to worsen . Here the measurement for each patient is equal to the time elapsed from the start of treatment in a patient in a test - as defined in the test protocol - until the detection of a tumor progression - as defined in the test protocol - or the appearance of any fatality, whichever comes first. If patient observation was stopped (for example, at the end of the study) after a period and no event was observed, then this observation time is called "censored."
El término "supervivencia global" (OS)" es un sinónimo para "tiempo hasta la muerte (TTD)" o "tasa de supervivencia" y se refiere al porcentaje de sujetos en un estudio que han sobrevivido durante un periodo definido de tiempo. Este porcentaje se indica habitualmente junto con el periodo de tiempo desde la fecha del diagnóstico o tratamiento. Habitualmente, la supervivencia global se mide desde el día de la cirugía hasta la muerte relacionada con el tumor. Aquí, la medición para cada paciente es igual al tiempo transcurrido desde el inicio del tratamiento de un paciente en una prueba -tal como se define en el protocolo- hasta la aparición de cualquier fatalidad. Si se detuvo la observación del paciente, por ejemplo, al final del estudio, después de un periodo t y el paciente sobrevivió a este tiempo, entonces este tiempo de observación t se denomina "censurado".The term "global survival" (OS) "is a synonym for" time to death (TTD) "or" rate of survival "and refers to the percentage of subjects in a study who have survived for a defined period of time. This percentage is usually indicated along with the period of time from the date of diagnosis or treatment. Usually, overall survival is measured from the day of the surgery until the death related to the tumor Here, the measurement for each patient is equal to the time elapsed from the beginning of the treatment of a patient in a test -as defined in the protocol- until the appearance of any fatality If the observation of the patient was stopped, for example, at the end of the study, after a period t and the patient survived this time, then this observation time t is called "censored".
El término "sujeto" se refiere a un organismo afectado de cáncer de pulmón, particularmente cáncer de pulmón de célula no pequeña, más particularmente adenocarcinoma de pulmón. Este sujeto puede ser un mamífero, preferiblemente un humano, rata, ratón, o un primate no humano. Habitualmente el sujeto es un paciente.The term "subject" refers to an organism affected by lung cancer, particularly non-small cell lung cancer, more particularly lung adenocarcinoma. This subject may be a mammal, preferably a human, rat, mouse, or a non-human primate. Usually the subject is a patient.
El término "metástasis" se refiere al movimiento o expansión de células cancerígenas de un órgano o tejido a otro. Las células cancerígenas se expanden normalmente a través del torrente sanguíneo o el sistema linfático. El término "metástasis" se refiere también a la formación de focos de tumores secundarios en crecimiento progresivo en puntos discontinuos a la lesión primaria. El proceso metastático es un mecanismo con múltiples etapas en que una célula de cáncer metastático se escapa del tumor primario, entra en la circulación, invade un punto de tejido distante y desarrolla un tumor macroscópico en el sitio diana. El término "actividad neoplásica" se refiere a una proliferación celular no regulada o anormalmente regulada y el proceso de angiogénesis o la formación de nueva vasculatura que da soporte a un neoplasma en el microambiente endotelial alrededor del neoplasma. El término "neoplasma" se refiere a células tumorales, células que presentan una proliferación no regulada o anormalmente regulada como resultado de la inestabilidad o mutación genética, y un endotelio en el que las células endoteliales presentan una proliferación no regulada o anormalmente regulada como resultado de un estado patogénico .The term "metastasis" refers to the movement or expansion of cancer cells from one organ or tissue to another. Cancer cells normally expand through the bloodstream or lymphatic system. The term "metastasis" also refers to the formation of foci of secondary tumors in progressive growth at discontinuous points to the primary lesion. The metastatic process is a multi-stage mechanism in which a metastatic cancer cell escapes from the primary tumor, enters the circulation, invades a distant tissue point and develops a macroscopic tumor at the target site. The term "neoplastic activity" refers to an unregulated or abnormally regulated cell proliferation and the process of angiogenesis or the formation of new vasculature that supports a neoplasm in the endothelial microenvironment around the neoplasm. The term "neoplasm" refers to tumor cells, cells that have an unregulated or abnormally regulated proliferation as a result of instability or genetic mutation, and an endothelium in which endothelial cells have an unregulated or abnormally regulated proliferation as a result of a pathogenic state.
El término "muestra", tal como se utiliza en la presente invención, se refiere a una composición que se obtiene o deriva de un sujeto de interés que contienen una entidad celular y/u otra entidad molecular que va a caracterizarse y/o identificarse en base a, por ejemplo, características físicas, bioquímicas, químicas y/o fisiológicas. Cualquier muestra adecuada en la que se puede determinar la sobreexpresión del gen de RhoB o el número de copias del mismo está incluida en el alcance de la presente invención .The term "sample", as used in the present invention, refers to a composition that is obtained or derived from a subject of interest that contains a cellular entity and / or another molecular entity to be characterized and / or identified in based on, for example, physical, biochemical, chemical and / or physiological characteristics. Any suitable sample in which the overexpression of the RhoB gene or the number of copies thereof can be determined is included within the scope of the present invention.
En una realización, la muestra es un material biológico obtenido de un sujeto, y puede ser cualquier muestra orgánica, muestra de tejido, muestra de célula, fluido corporal, precipitado de fluido corporal, o una muestra de lavado aislada de un sujeto que tiene un cáncer de pulmón o con el riesgo de un cáncer de pulmón (por ejemplo, en base al historial familiar o al historial personal, tal como fumador crónico) . Una muestra se puede obtener de un sujeto de cualquiera de las maneras utilizadas en dispositivos clínicos para obtener una muestra que comprende la célula o ácido nucleico requerido, es decir ADN o ARN. Por ejemplo, las muestras se pueden obtener a partir de muestras quirúrgicas recientes, congeladas o muestras en parafina o biopsias de un órgano o tejido que comprenden el ácido nucleico o célula adecuados a ensayar. Las muestras adecuadas utilizadas en procedimientos para la detección o pronóstico del cáncer se pueden obtener a partir de una muestra de mama, conductos o lóbulos de los tejidos mamarios, muestra del tracto gastrointestinal (por ejemplo, una biopsia de un pólipo) , una muestra de hígado, una muestra de tejido pancreático, una muestra de médula ósea, una muestra de piel, una muestra de nodulo linfático, una muestra de riñon, una muestra de pulmón, una muestra de músculo, una muestra de hueso, una muestra de cerebro, o similar. La muestra puede derivar de un fluido biológico, por ejemplo, sangre, médula ósea, fluido cerebroespinal, fluido peritoneal, fluido pleural, fluido linfático, suero, plasma, jugo pancreático, orina, quilo, heces, eyaculado, esputo, aspirado de los pezones, fluido de los conductos, saliva, muestras obtenidas con hisopos de conductos orgánicos, muestras de lavado de colon, y muestras de cepillado. Los tejidos y fluidos corporales se pueden recoger utilizando cualquiera de los procedimientos conocidos en la técnica. Si es apropiado, la muestra se puede obtener mediante la utilización de un procedimiento de aspiración.In one embodiment, the sample is a biological material obtained from a subject, and it can be any organic sample, tissue sample, cell sample, body fluid, body fluid precipitate, or an isolated wash sample from a subject having a lung cancer or with the risk of lung cancer (for example, based on family history or personal history, such as chronic smoker). A sample may be obtained from a subject in any of the ways used in clinical devices to obtain a sample comprising the required cell or nucleic acid, i.e. DNA or RNA For example, samples may be obtained from recent, frozen or paraffin surgical samples or biopsies of an organ or tissue comprising the nucleic acid or cell suitable for testing. Suitable samples used in procedures for the detection or prognosis of cancer can be obtained from a breast sample, ducts or lobes of the breast tissues, sample from the gastrointestinal tract (for example, a biopsy of a polyp), a sample of liver, a sample of pancreatic tissue, a bone marrow sample, a skin sample, a lymph node sample, a kidney sample, a lung sample, a muscle sample, a bone sample, a brain sample, or similar. The sample may be derived from a biological fluid, for example, blood, bone marrow, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymphatic fluid, serum, plasma, pancreatic juice, urine, kilo, feces, ejaculate, sputum, nipple suction , duct fluid, saliva, samples obtained with organic duct swabs, colon wash samples, and brush samples. Tissues and body fluids can be collected using any of the procedures known in the art. If appropriate, the sample can be obtained by using an aspiration procedure.
Entre las muestras habituales para la detección o el pronóstico de adenocarcinoma de pulmón se incluyen, sin limitación, células o tejido (por ejemplo, de una biopsia o autopsia) , tumores de pulmón sólidos, esputo, tos, lavado broncoalveolar, cepillados bronquiales, mucosa bucal, sangre periférica, sangre completa, concentrados de glóbulos rojos, concentrados de plaquetas, concentrados de leucocitos, proteínas de glóbulos rojos, plasma sanguíneo, plasma rico en plaquetas, un concentrado de plasma, un precipitado de cualquier fracción del plasma, un sobrenadante de cualquier fracción del plasma, fracciones de proteínas del plasma sanguíneo, proteínas u otros componentes sanguíneos purificados o parcialmente purificados, suero, tejido o muestras de biopsia con aguja fina y fluido pleural, etc. aislados de un sujeto con un cáncer de pulmón, o cualquier otro tumor, o cualquier otro extracto del mismo, obtenidos de un sujeto de prueba, voluntario sano o animal experimental.Typical samples for the detection or prognosis of lung adenocarcinoma include, without limitation, cells or tissue (for example, from a biopsy or autopsy), solid lung tumors, sputum, cough, bronchoalveolar lavage, bronchial brushes, mucosa buccal, peripheral blood, whole blood, red blood cell concentrates, platelet concentrates, leukocyte concentrates, red blood cell proteins, blood plasma, platelet rich plasma, a plasma concentrate, a precipitate of any fraction of the plasma, a supernatant of any fraction of the plasma, fractions of blood plasma proteins, proteins or other purified or partially purified blood components, serum, tissue or biopsy samples with fine needle and pleural fluid, etc. isolated from a subject with lung cancer, or any other tumor, or any other extract thereof, obtained from a test subject, healthy volunteer or experimental animal.
En algunas realizaciones, puede ser deseable separar las células de cáncer de pulmón de las células que no son de cáncer de pulmón en una muestra. En algunas realizaciones, puede ser deseable no separar las células de cáncer de pulmón de las células que no son de cáncer de pulmón en una muestra. En algunas realizaciones, una muestra puede estar sustancialmente libre de células tumorales, por ejemplo, contiene menos de un 1, 5, 10, 20, 30, 40, ó 50% de células tumorales en comparación con las células no tumorales en la muestra. En algunas realizaciones, las células no tumorales pueden ser células epiteliales o linfocitos. En el caso de evaluar el pronóstico de un sujeto con cáncer de pulmón, es preferible utilizar una muestra que contiene la célula de cáncer de pulmón. La muestra pueden ser células purificadas a partir de un tejido. Además, las muestras biológicas se pueden obtener de un sujeto o un sujeto en diferentes puntos de tiempo, incluyendo antes, durante y/o después de un tratamiento.In some embodiments, it may be desirable to separate lung cancer cells from non-lung cancer cells in a sample. In some embodiments, it may be desirable not to separate lung cancer cells from non-lung cancer cells in a sample. In some embodiments, a sample may be substantially free of tumor cells, for example, it contains less than 1, 5, 10, 20, 30, 40, or 50% of tumor cells compared to non-tumor cells in the sample. In some embodiments, the non-tumor cells may be epithelial cells or lymphocytes. In the case of evaluating the prognosis of a subject with lung cancer, it is preferable to use a sample containing the lung cancer cell. The sample can be purified cells from a tissue. In addition, biological samples can be obtained from a subject or a subject at different time points, including before, during and / or after a treatment.
Una muestra también puede incluir, sin limitación, productos producidos en cultivos celulares por células normales o transformadas, por ejemplo, a través de tecnología de ADN recombinante o anticuerpos monoclonales . Una muestra también puede ser una célula o una línea celular creada en condiciones experimentales que no se aislan directamente de un sujeto. Una muestra también puede estar libre de células, derivada o sintetizada artificialmente. Una muestra puede ser de una células o tejido conocido por ser canceroso, sospechoso de ser canceroso, o que se cree que no es canceroso (por ejemplo, normal o control) .A sample may also include, without limitation, products produced in cell cultures by normal or transformed cells, for example, through recombinant DNA technology or monoclonal antibodies. A sample can also be a cell or a cell line created under experimental conditions that are not isolate directly from a subject. A sample can also be cell free, derived or artificially synthesized. A sample may be from a cell or tissue known to be cancerous, suspected of being cancerous, or believed to be non-cancerous (for example, normal or control).
El término "expresión" del gen de RhoB se refiere al proceso de conversión de información genética codificada en el gen de RhoB en ARN, por ejemplo, ARNm, ARNr, ARNt, o ARNsn, a través de la transcripción del gen, es decir, a través de la acción enzimática de una ARN polimerasa, y para genes que codifican proteínas, en la proteina a través de la traducción del ARNm maduro. La expresión génica se puede regular en muchas etapas del proceso. La "regulación positiva" o "activación" se refiere a la regulación que aumenta la producción de los productos de expresión génica, es decir, ARN o proteina, mientras que la "regulación negativa" o "represión" se refiere a la regulación que disminuye la producción. Las moléculas, por ejemplo, factores de transcripción, que están implicados en la regulación positiva o la regulación negativa se denominan frecuentemente "activadores" y "represores", respectivamente .The term "expression" of the RhoB gene refers to the process of converting genetic information encoded in the RhoB gene into RNA, for example, mRNA, rRNA, tRNA, or mRNA, through gene transcription, that is, through the enzymatic action of an RNA polymerase, and for genes that encode proteins, in the protein through the translation of mature mRNA. Gene expression can be regulated in many stages of the process. "Positive regulation" or "activation" refers to regulation that increases the production of gene expression products, that is, RNA or protein, while "negative regulation" or "repression" refers to regulation that decreases the production. Molecules, for example, transcription factors, which are involved in positive regulation or negative regulation are often referred to as "activators" and "repressors," respectively.
La expresión del gen de RhoB se puede medir a nivel de proteina, a nivel de ARNm, o a nivel de ADNc.RhoB gene expression can be measured at the protein level, at the mRNA level, or at the cDNA level.
Medición a nivel de proteína La expresión de la proteina derivada de RhoB se puede medir utilizando cualquiera de los inmunoensayos disponibles por la técnica. Entre los ejemplos de inmunoensayos útiles en la determinación del nivel de expresión de la proteina se incluyen, pero no se limitan a, inmunoprecipitación, radioinmunoensayo, ensayo de transferencia mediante Western, ensayo inmunofluorescente, inmunoensayo enzimático, ensayo quimioluminiscente, ensayo inmunohistoquimico, y ensayo inmunoabsorbente unido a enzima (ELISA) . Además, los inmunoensayos anteriores se pueden utilizar combinados, tales como inmunoprecipitación seguido de transferencia mediante Western. Los procedimientos anteriores se describen en Principies and Practice of Immunoassay, Price y Newman, eds . , Stochton Press, última ed. Dichos ensayos pueden ser inmunoensayos directos, indirectos, competitivos, o no competitivos tal como se describen por la técnica (Oelbrick, N. (1984) J. Clin. Chem. Clin. Biochem., 22:895-904) . La proteina a analizar mediante dichos procedimientos se puede obtener directamente de la muestra. Alternativamente, la proteina se puede obtener como un lisado crudo o se puede purificar adicionalmente mediante procedimientos conocidos por los expertos en la materia, incluyendo la cromatografía de inmunoafinidad utilizando anticuerpos para la proteína RhoB (Sambrook, J. et al (2001) en "Molecular Cloning: A Laboratory Manual", CoId Spring Harbor Press, Plainview, N. Y.) . Alternativamente, los niveles de proteína RhoB se pueden detectar mediante immunohistoquímica de secciones de tumores fijadas o congeladas.Protein level measurement RhoB-derived protein expression can be measured using any of the immunoassays available in the art. Examples of immunoassays useful in determining the level of protein expression include, but are not limited to, immunoprecipitation, radioimmunoassay, assay of Western blotting, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay, and enzyme-linked immunoabsorbent assay (ELISA). In addition, the above immunoassays can be used in combination, such as immunoprecipitation followed by Western blotting. The above procedures are described in Principies and Practice of Immunoassay, Price and Newman, eds. , Stochton Press, last ed. Such assays may be direct, indirect, competitive, or non-competitive immunoassays as described by the technique (Oelbrick, N. (1984) J. Clin. Chem. Clin. Biochem., 22: 895-904). The protein to be analyzed by such procedures can be obtained directly from the sample. Alternatively, the protein can be obtained as a crude lysate or can be further purified by methods known to those skilled in the art, including immunoaffinity chromatography using antibodies to the RhoB protein (Sambrook, J. et al (2001) in "Molecular Cloning: A Laboratory Manual ", CoId Spring Harbor Press, Plainview, NY). Alternatively, RhoB protein levels can be detected by immunohistochemistry of fixed or frozen tumor sections.
Para la detección de proteína RhoB mediante inmunoensayo, se pueden utilizar anticuerpos policlonales o monoclonales anti-RhoB. Si se desean anticuerpos policlonales, se puede utilizar suero que contiene anticuerpos policlonales para la proteína RhoB o los anticuerpos policlonales se pueden purificar de antígenos presentes en el suero mediante cromatografía de inmunoafinidad. Alternativamente, los anticuerpos monoclonales dirigidos contra RhoB pueden ser producidos fácilmente por un experto en la materia. Los procedimientos de producción de anticuerpos monoclonales o policlonales son conocidos por un experto en la materia (Goding, J. W. (1996) Monoclonal antibodies: Principies and Practice, Plodermic Press, Inc., NY, N. Y., pág. 56-97; Hurn, B. A. L. et al. (1980) Meth. Enzymol., 70:104-141) .For the detection of RhoB protein by immunoassay, polyclonal or monoclonal anti-RhoB antibodies can be used. If polyclonal antibodies are desired, serum containing polyclonal antibodies to the RhoB protein can be used or the polyclonal antibodies can be purified from antigens present in the serum by immunoaffinity chromatography. Alternatively, monoclonal antibodies directed against RhoB can be easily produced by one skilled in the art. The methods of producing monoclonal or polyclonal antibodies are known to a person skilled in the art. (Goding, JW (1996) Monoclonal antibodies: Principies and Practice, Plodermic Press, Inc., NY, NY, pp. 56-97; Hurn, BAL et al. (1980) Meth. Enzymol., 70: 104-141) .
Entre los inmunógenos adecuados que se pueden utilizar para producir anticuerpos policlonales o monoclonales se incluyen Usados celulares preparados a partir de células transíectadas con una proteína RhoB recombinante, una proteína RhoB recombinante o nativa parcial o sustancialmente purificada, o péptidos derivados de la secuencia de aminoácidos de RhoB. Los vectores y su uso en la producción de proteínas recombinantes es conocida por los expertos en la materia (Sambrook, J. et al. (2001) in "Molecular Cloning, A Laboratory Manual", CoId Spring Harbor Press, Plainview, N. Y.) .Suitable immunogens that can be used to produce polyclonal or monoclonal antibodies include Cellular used prepared from cells transcribed with a recombinant RhoB protein, a partially or substantially purified recombinant or native RhoB protein, or peptides derived from the amino acid sequence of RhoB. Vectors and their use in the production of recombinant proteins are known to those skilled in the art (Sambrook, J. et al. (2001) in "Molecular Cloning, A Laboratory Manual", CoId Spring Harbor Press, Plainview, N. Y.).
Los anticuerpos o fragmentos de unión a antígeno también se pueden producir mediante ingeniería genética. La tecnología para la expresión de los genes de tanto la cadena pesada como la cadena ligera en E. coli es la materia de las solicitudes de patente PCT; publicación número WO 901443, WO 901443, y WO 9014424 y en Huse et al. (1989) Science, 246:1275-1281.Antibodies or antigen binding fragments can also be produced by genetic engineering. The technology for the expression of both heavy chain and light chain genes in E. coli is the subject of PCT patent applications; Publication number WO 901443, WO 901443, and WO 9014424 and in Huse et al. (1989) Science, 246: 1275-1281.
Alternativamente, los anticuerpos anti-RhoB se pueden inducir mediante la administración de anticuerpos antiidiotipos como inmunógenos. De forma práctica, se utiliza un anticuerpo anti-RhoB purificado para inducir un anticuerpo antiidiotipo en un animal huésped.Alternatively, anti-RhoB antibodies can be induced by administration of anti-idiotype antibodies as immunogens. Practically, a purified anti-RhoB antibody is used to induce an anti-idiotype antibody in a host animal.
Alternativamente a los inmunoensayos, los anticuerpos se pueden utilizar in situ para detectar la proteína RhoB en la muestra. Los anticuerpos se pueden utilizar en inmunofluorescencia directa o indirecta. Alternativamente, la proteína RhoB se puede detectar in situ mediante el uso de anticuerpo anti-RhoB radiomarcado o mediante el uso de un anticuerpo anti-RhoB no marcado seguido de un segundo anticuerpo radiomarcado reactivo al anticuerpo anti-RhoB.Alternatively to immunoassays, antibodies can be used in situ to detect RhoB protein in the sample. The antibodies can be used in direct or indirect immunofluorescence. Alternatively, RhoB protein can be detected in in situ by the use of radiolabeled anti-RhoB antibody or by the use of an unlabeled anti-RhoB antibody followed by a second radiolabeled antibody reactive to the anti-RhoB antibody.
Los anticuerpos también se pueden utilizar para inmunoprecipitar la proteina RhoB a partir de una mezcla de proteínas. El uso de la inmunoprecipitación como técnica sensible y específica para detectar y cuantificar antígenos diana en mezclas de proteínas es bien conocida para los expertos en la materia (véase, Molecular Cloning, A Laboratory Manual, 2d Edition, (2001) CoId Spring Harbor Press) .Antibodies can also be used to immunoprecipitate RhoB protein from a mixture of proteins. The use of immunoprecipitation as a sensitive and specific technique to detect and quantify target antigens in protein mixtures is well known to those skilled in the art (see, Molecular Cloning, A Laboratory Manual, 2d Edition, (2001) CoId Spring Harbor Press) .
Los anticuerpos también se pueden fijar a soportes sólidos para su uso en el aislamiento de la proteína Rhob mediante cromatografía de inmunoafinidad. Las técnicas para la cromatografía de inmunoafinidad son bien conocidas en la técnica (Harlow, E. y Lañe, D. (1888) "Antibodies : A Laboratory Manual", CoId Spring Harbor Laboratory, CoId Spring Harbor, N. Y.) e incluyen técnicas para fijar anticuerpos a soportes sólidos, de manera que mantienen su actividad inmunoselectiva; las técnicas utilizadas pueden ser aquellas en las que los anticuerpos se adsorben al soporte, así como aquellas en las que los anticuerpos están unidos covalentemente al soporte.The antibodies can also be fixed to solid supports for use in the isolation of Rhob protein by immunoaffinity chromatography. Techniques for immunoaffinity chromatography are well known in the art (Harlow, E. and Lañe, D. (1888) "Antibodies: A Laboratory Manual", CoId Spring Harbor Laboratory, CoId Spring Harbor, NY) and include techniques for fixing antibodies to solid supports, so that they maintain their immunoselective activity; The techniques used may be those in which the antibodies are adsorbed to the support, as well as those in which the antibodies are covalently bound to the support.
Un anticuerpo adecuado para la medición de la expresión del gen de Rho B a nivel de proteína es Rho B (119) sc-180 disponible comercialmente de Santa Cruz Biotechnology, Inc. Rho B (119) es un anticuerpo policlonal de conejo purificado por afinidad desarrollado contra un péptido que se mapea en una región interna de Rho B de origen humano.An antibody suitable for the measurement of Rho B gene expression at the protein level is Rho B (119) sc-180 commercially available from Santa Cruz Biotechnology, Inc. Rho B (119) is an affinity purified rabbit polyclonal antibody developed against a peptide that is mapped into an internal Rho B region of human origin.
Los anticuerpos descritos anteriormente y los fragmentos de unión a los mismos se pueden suministrar en un kit de diagnóstico útil para la detección de alteraciones en la expresión de proteina Rhob.The antibodies described above and the fragments Binding to them can be supplied in a diagnostic kit useful for the detection of alterations in Rhob protein expression.
Medición a nivel de ARN.RNA level measurement.
La expresión del gen de RhoB se puede controlar a nivel de ARN mensajero (ARNm) según los procedimientos conocidos en la técnica. Los procedimientos que utilizan la hibridación de sondas de ácido nucleico al transcrito de RhoB, incluyendo la tecnología de microarray, visualización en gel, y transferencia mediante Northern, se pueden utilizar para medir la presencia y/o nivel de ARNm de RhoB. El ARNm también se puede evaluar utilizando técnicas de amplificación, tales como RT-PCR o RT-PCR cuantitativa a tiempo real.RhoB gene expression can be controlled at the level of messenger RNA (mRNA) according to methods known in the art. Procedures that utilize hybridization of RhoB transcript nucleic acid probes, including microarray technology, gel visualization, and Northern blotting, can be used to measure the presence and / or level of RhoB mRNA. The mRNA can also be evaluated using amplification techniques, such as real-time quantitative RT-PCR or RT-PCR.
El análisis de hibridación, que se basa en la especificidad de las interacciones de nucleótidos, utiliza oligonucleótidos o ADNc para identificar de manera selectiva o capturar ADN o ARNm de la composición específica de secuencias. La cantidad de ARNm o ADNc hibridada a una secuencia de captura conocida se puede determinar cuantitativa o cualitativamente, proporcionando de este modo la información sobre la representación relativa de la proteína RhoB. También se puede realizar la hibridación a arrays o chips tanto de ADN, ADNc, oligonucleóticos o ARN , donde éstos se pueden producir según cualquier procedimiento adecuado conocido por técnica. Por ejemplo, los procedimientos de producción de grandes arrays de oligonucleótidos se describen en US5.134.854, y US5.445.934 utilizando técnicas de síntesis dirigidas por luz. Utilizando un sistema controlado por ordenador, se convierte un array heterogéneo de monómeros, a través del acoplamiento simultáneo en un número de sitios de reacción, en un array heterogéneo de polímeros. Alternativamente, los microarrays se generan mediante la deposición de oligonucleótidos presintetizados sobre un sustrato sólido, por ejemplo tal como se describe en WO95/35505.Hybridization analysis, which is based on the specificity of nucleotide interactions, uses oligonucleotides or cDNAs to selectively identify or capture DNA or mRNA from the specific sequence composition. The amount of mRNA or cDNA hybridized to a known capture sequence can be determined quantitatively or qualitatively, thereby providing information on the relative representation of the RhoB protein. Hybridization can also be performed to arrays or chips of both DNA, cDNA, oligonucleotides or RNA, where these can be produced according to any suitable procedure known in the art. For example, the methods of producing large oligonucleotide arrays are described in US5,134,854, and US5,445,934 using light-directed synthesis techniques. Using a computer-controlled system, a heterogeneous array of monomers is converted, through simultaneous coupling at a number of reaction sites, into a heterogeneous array of polymers. Alternatively, the microarrays are generated by the deposition of presynthesized oligonucleotides on a solid substrate, for example as described in WO95 / 35505.
Los procedimientos basados en secuenciación son una alternativa. Estos procedimientos empezaron con el uso de marcadores de secuencia expresada (ESTs), y ahora incluyen procedimientos basados en marcadores cortos, tales como el análisis en serie de la expresión génica (SAGE) y la MPSS (Massively Parallel Signature Sequencing) . Como tal, los niveles de expresión de ARNm en una muestra de prueba se pueden determinar mediante la generación de una biblioteca de ESTs a partir de dicha muestra de prueba. La enumeración de la representación relativa de ESTs en la biblioteca se puede utilizar para aproximar la representación relativa de un transcrito génico en la muestra de partida. Los resultados de los análisis de EST de una muestra de prueba se puede entonces comparar con el análisis de EST de una muestra de referencia que comprende células de control para determinar los niveles de expresión relativos de un polinucleótido que codifica RhoB.Sequencing-based procedures are an alternative. These procedures began with the use of expressed sequence markers (ESTs), and now include procedures based on short markers, such as serial analysis of gene expression (SAGE) and MPSS (Massively Parallel Signature Sequencing). As such, mRNA expression levels in a test sample can be determined by generating a library of ESTs from said test sample. The enumeration of the relative representation of ESTs in the library can be used to approximate the relative representation of a gene transcript in the starting sample. The results of the EST analyzes of a test sample can then be compared with the EST analysis of a reference sample comprising control cells to determine the relative expression levels of a polynucleotide encoding RhoB.
El SAGE (Velculescu et al., Science (1995) 270:484) implica el aislamiento de marcadores de secuencia cortos únicos de una localización especifica en cada transcrito. Los marcadores de secuencia se concatenan, clonan y secuencian. La frecuencia de transcritos particulares en la muestra de prueba de partida está reflejada por el número de veces que el marcador de secuencia asociado se encuentra en la población de secuencias.SAGE (Velculescu et al., Science (1995) 270: 484) involves the isolation of unique short sequence markers from a specific location in each transcript. Sequence markers are concatenated, cloned and sequenced. The frequency of particular transcripts in the starting test sample is reflected by the number of times the associated sequence marker is found in the sequence population.
La MPSS, descrita por Brenner et al., Nature Biotechnology 18:630-634 (2000), es una estrategia de secuenciación que combina secuenciación no basada en gel con la clonación in vitro de millones de plantillas en microesferas separadas de 5 μm de diámetro. En primer lugar, se construye mediante clonación in vitro una biblioteca de microesferas de plantillas de ADN. Esto va seguido del montaje de un array plano de las microesferas que contienen plantillas en una células de flujo a una densidad elevada (habitualmente superior a 3χlO6 microesferas/cm2) . Los extremos libres de las plantillas clonadas en cada microesfera se analizan simultáneamente, utilizando un procedimiento de secuenciación basado en fluorescencia que no requiere la separación de fragmentos de ADN. Esta técnica permite la identificación eficaz y el aislamiento de muchos cientos de miles de secuencias individuales, la generación de una "biblioteca" de ARNs pequeños. La abundancia o frecuencia de aparición de cada secuencia diferente de una "biblioteca" de ARNs pequeños es indicativo de la cantidad en la muestra original de la que se obtuvo el ARN. Además, mediante la comparación de las secuencias, que tienen habitualmente 17-20 nucleótidos de longitud, con una base de datos de ADN genómico, es posible determinar los puntos en el ADN que actúan como origen para los ARNs pequeños. Las comparaciones con las anotaciones del genoma, bases de datos de ADNc, y otros datos se pueden utilizar frecuentemente para identificar los precursores de ARN más grandes de los ARNs pequeños. De manera más significativa, MPSS proporciona la capacidad para dirigir la biología del ARN pequeño a una escala amplia del genoma.MPSS, described by Brenner et al., Nature Biotechnology 18: 630-634 (2000), is a sequencing strategy that combines non-gel sequencing with in vitro cloning of millions of templates in separate microspheres of 5 μm in diameter. First, a DNA template microsphere library is constructed by in vitro cloning. This is followed by the assembly of a flat array of microspheres containing templates in a flow cell at a high density (usually greater than 3χO 6 microspheres / cm 2 ). The free ends of the cloned templates in each microsphere are analyzed simultaneously, using a fluorescence-based sequencing procedure that does not require separation of DNA fragments. This technique allows the efficient identification and isolation of many hundreds of thousands of individual sequences, the generation of a "library" of small RNAs. The abundance or frequency of occurrence of each different sequence of a "library" of small RNAs is indicative of the amount in the original sample from which the RNA was obtained. In addition, by comparing the sequences, which are usually 17-20 nucleotides in length, with a genomic DNA database, it is possible to determine the points in the DNA that act as the origin for small RNAs. Comparisons with genome annotations, cDNA databases, and other data can often be used to identify the largest RNA precursors of small RNAs. More significantly, MPSS provides the ability to direct the biology of small RNA to a broad genome scale.
Medición a nivel de ADNcCDNA level measurement
La expresión del gen de RhoB también se puede controlar a nivel de ADNc. Los niveles de expresión del gen de RhoB se determinan utilizando la reacción en cadena de la polimerasa de transcriptasa inversa (RT-PCR) . La RT-PCR es una técnica bien conocida en el sector que se basa en que la enzima transcriptasa inversa transcribe de forma inversa el ARNm para formar ADNc, que a continuación se puede amplificar en una reacción PCR estándar. Los protocolos y kits para llevar a cabo la RT-PCR son bien conocidos por los expertos en la materia y están disponibles comercialmente .RhoB gene expression can also be controlled at the cDNA level. RhoB gene expression levels are determined using the reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR is a well known technique in the sector that is based on the fact that the enzyme reverse transcriptase reverse transcribes the mRNA to form cDNA, which can then be amplified in a standard PCR reaction. The protocols and kits for carrying out the RT-PCR are well known to those skilled in the art and are commercially available.
La RT-PCR se puede llevar a cabo de una manera no cuantitativa. Dicha RT-PCR de punto final mide los cambios en los niveles de expresión utilizando tres procedimientos diferentes: relativo, competitivo y comparativo. Estos procedimientos tradicionales son bien conocidos en la técnica, por ejemplo, visualización del amplicón en una electroforesis en gel.RT-PCR can be carried out in a non-quantitative manner. Said end-point RT-PCR measures changes in expression levels using three different procedures: relative, competitive and comparative. These traditional procedures are well known in the art, for example, visualization of the amplicon in a gel electrophoresis.
En una realización alternativa, la RT-PCR se lleva a cabo a tiempo real y de manera cuantitativa. La PCR a tiempo real es una técnica utilizada para controlar el progreso de una reacción PCR en tiempo real. Al mismo tiempo, se puede cuantificar una cantidad relativamente pequeña de producto de PCR (ADN, ADNc o ARN) . La PCR a tiempo real se basa en la detección de la fluorescencia producida por una molécula informadora que aumenta a medida que progresa la reacción. Esto ocurre debido a la acumulación del producto de la PCR con cada ciclo de amplificación. Estas moléculas informadoras fluorescentes incluyen fluorocromos que se unen al ADN de doble cadena o sondas de secuencia especifica .In an alternative embodiment, the RT-PCR is carried out in real time and quantitatively. Real-time PCR is a technique used to monitor the progress of a real-time PCR reaction. At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified. Real-time PCR is based on the detection of fluorescence produced by a reporter molecule that increases as the reaction progresses. This occurs due to the accumulation of the PCR product with each amplification cycle. These fluorescent reporter molecules include fluorochromes that bind to double stranded DNA or specific sequence probes.
La RT-PCT cuantitativa a tiempo real o la PCR a tiempo real han sido descritas ampliamente en la bibliografía (véase, Gibson et al para un ejemplo inicial de la técnica) y son posibles una serie de técnicas para controlar el producto de PCR. Estas técnicas incluyen el uso de sondas hidrolíticas (Taqman®) , sondas en horquilla ("hairpin") (Molecular Beacons), parejas de sondas para la técnica de FRET (LightCycler ) , cebadores que incorporan una sonda en horquilla (Scorpion), sistema Plexor™, cebadores en horquilla (Amplifluor) , cebadores que incorporan secuencias complementarias de ADNzimas que dividen un sustrato informador incluido en la mezcla de reacción (DzyNA) , o colorantes fluorescentes (SYBR Green etc.) - Todas estas sondas, cebadores, o fluorocromos están disponibles comercialmente y están bien caracterizados .Quantitative real-time RT-PCT or real-time PCR has been described extensively in the literature (see, Gibson et al for an initial example of the technique) and a number of techniques are possible to control the PCR product. These techniques include the use of hydrolytic probes (Taqman®), hairpin probes (Molecular Beacons), pairs of probes for the FRET technique (LightCycler), primers incorporating a hairpin probe (Scorpion), Plexor ™ system, primers in hairpin (Amplifluor), primers incorporating complementary sequences of ADNzymes that divide a reporter substrate included in the reaction mixture (DzyNA), or fluorescent dyes (SYBR Green etc.) - All these probes, primers, or fluorochromes are commercially available and They are well characterized.
En una realización, la presente invención proporciona cebadores con las SEC ID NO 4-6 para realizar la RT-PCR o PCR de tiempo real. Los cebadores con SEC ID NO 4 y 5 son particularmente adecuados para su uso en el perfil de expresión del gen de RhoB utilizando RT-PCRIn one embodiment, the present invention provides primers with SEQ ID NO 4-6 for performing real-time RT-PCR or PCR. Primers with SEQ ID NO 4 and 5 are particularly suitable for use in the expression profile of the RhoB gene using RT-PCR
Figure imgf000034_0001
Figure imgf000034_0001
Tal como se ha mencionado en la presente invención, la RT- PCR cuantitativa a tiempo real o PCR a tiempo real produce una lectura de fluorescencia que se puede controlar de manera continua. Las técnicas de tiempo real son ventajosas porque mantienen la reacción en un "único tubo". Esto significa que no existe la necesidad de un análisis ulterior para obtener resultados, lo cual conduce a resultados obtenidos más rápidamente. Además, manteniendo la reacción en un medio de "único tubo" se reduce el riesgo de contaminación cruzada y permite una salida cuantitativa a partir de los procedimientos de la presente invención. Esto puede ser particularmente importante en la disposición clínica de la presente invención .As mentioned in the present invention, quantitative real-time RT-PCR or real-time PCR produces a fluorescence reading that can be monitored continuously. Real-time techniques are advantageous because they keep the reaction in a "single tube." This means that there is no need for further analysis to obtain results, which leads to results obtained more quickly. In addition, keeping the reaction in a "single tube" medium reduces the risk of cross contamination and allows quantitative output from the procedures of the present invention. This may be particularly important in the clinical arrangement of the present invention.
El reconocimiento de múltiples alteraciones de ADN puede aumentar la identificación eficaz del cáncer. Las sondas permiten que se midan múltiples tipos de ADN en la misma muestra (PCR multiplex) , ya que los colorantes fluorescentes con diferentes espectros de emisión se pueden unir a las diferentes sondas. La PCR Multiplex permite que los controles internos se coamplifiquen. Estas sondas de hibridación permiten un nivel de discriminación imposible de obtener con SYBR Green, ya que solo se hibridarán a verdaderas dianas en una PCR y no a cebadores-dímeros u otros productos no específicos.The recognition of multiple DNA alterations can increase the effective identification of cancer. The probes allow multiple types of DNA to be measured in the same sample (multiplex PCR), since fluorescent dyes with different emission spectra can bind to different probes. Multiplex PCR allows internal controls to be co-amplified. These hybridization probes allow a level of discrimination impossible to obtain with SYBR Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or other non-specific products.
Las variantes de la técnica de PCR básica tales como PCR anidada, y similares también se incluyen en el alcance de la presente invención. Ente los ejemplos se incluyen técnicas de amplificación isotérmica tales como NASBA, 3SR, TMA, y triamplificación, todas ellas conocidas en el sector y disponibles comercialmente . Otros procedimientos adecuados de amplificación incluyen la reacción en cadena de la ligasa (LCR) (Barringer et al, 1990), ensayo de Amplificación de Sondas dependiente de Ligandos Múltiples Específico de Metilación (MS-MLPA) (Nygrenet al., Nucleic Acids Res. 2005 ;33(14) el28; www.mlpa.com), amplificación selectiva de secuencias de polinucleótidos diana (US6, 410, 276 ) , reacción en cadena de la polimerasa con cebado con secuencias de consenso (US4, 437, 975) , reacción en cadena de la polimerasa con cebado arbitrario (WO90/06995) , pirosecuenciación, y amplificación por desplazamiento de corte (WO2004/067726 ) . La expresión génica en una muestra de prueba también se puede analizar utilizando la metodología de "differential display" (DD) . Esta familia de técnicas se basa en la amplificación aleatoria de fragmentos de ADNc generados mediante digestión por restricción, donde los fragmentos de ADNc se utilizan como identificadores únicos de genes, acoplados con la información sobre la longitud del fragmento o la localización del fragmento en el gen expresado. La representación relativa de un gen expresado en una muestra se puede entonces estimar en base a la representación relativa del fragmento asociado con el gen que está en el grupo de todos los posibles fragmentos. Los procedimientos y composiciones para llevar a cabo DD son conocidos en la técnica, véase, por ejemplo, US5.776.683; y US5.807.680.Variants of the basic PCR technique such as nested PCR, and the like are also included within the scope of the present invention. Examples include isothermal amplification techniques such as NASBA, 3SR, TMA, and triamplification, all known in the industry and commercially available. Other suitable amplification procedures include ligase chain reaction (CSF) (Barringer et al, 1990), Methylation Specific Multiple Ligand-dependent Probe Amplification assay (MS-MLPA) (Nygrenet al., Nucleic Acids Res. 2005; 33 (14) el28; www.mlpa.com), selective amplification of target polynucleotide sequences (US6, 410, 276), polymerase chain reaction with priming with consensus sequences (US4, 437, 975), Polymerase chain reaction with arbitrary priming (WO90 / 06995), pyrosequencing, and shear shift amplification (WO2004 / 067726). Gene expression in a test sample can also be analyzed using the "differential display" (DD) methodology. This family of techniques is based on the random amplification of cDNA fragments generated by restriction digestion, where cDNA fragments are used as unique gene identifiers, coupled with information about the fragment length or the location of the fragment in the gene expressed. The relative representation of a gene expressed in a sample can then be estimated based on the relative representation of the fragment associated with the gene that is in the group of all possible fragments. Methods and compositions for carrying out DD are known in the art, see, for example, US 5,776,683; and US5,807,680.
Las secuencias del ADN genómico, proteína, ARNm, y ADNc del gen de RhoB, o una región de las mismas, se proporcionan a continuación. De la misma manera, los cebadores y sondas descritas en la presente invención se proporcionan como las SEC ID No. 4-6.The genomic DNA, protein, mRNA, and cDNA sequences of the RhoB gene, or a region thereof, are provided below. In the same manner, the primers and probes described in the present invention are provided as SEQ ID NO: 4-6.
La secuencia de nucleótidos del ADN genómico de RhoB humana (NC_000002.10 ) (5'→3', 2367 pb) se proporciona en la siguiente secuencia SEC ID No. 1:The nucleotide sequence of the human RhoB genomic DNA (NC_000002.10) (5 '→ 3', 2367 bp) is provided in the following sequence SEQ ID NO: 1:
ATCTGCCACCGCAGTCTGGTTGGAGCTGTTGTCTTGTATGCTCAGCGAGGCCCGGAGA GACCCGGGAGAGAGCTAGGCCGAGTCCACCGCCCGAGTCTGCTGCCCGAGCCCGCGTT ACGCACAAAGCCGCCGATCCCCGGCCTGGGGTGAGCAGAGCGACCACCGCCCGGGAGC AGCGCGGCGAGACGCACGGTGCGCCCTATGCCCCCGCGCCCCCACCGCCCCCGCCGCG GCAGCCGAAGCGCAGCGAGAGAACGCGCCACCGCGGGGCCCGGGTGCAGCTAGCGACC CTCTCGCCACCTGCGCGCAGCCCGAGGTGAGCAGTGAGCGGCGAGCGGGAGGGCAGCG AGGCGTTCGCGGGCCCCCTCCTGCTGCCCGGGCCCGGCCCGCTCATGGCGGCCATCCG CAAGAAGCTGGTGGTGGTGGGCGACGGCGCGTGTGGCAAGACGTGCCTGCTGATCGTG TTCAGTAAGGACGAGTTCCCCGAGGTGTACGTGCCCACCGTCTTCGAGAACTATGTGG CCGACATTGAGGTGGACGGCAAGCAGGTGGAGCTGGCGCTGTGGGACACGGCGGGCCA GGAGGACTACGACCGCCTGCGGCCGCTCTCCTACCCGGACACCGACGTCATTCTCATG TGCTTCTCGGTGGACAGCCCGGACTCGCTGGAGAACATCCCCGAGAAGTGGGTCCCCG AGGTGAAGCACTTCTGTCCCAATGTGCCCATCATCCTGGTGGCCAACAAAAAAGACCT GCGCAGCGACGAGCATGTCCGCACAGAGCTGGCCCGCATGAAGCAGGAACCCGTGCGC ACGGATGACGGCCGCGCCATGGCCGTGCGCATCCAAGCCTACGACTACCTCGAGTGCT CTGCCAAGACCAAGGAAGGCGTGCGCGAGGTCTTCGAGACGGCCACGCGCGCCGCGCT GCAGAAGCGCTACGGCTCCCAGAACGGCTGCATCAACTGCTGCAAGGTGCTATGAGGG CCGCGCCCGTCGCGCCTGCCCCTGCCGGCACGGCTCCCCCTCCTGGACCAGTCCCCCG CGAGCCCGGAGAAGGGGAGACCCGTGTCCCACAAGGACCCCACCGGCCTGCCTGGCAT CTGTCTGCTGACGCCTCTGGCTTGCGCCAGGACTTGGCGTGGGCACCGGGCGCCCCCA TCCCAGTGTCTGTGTGCGTCCAGCTGTGTTGCACAGGCCTGGGCTCCCCACTGAGTGC CAAGGGTCCCCTGAGCATGCTTTTCTGAAGAGCCGGGCCTCAGAGTGTGTGGCTGTGT GTCTGTTCGACTCCCCTCGCCCCATTTTCACCCCACCCCCGCCTCTGATCCCCGGGGG CGAGATTGGCGCGGGAGTGTGGCCGCGCCCCATCAGATGTTCGCCCTTCACCAGCGGG AGCTTGATATCCCTTGTCTGTAACATAGACCCCGGGTACTGCGGGAGGGGAGGGCTGC TGGGGAGGATGGGGGGATGTTATATAAATATAGATATAATTTTATTTTCGGAGCTAAG ATGGTGTTATTTAAGGGTGGTGATGGGTGAGCGCTCTGGCCCAGGCTGGGCCAGACTC CCGCCCAAGCATGAACAGGACTTGACCATCTTTCCAACCCCTGGGGAAGACATTTGCA ACTGACTTGGGGAGGACACAGCTTCAGCACAGCCTCTCCTGCGGGCCAGCCCGCTGCG AACCCTCCACCAGCTACCGGAGGGAGGAGGGAGGATGCGCTGTGGGGTTGTTTTTGCC ATAAGCGAACTTTGTGCCTGTCCTAGAAGTGAAAATTGTTCAGTCCAAGAAACTGATG TTATTTGATTTATTTAAAGGCTAAAATTTGTTTTTTTATTCTTTGCACAATTGTTTCA TTGTTTGACACTTAATGCACTCGTCATTTGCATACGACAGTAGCATTCTGACCACACT TGTACGCTGTAACCTCATCTACTTCTGATGTTTTTAAAAAATGACTTTTAACAAGGAG AGGGAAAAGAAACCCACTAAATTTTGCTTTGTTTCCTTGAAGAATGTGGCAACACTGT TTTGTGATTTTATTTGTGCAGGTCATGCACACAGTTTTGATAAAGGGCAGTAACAAGT ATTGGGGCCTATTTTTTTTTTTTCCACAAGGCATTCTCTAAAGCTATGTGAAATTTTC TCTGCACCTCTGTACAGAGAATACACCTGCCCCTGTATATCCTTTTTTCCCCTCCCCT CCCTCCCAGTGGTACTTCTACTAAATTGTTGTCTTGTTTTTTATTTTTTAAATAAACT GACAAATGACAAAATGGTGAGCTTATGATGTTTACATAAAAGTTCTATAAGCTGTGTA TACAGTTTTTTATGTAAAATATTAAAAGACTATGATGATGACATTTAATCTGCCACCGCAGTCTGGTTGGAGCTGTTGTCTTGTATGCTCAGCGAGGCCCGGAGA GACCCGGGAGAGAGCTAGGCCGAGTCCACCGCCCGAGTCTGCTGCCCGAGCCCGCGTT ACGCACAAAGCCGCCGATCCCCGGCCTGGGGTGAGCAGAGCGACCACCGCCCGGGAGC AGCGCGGCGAGACGCACGGTGCGCCCTATGCCCCCGCGCCCCCACCGCCCCCGCCGCG GCAGCCGAAGCGCAGCGAGAGAACGCGCCACCGCGGGGCCCGGGTGCAGCTAGCGACC CTCTCGCCACCTGCGCGCAGCCCGAGGTGAGCAGTGAGCGGCGAGCGGGAGGGCAGCG AGGCGTTCGCGGGCCCCCTCCTGCTGCCCGGGCCCGGCCCGCTCATGGCGGCCATCCG CAAGAAGCTGGTGGTGGTGGGCGACGGCGCGTGTGGCAAGACGTGCCTGCTGATCGTG TTCAGTAAGGACGAGTTCCCCGAGGTGTACGTGCCCACCGTCTTCGAGAACTATGTGG CCGACATTGAGGTGGACGGCAAGCAGGTGGAGCTGGCGCTGTGGGACACGGCGGGCCA GGAGGACTACGACCGCCTGCGGCCGCTCTCCTACCCGGACACCGACGTCATTCTCATG TGCTTCTCGGTGGACAGCCCGGACTCGCTGGAGAACATCCCCGAGAAGTGGGTCCCCG AGGTGAAGCACTTCTGTCCCAATGTGCCCATCATCCTGGTGGCCAACAAAAAAGACCT GCGCAGCGACGAGCATGTCCGCACAGAGCTGGCCCGCATGAAGCAGGAACCCGTGCGC ACGGATGACGGCCGCGCCATGGCCGTGCGCATCCAAGCCTACGACTACCTCGAGTGCT CTGCCAAGACCAAGGAAGGCGTGCGCGAGGTCTTCGAGACGGCCACGCGCGCCGCGCT GCAGAAGCGCTACGGCTCCCAGAACGGCTGCATCAACTGCTGCAAGGTGCTATGAGGG CCGCGCCCGTCGCGCCTGCCCCTGCCGGCACGGCTCCCCCTCCTGGACCAGTCCCCCG CGAGCCCGGAGAAGGGGAGACCCGTGTCCCACAAGGACCCCACCGGCCTGCCTGGCAT CTGTCTGCTGACGCCTCTGGCTTGCGCCAGGACTTGGCGTGGGCACCGGGCGCCCCCA TCCCAGTGTCTGTGTGCGTCCAGCTGTGTTGCACAGGCCTGGGCTCCCCACTGAGTGC CAAGGGTCCCCTGAGCATGCTTTTCTGAAGAGCCGGGCCTCAGAGTGTGTGGCTGTGT GTCTGTTCGACTCCCCTCGCCCCATTTTCACCCCACCCCCGCCTCTGATCCCCGGGGG CGAGATTGGCGCGGGAGTGTGGCCGCGCCCCATCAGATGTTCGCCCTTCACCAGCGGG AGCTTGATATCCCTTGTCTGTAACATAGACCCCGGGTACTGCGGGAGGGGAGGGCTGC TGGGGAGGATGGGGGGATGTTATATAAATATAGATATAATTTTATTTTCGGAGCTAAG ATGGTGTTATTTAAGGGTGGTGATGGGTGAGCGCTCTGGCCCAGGCTGGGCCAGAC TC CCGCCCAAGCATGAACAGGACTTGACCATCTTTCCAACCCCTGGGGAAGACATTTGCA ACTGACTTGGGGAGGACACAGCTTCAGCACAGCCTCTCCTGCGGGCCAGCCCGCTGCG AACCCTCCACCAGCTACCGGAGGGAGGAGGGAGGATGCGCTGTGGGGTTGTTTTTGCC ATAAGCGAACTTTGTGCCTGTCCTAGAAGTGAAAATTGTTCAGTCCAAGAAACTGATG TTATTTGATTTATTTAAAGGCTAAAATTTGTTTTTTTATTCTTTGCACAATTGTTTCA TTGTTTGACACTTAATGCACTCGTCATTTGCATACGACAGTAGCATTCTGACCACACT TGTACGCTGTAACCTCATCTACTTCTGATGTTTTTAAAAAATGACTTTTAACAAGGAG AGGGAAAAGAAACCCACTAAATTTTGCTTTGTTTCCTTGAAGAATGTGGCAACACTGT TTTGTGATTTTATTTGTGCAGGTCATGCACACAGTTTTGATAAAGGGCAGTAACAAGT ATTGGGGCCTATTTTTTTTTTTTCCACAAGGCATTCTCTAAAGCTATGTGAAATTTTC TCTGCACCTCTGTACAGAGAATACACCTGCCCCTGTATATCCTTTTTTCCCCTCCCCT CCCTCCCAGTGGTACTTCTACTAAATTGTTGTCTTGTTTTTTATTTTTTAAATAAACT GACAAATGACAAAATGGTGAGCTTATGATGTTTACATAAAAGTTCTATAAGCTGTGTA TACAGTTTTTTATGTAAAATATTAAAAGACTATGATGATGACATTTA
La secuencia de aminoácidos de la proteína RhoB humana (NP_004031.1 ) (196 aa) se proporciona en la siguiente secuencia SEC ID No. 2: MAAIRKKLVVVGDGACGKTCLLIVFSKDEFPEVYVPTVFENYVADIEVDGKQVELALW DTAGQEDYDRLRPLSYPDTDVILMCFSVDSPDSLENIPEKWVPEVKHFCPNVPIILVA NKKDLRSDEHVRTELARMKQEPVRTDDGRAMAVRIQAYDYLECSAKTKEGVREVFETA TRAALQKRYGSQNGCINCCKVLThe amino acid sequence of the human RhoB protein (NP_004031.1) (196 aa) is provided in the following sequence SEQ ID NO: 2: MAAIRKKLVVVGDGACGKTCLLIVFSKDEFPEVYVPTVFENYVADIEVDGKQVELALW DTAGQEDYDRLRPLSYPDTDVILMCFSVDSPDSLENIPEKWVPEVKHFCPNVPIILVA NKKDLRSKTQRKVKVKVKVKVKVKVKTKVKVKVKVKTKRKVKVKVKVKVKTKRKVKVKTKRKVKVKTKRKVGKRKVKG
La secuencia de nucleótidos del ARNm de RhoB humana (NM_004040.2) (5'→3', 2384 pb) se proporciona en la siguiente secuencia SEC ID No. 7: ATCTGCCACCGCAGTCTGGTTGGAGCTGTTGTCTTGTATGCTCAGCGAGGCCCGGAGA GACCCGGGAGAGAGCTAGGCCGAGTCCACCGCCCGAGTCTGCTGCCCGAGCCCGCGTT ACGCACAAAGCCGCCGATCCCCGGCCTGGGGTGAGCAGAGCGACCACCGCCCGGGAGC AGCGCGGCGAGACGCACGGTGCGCCCTATGCCCCCGCGCCCCCACCGCCCCCGCCGCG GCAGCCGAAGCGCAGCGAGAGAACGCGCCACCGCGGGGCCCGGGTGCAGCTAGCGACC CTCTCGCCACCTGCGCGCAGCCCGAGGTGAGCAGTGAGCGGCGAGCGGGAGGGCAGCG AGGCGTTCGCGGGCCCCCTCCTGCTGCCCGGGCCCGGCCCGCTCATGGCGGCCATCCG CAAGAAGCTGGTGGTGGTGGGCGACGGCGCGTGTGGCAAGACGTGCCTGCTGATCGTG TTCAGTAAGGACGAGTTCCCCGAGGTGTACGTGCCCACCGTCTTCGAGAACTATGTGG CCGACATTGAGGTGGACGGCAAGCAGGTGGAGCTGGCGCTGTGGGACACGGCGGGCCA GGAGGACTACGACCGCCTGCGGCCGCTCTCCTACCCGGACACCGACGTCATTCTCATG TGCTTCTCGGTGGACAGCCCGGACTCGCTGGAGAACATCCCCGAGAAGTGGGTCCCCG AGGTGAAGCACTTCTGTCCCAATGTGCCCATCATCCTGGTGGCCAACAAAAAAGACCT GCGCAGCGACGAGCATGTCCGCACAGAGCTGGCCCGCATGAAGCAGGAACCCGTGCGC ACGGATGACGGCCGCGCCATGGCCGTGCGCATCCAAGCCTACGACTACCTCGAGTGCT CTGCCAAGACCAAGGAAGGCGTGCGCGAGGTCTTCGAGACGGCCACGCGCGCCGCGCT GCAGAAGCGCTACGGCTCCCAGAACGGCTGCATCAACTGCTGCAAGGTGCTATGAGGG CCGCGCCCGTCGCGCCTGCCCCTGCCGGCACGGCTCCCCCTCCTGGACCAGTCCCCCG CGAGCCCGGAGAAGGGGAGACCCGTGTCCCACAAGGACCCCACCGGCCTGCCTGGCAT CTGTCTGCTGACGCCTCTGGCTTGCGCCAGGACTTGGCGTGGGCACCGGGCGCCCCCA TCCCAGTGTCTGTGTGCGTCCAGCTGTGTTGCACAGGCCTGGGCTCCCCACTGAGTGC CAAGGGTCCCCTGAGCATGCTTTTCTGAAGAGCCGGGCCTCAGAGTGTGTGGCTGTGT GTCTGTTCGACTCCCCTCGCCCCATTTTCACCCCACCCCCGCCTCTGATCCCCGGGGG CGAGATTGGCGCGGGAGTGTGGCCGCGCCCCATCAGATGTTCTCCCTTCACCAGCGGG AGCTTGATATCCCTTGTCTGTAACATAGACCCCGGGTACTGCGGGAGGGGAGGGCTGC TGGGGAGGATGGGGGGATGTTATATAAATATAGATATAATTTTATTTTCGGAGCTAAG ATGGTGTTATTTAAGGGTGGTGATGGGTGAGCGCTCTGGCCCAGGCTGGGCCAGACTC CCGCCCAAGCATGAACAGGACTTGACCATCTTTCCAACCCCTGGGGAAGACATTTGCA ACTGACTTGGGGAGGACACAGCTTCAGCACAGCCTCTCCTGCGGGCCAGCCCGCTGCG AACCCTCCACCAGCTACCGGAGGGAGGAGGGAGGATGCGCTGTGGGGTTGTTTTTGCC ATAAGCGAACTTTGTGCCTGTCCTAGAAGTGAAAATTGTTCAGTCCAAGAAACTGATG TTATTTGATTTATTTAAAGGCTAAAATTTGTTTTTTTATTCTTTGCACAATTGTTTCA TTGTTTGACACTTAATGCACTCGTCATTTGCATACGACAGTAGCATTCTGACCACACT TGTACGCTGTAACCTCATCTACTTCTGATGTTTTTAAAAAATGACTTTTAACAAGGAG AGGGAAAAGAAACCCACTAAATTTTGCTTTGTTTCCTTGAAGAATGTGGCAACACTGT TTTGTGATTTTATTTGTGCAGGTCATGCACACAGTTTTGATAAAGGGCAGTAACAAGT ATTGGGGCCTATTTTTTTTTTTTCCACAAGGCATTCTCTAAAGCTATGTGAAATTTTC TCTGCACCTCTGTACAGAGAATACACCTGCCCCTGTATATCCTTTTTTCCCCTCCCCT CCCTCCCAGTGGTACTTCTACTAAATTGTTGTCTTGTTTTTTATTTTTTAAATAAACT GACAAATGACAAAATGGTGAGCTTATGATGTTTACATAAAAGTTCTATAAGCTGTGTA TACAGTTTTTTATGTAAAATATTAAAAGACTATGATGATGACATTTAAAAAAAAAAAA AAAAAAThe nucleotide sequence of human RhoB mRNA (NM_004040.2) (5 '→ 3', 2384 bp) is provided in the following sequence SEQ ID No. 7: ATCTGCCACCGCAGTCTGGTTGGAGCTGTTGTCTTGTATGCTCAGCGAGGCCCGGAGA GACCCGGGAGAGAGCTAGGCCGAGTCCACCGCCCGAGTCTGCTGCCCGAGCCCGCGTT ACGCACAAAGCCGCCGATCCCCGGCCTGGGGTGAGCAGAGCGACCACCGCCCGGGAGC AGCGCGGCGAGACGCACGGTGCGCCCTATGCCCCCGCGCCCCCACCGCCCCCGCCGCG GCAGCCGAAGCGCAGCGAGAGAACGCGCCACCGCGGGGCCCGGGTGCAGCTAGCGACC CTCTCGCCACCTGCGCGCAGCCCGAGGTGAGCAGTGAGCGGCGAGCGGGAGGGCAGCG AGGCGTTCGCGGGCCCCCTCCTGCTGCCCGGGCCCGGCCCGCTCATGGCGGCCATCCG CAAGAAGCTGGTGGTGGTGGGCGACGGCGCGTGTGGCAAGACGTGCCTGCTGATCGTG TTCAGTAAGGACGAGTTCCCCGAGGTGTACGTGCCCACCGTCTTCGAGAACTATGTGG CCGACATTGAGGTGGACGGCAAGCAGGTGGAGCTGGCGCTGTGGGACACGGCGGGCCA GGAGGACTACGACCGCCTGCGGCCGCTCTCCTACCCGGACACCGACGTCATTCTCATG TGCTTCTCGGTGGACAGCCCGGACTCGCTGGAGAACATCCCCGAGAAGTGGGTCCCCG AGGTGAAGCACTTCTGTCCCAATGTGCCCATCATCCTGGTGGCCAACAAAAAAGACCT GCGCAGCGACGAGCATGTCCGCACAGAGCTGGCCCGCATGAAGCAGGAACCCGTGCGC ACGGATGACGGCCGCGCCATGGCCGTGCGCATC CAAGCCTACGACTACCTCGAGTGCT CTGCCAAGACCAAGGAAGGCGTGCGCGAGGTCTTCGAGACGGCCACGCGCGCCGCGCT GCAGAAGCGCTACGGCTCCCAGAACGGCTGCATCAACTGCTGCAAGGTGCTATGAGGG CCGCGCCCGTCGCGCCTGCCCCTGCCGGCACGGCTCCCCCTCCTGGACCAGTCCCCCG CGAGCCCGGAGAAGGGGAGACCCGTGTCCCACAAGGACCCCACCGGCCTGCCTGGCAT CTGTCTGCTGACGCCTCTGGCTTGCGCCAGGACTTGGCGTGGGCACCGGGCGCCCCCA TCCCAGTGTCTGTGTGCGTCCAGCTGTGTTGCACAGGCCTGGGCTCCCCACTGAGTGC CAAGGGTCCCCTGAGCATGCTTTTCTGAAGAGCCGGGCCTCAGAGTGTGTGGCTGTGT GTCTGTTCGACTCCCCTCGCCCCATTTTCACCCCACCCCCGCCTCTGATCCCCGGGGG CGAGATTGGCGCGGGAGTGTGGCCGCGCCCCATCAGATGTTCTCCCTTCACCAGCGGG AGCTTGATATCCCTTGTCTGTAACATAGACCCCGGGTACTGCGGGAGGGGAGGGCTGC TGGGGAGGATGGGGGGATGTTATATAAATATAGATATAATTTTATTTTCGGAGCTAAG ATGGTGTTATTTAAGGGTGGTGATGGGTGAGCGCTCTGGCCCAGGCTGGGCCAGACTC CCGCCCAAGCATGAACAGGACTTGACCATCTTTCCAACCCCTGGGGAAGACATTTGCA ACTGACTTGGGGAGGACACAGCTTCAGCACAGCCTCTCCTGCGGGCCAGCCCGCTGCG AACCCTCCACCAGCTACCGGAGGGAGGAGGGAGGATGCGCTGTGGGGTTGTTTTTGCC ATAAGCGAACTTTGTGCCTGTCCTAGAAGTGAAAATTGTTCAGTCCAAGAAACTGATG TTATTTGATTTATTTAAAGGCTAAAATTTGTTTTTTTATTCTTTGCACAATTGTTTCA TTGTTTGACACTTAATGCACTCGTCATTTGCATACGACAGTAGCATTCTGACCACACT TGTACGCTGTAACCTCATCTACTTCTGATGTTTTTAAAAAATGACTTTTAACAAGGAG AGGGAAAAGAAACCCACTAAATTTTGCTTTGTTTCCTTGAAGAATGTGGCAACACTGT TTTGTGATTTTATTTGTGCAGGTCATGCACACAGTTTTGATAAAGGGCAGTAACAAGT ATTGGGGCCTATTTTTTTTTTTTCCACAAGGCATTCTCTAAAGCTATGTGAAATTTTC TCTGCACCTCTGTACAGAGAATACACCTGCCCCTGTATATCCTTTTTTCCCCTCCCCT CCCTCCCAGTGGTACTTCTACTAAATTGTTGTCTTGTTTTTTATTTTTTAAATAAACT GACAAATGACAAAATGGTGAGCTTATGATGTTTACATAAAAGTTCTATAAGCTGTGTA TACAGTTTTTTATGTAAAATATTAAAAGACTATGATGATGACATTTAAAAAAAAAAAA AAAAAA
La secuencia de nucleótidos del ADNc de RhoB humana (CCDS1699.1) (5'→3', 591 pb) se proporciona en la siguiente secuencia SEC ID No. 8:The nucleotide sequence of the human RhoB cDNA (CCDS1699.1) (5 '→ 3', 591 bp) is provided in the following sequence SEQ ID NO: 8:
ATGGCGGCCATCCGCAAGAAGCTGGTGGTGGTGGGCGACGGCGCGTGTGGCAAGACGT GCCTGCTGATCGTGTTCAGTAAGGACGAGTTCCCCGAGGTGTACGTGCCCACCGTCTT CGAGAACTATGTGGCCGACATTGAGGTGGACGGCAAGCAGGTGGAGCTGGCGCTGTGG GACACGGCGGGCCAGGAGGACTACGACCGCCTGCGGCCGCTCTCCTACCCGGACACCG ACGTCATTCTCATGTGCTTCTCGGTGGACAGCCCGGACTCGCTGGAGAACATCCCCGA GAAGTGGGTCCCCGAGGTGAAGCACTTCTGTCCCAATGTGCCCATCATCCTGGTGGCC AACAAAAAAGACCTGCGCAGCGACGAGCATGTCCGCACAGAGCTGGCCCGCATGAAGC AGGAACCCGTGCGCACGGATGACGGCCGCGCCATGGCCGTGCGCATCCAAGCCTACGA CTACCTCGAGTGCTCTGCCAAGACCAAGGAAGGCGTGCGCGAGGTCTTCGAGACGGCC ACGCGCGCCGCGCTGCAGAAGCGCTACGGCTCCCAGAACGGCTGCATCAACTGCTGCA AGGTGCTATGAATGGCGGCCATCCGCAAGAAGCTGGTGGTGGTGGGCGACGGCGCGTGTGGCAAGACGT GCCTGCTGATCGTGTTCAGTAAGGACGAGTTCCCCGAGGTGTACGTGCCCACCGTCTT CGAGAACTATGTGGCCGACATTGAGGTGGACGGCAAGCAGGTGGAGCTGGCGCTGTGG GACACGGCGGGCCAGGAGGACTACGACCGCCTGCGGCCGCTCTCCTACCCGGACACCG ACGTCATTCTCATGTGCTTCTCGGTGGACAGCCCGGACTCGCTGGAGAACATCCCCGA GAAGTGGGTCCCCGAGGTGAAGCACTTCTGTCCCAATGTGCCCATCATCCTGGTGGCC AACAAAAAAGACCTGCGCAGCGACGAGCATGTCCGCACAGAGCTGGCCCGCATGAAGC AGGAACCCGTGCGCACGGATGACGGCCGCGCCATGGCCGTGCGCATCCAAGCCTACGA CTACCTCGAGTGCTCTGCCAAGACCAAGGAAGGCGTGCGCGAGGTCTTCGAGACGGCC ACGCGCGCCGCGCTGCAGAAGCGCTACGGCTCCCAGAACGGCTGCATCAACTGCTGCA AGGTGCTATGA
Las secuencias del ADN genómico, proteína, ARNm, ADNc, cebadores, y sondas presentadas en la presente invención también comprenden aquellas secuencias que tienen por lo menos un 70% de homología con respecto a las secuencias en SEC ID NO. 1-8, preferiblemente por lo menos un 80%, y más preferiblemente por lo menos un 90%, 95%, 96%, 97%, 98%, 99% de homología. Alternativamente, las formas generadas por splicing alternativo, también están comprendidas en el alcance de la presente invención.The sequences of genomic DNA, protein, mRNA, cDNA, primers, and probes presented in the present invention also comprise those sequences that have minus 70% homology with respect to the sequences in SEQ ID NO. 1-8, preferably at least 80%, and more preferably at least 90%, 95%, 96%, 97%, 98%, 99% homology. Alternatively, the forms generated by alternative splicing are also within the scope of the present invention.
El término "homología" se refiere al nivel de similitud entre las secuencias de ácido nucleico o aminoácidos en términos de porcentaje de identidad posicional de nucleótidos o aminoácidos, respectivamente, es decir, la similitud o identidad de las secuencias. El porcentaje de homología entre dos secuencias se puede calcular según los algoritmos FASTA o BLAST (Altschul et al. "Basic local alignment search tool" J. Mol. Biol. 1990, 215, 403-410, http://www.ncbi.nlm.nih.gov/blast; Lipman et al. "Rapid and sensitive protein similarity searches". Science 1985, 227, 1435-1441. http://www.ebi.ac.uk), que se incorporan en la presente invención por referencia.The term "homology" refers to the level of similarity between the nucleic acid or amino acid sequences in terms of percentage of positional identity of nucleotides or amino acids, respectively, that is, the similarity or identity of the sequences. The percentage of homology between two sequences can be calculated according to the FASTA or BLAST algorithms (Altschul et al. "Basic local alignment search tool" J. Mol. Biol. 1990, 215, 403-410, http: //www.ncbi. nlm.nih.gov/blast; Lipman et al. "Rapid and sensitive protein similarity searches". Science 1985, 227, 1435-1441. http://www.ebi.ac.uk), which are incorporated into the present invention by reference
Cualquier otra técnica adecuada para medir la expresión del gen de Rhob a nivel de proteína, ARNm, o ADNc también está comprendida en el alcance de la presente invención.Any other suitable technique for measuring Rhob gene expression at the level of protein, mRNA, or cDNA is also within the scope of the present invention.
En una realización alternativa de la presente invención, el procedimiento de pronóstico de la evolución de la enfermedad en un sujeto con adenocarcinoma de pulmón comprende la medición del número de copias del gen de RhoB.In an alternative embodiment of the present invention, the method of prognosis of disease progression in a subject with lung adenocarcinoma comprises measuring the copy number of the RhoB gene.
Medición del número de copias del gen de RhoB Una metodología para determinar el número de copias del gen de RhoB en una muestra es la hibridación in situ, por ejemplo, hibridación in situ por fluorescencia (FISH) (véase, Angerer, 1987 Meth. Enzymol 152: 649) . Generalmente, la hibridación in situ comprende las siguientes etapas principales: (1) fijación del tejido o estructura biológica a analizar; (2) tratamiento de prehibridización de la estructura biológica para aumentar la accesibilidad al ADN diana, y para reducir la unión no especifica; (3) hibridación de la mezcla de ácidos nucleicos al ácido nucleico en la estructura biológica o tejido; (4) lavados posteriores a la hibridación para eliminar los fragmentos de ácidos nucleicos no unidos en la hibridación, y (5) la detección de los fragmentos de ácido nucleico hibridados. Las sondas utilizadas en dichas aplicaciones habitualmente se marcan, por ejemplo, con radioisótopos o informadores fluorescentes. Las sondas preferidas son suficientemente largas, por ejemplo, de aproximadamente 50, 100, ó 200 nucleótidos hasta aproximadamente 1000 o más nucleótidos, para permitir la hibridación especifica con el ácido o ácidos nucleicos diana en condiciones rigurosas.Measurement of the number of copies of the RhoB gene One methodology for determining the number of copies of the RhoB gene in a sample is in situ hybridization, for example, fluorescence in situ hybridization (FISH) (see, Angerer, 1987 Meth. Enzymol 152: 649). Generally, in situ hybridization comprises the following main stages: (1) fixation of the tissue or biological structure to be analyzed; (2) Prehybridization treatment of the biological structure to increase accessibility to the target DNA, and to reduce non-specific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove fragments of unbound nucleic acids in hybridization, and (5) detection of hybridized nucleic acid fragments. The probes used in such applications are usually labeled, for example, with radioisotopes or fluorescent reporters. Preferred probes are long enough, for example, from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to allow specific hybridization with the target acid or nucleic acids under stringent conditions.
Otra metodología alternativa para determinar el número de copias de ADN es la hibridación genómica comparativa (CGH) . En los procedimientos de hibridación genómica comparativa, se marca un grupo de "prueba" de ácidos nucleicos con un primer marcador, mientras que un segundo grupo de prueba (por ejemplo, de una célula o tejido normal) se marca con un segundo marcador. La proporción de hibridación de los ácidos nucleicos se determina mediante la proporción de la unión del primer y segundo marcadores a cada sonda de un grupo. Se detectan las diferencias en la proporción de las señales de los dos marcadores, por ejemplo, debido a la amplificación de genes en el grupo de prueba, y la proporción proporciona una medición del número de copias del gen de RhoB, correspondiente a la sonda específica utilizada. Se puede generar una representación citogenética de la variación del número de copias de ADN mediante CGH, lo cual proporciona unas relaciones de fluorescencia a lo largo de la longitud de los cromosomas de los ADN de prueba marcados de manera diferencial y ADN genómico de referencia.Another alternative methodology for determining the number of DNA copies is comparative genomic hybridization (CGH). In comparative genomic hybridization procedures, a "test" group of nucleic acids is labeled with a first marker, while a second test group (for example, of a normal cell or tissue) is labeled with a second marker. The proportion of hybridization of nucleic acids is determined by the ratio of the binding of the first and second markers to each probe in a group. Differences in the proportion of the signals of the two markers are detected, for example, due to gene amplification in the test group, and the proportion provides a measurement of the copy number of the RhoB gene, corresponding to the specific probe used A cytogenetic representation of the variation in the number of DNA copies by CGH, which provides fluorescence ratios along the length of the chromosomes of differentially labeled test DNAs and reference genomic DNA.
Los protocolos de hibridación adecuados para su uso con los procedimientos de la presente invención se describen, por ejemplo, en Albertson (1984) EMBO J. 3:1227-1234; Pinkel (1988) Proc. Nati. Acad. Sci. USA 85:9138-9142; 430402EPAE PO Pub . No. 430:402; Methods in Molecular Biology, VoI. 33 : In Situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, NJ (1994) .Hybridization protocols suitable for use with the methods of the present invention are described, for example, in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Nati Acad. Sci. USA 85: 9138-9142; 430402EPAE PO Pub. No. 430: 402; Methods in Molecular Biology, VoI. 33: In Situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, NJ (1994).
Los ensayos basados en la amplificación también se pueden utilizar para medir el número de copias del gen de RhoB. En dichos ensayos, la correspondiente secuencia de ácidos nucleicos de RhoB actúa como plantilla en una reacción de amplificación (por ejemplo, Reacción en Cadena de la Polimerasa o PCR) . En una amplificación cuantitativa, la cantidad de producto de amplificación será proporcional a la cantidad de plantilla en la muestra original. La comparación con controles apropiados proporciona una medición del número de copias del gen de RhoB, correspondiente a la sonda especifica utilizada, según el principio descrito anteriormente. Los procedimientos de PCR cuantitativa a tiempo real que utilizan sondas Taqman son conocidos en la técnica. Se proporcionan protocolos detallados para PCR cuantitativa a tiempo real, por ejemplo, para ARN en: Gibson et al., 1996, A novel method for real time quantitative RT-PCR. Genome Res. 10:995- 1001; y para ADN en: Heid et al., 1996, Real time quantitative PCR. Genome Res. 10:986-994.Amplification-based assays can also be used to measure the copy number of the RhoB gene. In such tests, the corresponding RhoB nucleic acid sequence acts as a template in an amplification reaction (for example, Polymerase Chain Reaction or PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison with appropriate controls provides a measure of the copy number of the RhoB gene, corresponding to the specific probe used, according to the principle described above. Real-time quantitative PCR procedures using Taqman probes are known in the art. Detailed protocols for real-time quantitative PCR are provided, for example, for RNA in: Gibson et al., 1996, A novel method for real time quantitative RT-PCR. Genome Res. 10: 995-1001; and for DNA in: Heid et al., 1996, Real time quantitative PCR. Genome Res. 10: 986-994.
La transferencia Southern también se puede utilizar para medir el número de copias del gen de RhoB. Con esta metodología, se analiza el ADN en geles de agarosa o acrilamida para fraccionar el ADN según el tamaño seguido de la transferencia del ADN desde el gel hasta un soporte sólido, tal como nitrocelulosa o una membrana de nylon. A continuación, el ADN inmovilizado se hibrida con una sonda marcada para detectar las muestras de ADN complementarias a la sonda utilizada. El ADN se puede dividir con enzimas de restricción antes de la electroforesis . Después de la electroforesis, el ADN se puede despurinar parcialmente y desnaturalizar antes o durante la transferencia al soporte sólido. Las transferencias Southern son una herramienta habitual de los biólogos moleculares (J. Sambrook et al., Molecular Cloning: A Laboratory Manual, CoId Spring Harbor Press, NY, pág. 9.31-9.58 [1989]) .Southern blotting can also be used to measure the copy number of the RhoB gene. With this methodology, the DNA is analyzed in agarose or acrylamide gels to fractionate the DNA according to the size followed by the transfer of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. Then, the immobilized DNA is hybridized with a labeled probe to detect DNA samples complementary to the probe used. The DNA can be divided with restriction enzymes before electrophoresis. After electrophoresis, the DNA can be partially stripped and denatured before or during the transfer to the solid support. Southern blots are a common tool for molecular biologists (J. Sambrook et al., Molecular Cloning: A Laboratory Manual, CoId Spring Harbor Press, NY, p. 9.31-9.58 [1989]).
Un procedimiento potente para la determinación del número de copias de ADN utiliza plataformas basadas en microarrays. La tecnología de microarrays se puede utilizar porque ofrece una resolución elevada. Por ejemplo, la CGH tradicional tiene generalmente una resolución del mapeo limitada a 20 Mb; mientras que en CGH basada en arrays, las relaciones de fluorescencia de los ADN de prueba marcados de manera diferencial y ADN genómico de referencia proporcionan una medición "locus por locus" de la variación el número de copias de ADN, consiguiendo de esta manera una mayor resolución de mapeo. Los detalles de un procedimiento de microarray se puede hallar en la bibliografía. Véase, por ejemplo, 6232068USB US 6,232,068; Pollack et al., Nat Genet,1999, 23(l) :41-6 .A powerful procedure for determining the number of copies of DNA uses microarray-based platforms. Microarray technology can be used because it offers high resolution. For example, traditional CGH generally has a mapping resolution limited to 20 Mb; whereas in CGH based on arrays, the fluorescence ratios of the differentially labeled test DNAs and genomic reference DNA provide a "locus per locus" measurement of the variation in the number of copies of DNA, thus achieving greater mapping resolution Details of a microarray procedure can be found in the literature. See, for example, 6232068USB US 6,232,068; Pollack et al., Nat Genet, 1999, 23 (l): 41-6.
Cualquier otra técnica adecuada para medir el número de copias del gen de RhoB también está comprendida en el alcance de la presente invención.Any other suitable technique for measuring the copy number of the RhoB gene is also within the scope of the present invention.
Se ha observado que la sobreexpresión o aumento en el número de copias del gen de RhoB en relación con la expresión o el número de copias del mismo gen en una muestra de control son marcadores útiles para el pronóstico de adenocarcinoma de pulmón. En particular, dicha sobreexpresión o aumento en el número de copias del gen de RhoB es indicativo del riego de metástasis, o reaparición o reincidencia de actividad neoplásica.It has been observed that overexpression or increase in Number of copies of the RhoB gene in relation to the expression or number of copies of the same gene in a control sample are useful markers for the prognosis of lung adenocarcinoma. In particular, said overexpression or increase in the number of copies of the RhoB gene is indicative of the risk of metastasis, or recurrence or recurrence of neoplastic activity.
La "sobreexpresión" de un gen de RhoB o un "mayor" nivel o un nivel "elevado" de un polinucleótido o proteina RhoB se refiere a un nivel de polinucleótido o proteina RhoB que, en comparación con el nivel de control de RhoB, es de forma detectable más elevado. De manera similar, unThe "overexpression" of a RhoB gene or a "higher" level or "high" level of a RhoB polynucleotide or protein refers to a level of RhoB polynucleotide or protein which, compared to the RhoB control level, is in a higher detectable way. Similarly, a
"aumento" en el número de copias del gen de RhoB se refiere a un nivel del número de copias de RhoB que, en comparación con un nivel de control de RhoB, es de forma detectable más elevado."increase" in the number of copies of the RhoB gene refers to a level of the number of copies of RhoB which, in comparison to a level of RhoB control, is detectably higher.
La sobreexpresión o aumento en el número de copias del gen de RhoB son términos relativos que significan que se encuentra una diferencia detectable (más allá de la contribución del ruido en el sistema utilizado para medirlo) en la cantidad de expresión o número de copias del gen de RhoB relativo a cierta linea base. En la presente invención, la linea base es la expresión génica medida o el número de copias de una muestra de control, cuyo término se define a continuación.Overexpression or increase in the number of copies of the RhoB gene are relative terms that mean that a detectable difference (beyond the contribution of noise in the system used to measure it) is found in the amount of expression or number of copies of the gene of RhoB relative to a certain baseline. In the present invention, the baseline is the measured gene expression or the number of copies of a control sample, the term of which is defined below.
La comparación se puede llevar a cabo mediante análisis estadístico sobre las mediciones numéricas de la expresión o número de copias del gen de RhoB; o, se puede realizar a través del examen visual de los resultados experimentales por investigadores cualificados.The comparison can be carried out by statistical analysis on the numerical measurements of the expression or number of copies of the RhoB gene; or, it can be done through visual examination of experimental results by qualified researchers.
Por ejemplo, las intensidades de expresión génica de un tejido enfermo se pueden comparar con las intensidades de expresión generadas de tejido normal del mismo tipo (por ejemplo, muestra de tejido de pulmón enfermo vs . muestra de tejido de pulmón normal) . Una relación de estas intensidades de expresión indica las veces que cambia la expresión génica entre las muestras de prueba y de control .For example, the gene expression intensities of a Sick tissue can be compared with the expression intensities generated from normal tissue of the same type (for example, sample of diseased lung tissue vs. sample of normal lung tissue). A relationship of these expression intensities indicates the times that gene expression changes between the test and control samples.
En una realización de la presente invención, la sobreexpresión del gen de RhoB se determina cuando existe por lo menos una diferencia de 1,5 veces en su expresión en relación con la expresión del mismo gen en una muestra de control. En otra realización de la presente invención, un aumento en el número de copias del gen de RhoB se determina cuando existe por lo menos una diferencia de 1,5 veces en su número de copias en relación con el número de copias del mismo gen en una muestra de control.In one embodiment of the present invention, overexpression of the RhoB gene is determined when there is at least a 1.5-fold difference in its expression relative to the expression of the same gene in a control sample. In another embodiment of the present invention, an increase in the number of copies of the RhoB gene is determined when there is at least a 1.5-fold difference in its number of copies in relation to the number of copies of the same gene in a control sample.
El test t de Student es un ejemplo de test estadístico robusto que se puede utilizar para buscar diferencias significativas entre dos grupos. Cuanto más bajo es el valor de p, es más concluyente la evidencia de que el gen muestra una diferencia entre los diferentes grupos. Un valor de p inferior a 0,05 mediante el test t pone de manifiesto que el gen es significativamente diferente.The Student t test is an example of a robust statistical test that can be used to find significant differences between two groups. The lower the p-value, the more conclusive is the evidence that the gene shows a difference between the different groups. A p value of less than 0.05 by means of the t test shows that the gene is significantly different.
Una evidencia más concluyente es un valor de p inferior aA more conclusive evidence is a value of p less than
0,05 después de tener en cuenta la corrección de Sidak.0.05 after taking into account the correction of Sidak.
Para un número amplio de muestras en cada grupo, un valor de p inferior a 0,05 después del test de aleatorización/permutación es la evidencia más concluyente de una diferencia significativa.For a large number of samples in each group, a p-value of less than 0.05 after the randomization / permutation test is the most conclusive evidence of a significant difference.
Una "muestra de control" se refiere a una muestra de material biológico representativo de sujetos sanos sin cáncer. El nivel de expresión del gen de RhoB o el número de copias del gen de RhoB en una muestra de control es de manera deseable habitual de la población general de sujetos normales sin cáncer de la misma especie. Esta muestra se puede recoger de un sujeto con el objetivo de ser utilizada en los procedimientos descritos en la presente invención o, puede ser cualquier material biológico representativo de sujetos normales sin cáncer obtenido para otras razones, pero sin embargo, adecuado para su uso en los procedimientos de la presente invención. También se puede obtener una muestra de control a partir de tejido normal del sujeto que tiene cáncer o es sospechoso de tener cáncer. Una muestra de control también se puede referir a un nivel determinado de expresión del gen de RhoB o el número de copias del gen de RhoB representativos de la población sin cáncer, que se han establecido previamente en base a las mediciones de sujetos normales sin cáncer. Como tal, una muestra de control no necesita ser un material, sino que pueden ser datos o información relacionada con una muestra de control. Estos datos o información se almacenan habitualmente en forma electrónico o papel. Alternativamente, una muestra biológica de control puede referirse a una muestra que se obtiene de un individuo diferente o puede ser un valor normalizado basado en los valores de la linea base hallados en una población. Además, una muestra de control se puede definir mediante una edad, sexo, etnia u otros parámetros demográficos específicos. En algunas situaciones, el control está implícito en la medición concreta. Por ejemplo, se considera un procedimiento de detección que sólo puede detectar RhoB o el número de copias del gen de RhoB cuando está presente un nivel más elevado de lo habitual en un sujeto normal sin cáncer, por ejemplo, un ensayo inmunohistoquímico, para evaluar el nivel de RhoB o el número de copias del gen de RhoB en comparación con el nivel de control o el número de copias del gen RhoB, ya que el nivel de control o el número de copias es natural y conocidos en el ensayo.A "control sample" refers to a sample of biological material representative of healthy subjects without cancer. The expression level of the RhoB gene or the number Copies of the RhoB gene in a control sample are desirably customary for the general population of normal subjects without cancer of the same species. This sample can be collected from a subject for the purpose of being used in the procedures described in the present invention or, it can be any biological material representative of normal subjects without cancer obtained for other reasons, but nevertheless, suitable for use in Procedures of the present invention. A control sample can also be obtained from normal tissue of the subject who has cancer or is suspected of having cancer. A control sample can also refer to a certain level of RhoB gene expression or the number of RhoB gene copies representative of the non-cancer population, which have been previously established based on measurements of normal subjects without cancer. As such, a control sample does not need to be a material, but can be data or information related to a control sample. This data or information is usually stored in electronic form or paper. Alternatively, a biological control sample may refer to a sample that is obtained from a different individual or it may be a normalized value based on baseline values found in a population. In addition, a control sample can be defined by age, sex, ethnicity or other specific demographic parameters. In some situations, control is implicit in the concrete measurement. For example, it is considered a detection procedure that can only detect RhoB or the number of copies of the RhoB gene when a higher level than usual is present in a normal subject without cancer, for example, an immunohistochemical test, to evaluate the RhoB level or the number of copies of the RhoB gene compared to the level of control or the number of copies of the RhoB gene, since the level of control or the number of copies is natural and known in the assay.
La presente invención se refiere además al uso de un gen de RhoB, o una región del mismo, como marcador de pronóstico de la metástasis, o la reaparición o la reincidencia de actividad neoplásica en un sujeto con adenocarcinoma de pulmón.The present invention further relates to the use of a RhoB gene, or a region thereof, as a prognostic marker of metastasis, or the recurrence or recurrence of neoplastic activity in a subject with lung adenocarcinoma.
La presente invención proporciona además un procedimiento para el screening de fármacos activos contra el adenocarcinoma de pulmón, comprendiendo dicho procedimiento : a) la medición de la expresión del gen de RhoB, o la medición del número de copias del gen de RhoB, en una muestra con células de adenocarcinoma de pulmón en presencia de un fármaco, o la medición de la afinidad de unión ente un fármaco y un polipéptido RhoB; b) la comparación de la expresión o números de copias medidas del gen de RhoB, o la afinidad de unión de la etapa a) a una muestra de control; caracterizado porque una expresión o número de copias reducido del gen de RhoB, o una mayor actividad enzimática en la etapa a) , en relación con la expresión o el número de copias del mismo gen o la afinidad de unión del mismo polipéptido en una muestra de control es indicativa de la actividad del fármaco contra el adenocarcinoma de pulmón.The present invention further provides a method for screening active drugs against lung adenocarcinoma, said method comprising: a) measuring the expression of the RhoB gene, or measuring the number of copies of the RhoB gene, in a sample with lung adenocarcinoma cells in the presence of a drug, or the measurement of binding affinity between a drug and a RhoB polypeptide; b) comparing the expression or measured copy numbers of the RhoB gene, or the binding affinity of step a) to a control sample; characterized in that a reduced expression or copy number of the RhoB gene, or a greater enzymatic activity in step a), in relation to the expression or the number of copies of the same gene or the binding affinity of the same polypeptide in a sample of Control is indicative of the activity of the drug against lung adenocarcinoma.
Como tal, la presente invención proporciona procedimientos para identificar compuestos o agentes que se pueden utilizar para tratar trastornos caracterizados por, o asociados con una expresión o número de copias reducido del gen de RhoB. Estos procedimientos incluyen habitualmente la etapa de screening de un candidato, compuesto de prueba o agente para identificar compuestos que son un agonista o antagonista de un polipéptido RhoB, y específicamente por la capacidad de interaccionar con (por ejemplo, unirse a) un polipéptido RhoB, para modular la interacción de un polipéptido RhoB y una molécula diana, y/o para modular la expresión de ácido nucleico de RhoB y/o la actividad del polipéptido RhoB. Los candidatos, compuestos de prueba o agentes que tienen una o más de estas capacidades se pueden utilizar como fármacos para tratar trastornos caracterizados por una expresión o número de copias reducido del gen de RhoB.As such, the present invention provides methods for identifying compounds or agents that can be used to treat disorders characterized by, or associated with, a reduced expression or number of copies of the RhoB gene. These procedures usually include the screening stage of a candidate, test compound or agent for identifying compounds that are an agonist or antagonist of a RhoB polypeptide, and specifically for the ability to interact with (for example, bind to) a RhoB polypeptide, to modulate the interaction of a RhoB polypeptide and a target molecule , and / or to modulate RhoB nucleic acid expression and / or RhoB polypeptide activity. Candidates, test compounds or agents that have one or more of these capabilities can be used as drugs to treat disorders characterized by a reduced expression or number of copies of the RhoB gene.
Una expresión o número de copias reducida del marcador del gen de RhoB en un adenocarcinoma de pulmón también puede servir para predecir la respuesta de ese cáncer a terapias específicas. Este marcador molecular se puede utilizar para seleccionar la terapia más apropiada para el sujeto.A reduced expression or number of copies of the RhoB gene marker in a lung adenocarcinoma can also help predict that cancer's response to specific therapies. This molecular marker can be used to select the most appropriate therapy for the subject.
Como tal, la presente invención proporciona además un procedimiento para determinar la eficacia de una pauta de tratamiento terapéutico en un sujeto con adenocarcinoma de pulmón, comprendiendo dicho procedimiento a) la medición de la expresión del gen de RhoB, o la medición del número de copias del gen de RhoB, en una muestra con células de adenocarcinoma de pulmón de dicho sujeto, generando de este modo un nivel inicial; b) la medición de la expresión del gen de RhoB, o la medición del número de copias del gen de RhoB, en una segunda muestra con células de adenocarcinoma de pulmón del mismo sujeto en un momento posterior a la administración de la pauta de tratamiento, obteniendo de este modo un nivel de prueba; y b) la comparación de los niveles inicial y de prueba; caracterizado porque una expresión o número de copias reducido del gen de RhoB en el nivel de prueba en relación con el nivel inicial es indicativa de que la pauta de tratamiento es eficaz en el sujeto.As such, the present invention further provides a method for determining the efficacy of a therapeutic treatment regimen in a subject with lung adenocarcinoma, said method comprising a) measuring RhoB gene expression, or measuring copy number of the RhoB gene, in a sample with lung adenocarcinoma cells of said subject, thereby generating an initial level; b) the measurement of the RhoB gene expression, or the measurement of the copy number of the RhoB gene, in a second sample with lung adenocarcinoma cells of the same subject at a time after the administration of the treatment regimen, thus obtaining a test level; and b) comparison of initial and test levels; characterized in that a reduced expression or number of copies of the RhoB gene at the level of test in relation to With the initial level it is indicative that the treatment regimen is effective in the subject.
La presente invención se refiere además al uso de una molécula de nucleótido o anticuerpo que interaccionan con ADN, ARN, proteina de RhoB, o un fragmento de los mismos, para el pronóstico de la evolución de la enfermedad en un sujeto con adenocarcinoma de pulmón, para el screening de fármacos activos contra el adenocarcinoma de pulmón, o para determinar la eficacia de una pauta de tratamiento terapéutico en un sujeto con adenocarcinoma de pulmón. Dicha molécula de nucleótido es un nucleótido que tiene por lo menos un 70% de homología con respecto a la SEC ID NO. 4-6, preferiblemente por lo menos aproximadamente un 80, 85, 90, 95, 96, 97, 98, 99, o 100% de homología con respecto a la SEC ID NO. 4-6. La molécula de anticuerpo que interacciona con ADN, ARN, proteína de Rhob, o un fragmento de los mismos, puede ser monoclonal, policlonal, quimérico, humanizado o biespecífico, o está conjugado a un segundo anticuerpo, o un fragmento del mismo. Particularmente, la molécula de anticuerpo es el anticuerpo policlonal Rho B (119), o una molécula de anticuerpo que tiene preferiblemente por lo menos un 80% y más preferiblemente por lo menos un 85%, 90%, 95%, 96%, 97%, 98%, 99% ó 100% de homología con el anticuerpo policlonal Rho B (119) .The present invention further relates to the use of a nucleotide molecule or antibody that interacts with DNA, RNA, RhoB protein, or a fragment thereof, for the prognosis of disease progression in a subject with lung adenocarcinoma, for screening active drugs against lung adenocarcinoma, or to determine the efficacy of a therapeutic treatment regimen in a subject with lung adenocarcinoma. Said nucleotide molecule is a nucleotide that has at least 70% homology with respect to SEQ ID NO. 4-6, preferably at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% homology with respect to SEQ ID NO. 4-6. The antibody molecule that interacts with DNA, RNA, Rhob protein, or a fragment thereof, can be monoclonal, polyclonal, chimeric, humanized or bispecific, or is conjugated to a second antibody, or a fragment thereof. Particularly, the antibody molecule is the polyclonal Rho B (119) antibody, or an antibody molecule that preferably has at least 80% and more preferably at least 85%, 90%, 95%, 96%, 97 %, 98%, 99% or 100% homology with the polyclonal antibody Rho B (119).
Un "fragmento" es una parte de una secuencia de Rhob madura natural, nativa o endógena de longitud completa que tiene uno o más residuos de aminoácidos eliminados. El residuo o residuos de aminoácidos eliminados pueden estar en cualquier parte del polipéptido, incluyendo en el extremo N-terminal o el extremo C-terminal, o internamente. Dicho fragmento tendrá por lo menos una propiedad biológica en común con RhoB. Los fragmentos de RhoB tendrán habitualmente una secuencia consecutiva de por lo menos 10, 15, 20, 25, 30, 40, 50 ó 60 residuos de aminoácidos que son idénticos a las secuencias de la RhoB aislada de un mamífero, incluyendo RhoB de SEC ID NO: 2.A "fragment" is a part of a full length natural, native or endogenous mature Rhob sequence that has one or more amino acid residues removed. The amino acid residue or residues removed may be anywhere in the polypeptide, including at the N-terminal or C-terminal end, or internally. Said fragment will have at least one biological property in common with RhoB. The fragments of RhoB will usually have a consecutive sequence of at least 10, 15, 20, 25, 30, 40, 50 or 60 amino acid residues that are identical to the isolated RhoB sequences of a mammal, including RhoB of SEQ ID NO: 2 .
Tal como se utiliza en la presente invención, el término "anticuerpo" es intercambiable con "inmunoglobulina" y se refiere a inmunoglobulinas de la forma general hallada en especies vertebradas, incluyendo mamíferos, tales como humanos, primates, roedores, conejos y muchas otras especies en las que se han identificado dichas inmunoglobulinas. "Anticuerpo" significa moléculas que corresponden a inmunoglobulinas completas, así como fragmentos de las mismas, tales como Fab, F(ab')2, y Fv, que son capaces de unirse al determinante epitópico.As used in the present invention, the term "antibody" is interchangeable with "immunoglobulin" and refers to immunoglobulins in the general form found in vertebrate species, including mammals, such as humans, primates, rodents, rabbits and many other species. in which said immunoglobulins have been identified. "Antibody" means molecules that correspond to complete immunoglobulins, as well as fragments thereof, such as Fab, F (ab ') 2, and Fv, which are capable of binding to the epitopic determinant.
El término "anticuerpo monoclonal" se refiere a una población de moléculas de anticuerpo que contienen sólo una especie de un sitio de unión a antígeno capaz de inmunorreaccionar con un epítopo particular de un antígeno, mientras que el término "anticuerpo policlonal" se refiere a una población de moléculas de anticuerpo que contienen múltiples especies de sitios de unión a antígeno capaces de interaccionar con un antígeno particular. De este modo, una composición con anticuerpos monoclonales muestra habitualmente una única afinidad de unión para un antígeno particular con el que inmunorreacciona .The term "monoclonal antibody" refers to a population of antibody molecules that contain only a species from an antigen binding site capable of immunoreacting with a particular epitope of an antigen, while the term "polyclonal antibody" refers to a population of antibody molecules that contain multiple species of antigen binding sites capable of interacting with a particular antigen. Thus, a composition with monoclonal antibodies usually shows a unique binding affinity for a particular antigen with which it immunoreacts.
Los procedimientos para producir y cribar anticuerpos específicos utilizando tecnología de hibridomas son rutinarios y conocidos en la técnica. En un ejemplo no limitante, se pueden inmunizar ratones con un polipéptido de la presente invención o una célula que expresa dicho péptido. Una vez se detecta una respuesta inmune, por ejemplo, se detectan anticuerpos específicos para el antígeno en el suero de ratón, se recoge el bazo del ratón y se aislan los esplenocitos . A continuación, los esplenocitos se fusionan mediante técnicas conocidas a cualquier célula de mieloma adecuada. Los hibridomas se seleccionan y se clonan mediante dilución limitada. A continuación, los clones de hibridomas se ensayan mediante procedimientos conocidos en la técnica para células que secretan anticuerpos capaces de unirse a un polipéptido de la presente invención. Los fluidos asciticos, que contienen en general niveles elevados de anticuerpos, se pueden generar mediante la inmunización de ratones con clones de hibridomas positivos.Procedures for producing and screening specific antibodies using hybridoma technology are routine and known in the art. In a non-limiting example, mice can be immunized with a polypeptide of the present invention or a cell expressing said peptide. Once an immune response is detected, for example, antibodies specific to the antigen in the mouse serum, the mouse spleen is collected and the splenocytes are isolated. The splenocytes are then fused by known techniques to any suitable myeloma cell. The hybridomas are selected and cloned by limited dilution. Next, hybridoma clones are assayed by methods known in the art for cells that secrete antibodies capable of binding to a polypeptide of the present invention. Ascetic fluids, which generally contain high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
Los anticuerpos policlonales se producen habitualmente mediante la inmunización de un mamífero adecuado, tal como un ratón, conejo o cabra. Frecuentemente se prefieren mamíferos más grandes, ya que la cantidad de suero que se puede recoger es mayor. Se inyecta un antigeno al mamífero. Esto induce a que los linfocitos B produzcan inmunoglobulinas IgG especificas para el antigeno. Esta IgG policlonal se purifica del suero de mamífero. En cambio, los anticuerpos monoclonales derivan de una única linea celular.Polyclonal antibodies are usually produced by immunization of a suitable mammal, such as a mouse, rabbit or goat. Frequently larger mammals are preferred, since the amount of serum that can be collected is greater. An antigen is injected into the mammal. This induces B lymphocytes to produce specific IgG immunoglobulins for the antigen. This polyclonal IgG is purified from mammalian serum. Instead, monoclonal antibodies are derived from a single cell line.
El término "anticuerpo quimérico" se refiere a un anticuerpo en el que la región constante proviene de un anticuerpo de una especie (habitualmente humana) y la región variable proviene de un anticuerpo de otra especie (habitualmente roedor) . Los procedimientos para producir anticuerpos quiméricos son conocidos en la técnica.The term "chimeric antibody" refers to an antibody in which the constant region comes from an antibody of one species (usually human) and the variable region comes from an antibody of another species (usually rodent). Methods for producing chimeric antibodies are known in the art.
El término "anticuerpo humanizado" tal como se utiliza en la presente invención, se refiere a moléculas de anticuerpo en las que se han sustituido aminoácidos en las regiones que no son de unión a antigeno con el fin de que se asemeje lo más posible a un anticuerpo humano, mientras mantiene aún la capacidad de unión original. Dependiendo del contexto, esta expresión también puede incluir anticuerpos "de primate", en los que un primer anticuerpo obtenido de un organismo que no es primate ha sido modificado para asemejarse lo más posible a una inmunoglobulina de primate.The term "humanized antibody", as used in the present invention, refers to antibody molecules in which amino acids have been substituted in the regions that are not antigen-binding in order that it resembles as much as possible a human antibody, while still maintaining the original binding capacity. Depending on the context, this expression may also include "primate" antibodies, in which a first antibody obtained from an organism that is not primate has been modified to closely resemble a primate immunoglobulin.
Un anticuerpo "biespecífico" o bifuncional es un anticuerpo híbrido artificial que tiene dos parejas de cadena pesada/ligera diferentes y dos sitios de unión diferentes. Los anticuerpos biespecíficos se pueden producir mediante una serie de procedimientos incluyendo la fusión de hibridomas o la unión de fragmentos Fab'. Véase, por ejemplo, Songsivilai & Lachmann, Clin Exp.A "bispecific" or bifunctional antibody is an artificial hybrid antibody that has two different heavy / light chain partners and two different binding sites. Bispecific antibodies can be produced by a series of procedures including hybridoma fusion or Fab 'fragment binding. See, for example, Songsivilai & Lachmann, Clin Exp.
Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol.Immunol 79: 315-321 (1990); Kostelny et al., J. Immunol.
148, 1547-1553 (1992) .148, 1547-1553 (1992).
Una realización adicional de la presente invención proporciona un kit para el pronóstico de la evolución de la enfermedad en un sujeto con adenocarcinoma de pulmón que comprende reactivos para medir la expresión o el número de copias del gen de RhoB. Dicho kit puede comprender adicionalmente reactivos adecuados para realizar una análisis con microarray. En particular, el kit según la presente invención comprende Rho B (119) :sc- 180 Santa Cruz Biotechnology, Inc. Rho B (119) es un anticuerpo policlonal de conejo purificado por afinidad desarrollado contra un péptido que se mapea en una región interna de RhoB de origen humano. Alternativamente, el kit según la presente invención comprende cebadores con SEC ID No. 4-6 para realizar la RT-PCR o PCR cuantitativa a tiempo real.A further embodiment of the present invention provides a kit for the prognosis of disease progression in a subject with lung adenocarcinoma comprising reagents for measuring expression or copy number of the RhoB gene. Said kit may additionally comprise suitable reagents for performing a microarray analysis. In particular, the kit according to the present invention comprises Rho B (119): sc-180 Santa Cruz Biotechnology, Inc. Rho B (119) is an affinity purified rabbit polyclonal antibody developed against a peptide that maps to an internal region of RhoB of human origin. Alternatively, the kit according to the present invention comprises primers with SEQ ID No. 4-6 for performing real-time quantitative RT-PCR or PCR.
La presente invención proporciona además un amplicón de gen de RhoB aislado, donde el amplicón comprende más de una copia de un polinucleótido seleccionado entre: a) un polinucleótido que codifica el polipéptido establecido en la SEC ID NO. 2 ; b) un polinucleótido establecido en la SEC ID NO. 1 ; c) un polinucleótido que tiene por lo menos un 70% de homología con el polinucleótido de a) o b) ; y d) un polinucleótido que se sobreexpresa en células de adenocarcinoma de pulmón que tiene por lo menos un 70% de homología con el polinucleótido de a) o b) .The present invention further provides an amplicon of Isolated RhoB gene, where the amplicon comprises more than one copy of a polynucleotide selected from: a) a polynucleotide encoding the polypeptide set forth in SEQ ID NO. 2 ; b) a polynucleotide established in SEQ ID NO. one ; c) a polynucleotide having at least 70% homology with the polynucleotide of a) or b); and d) a polynucleotide that is overexpressed in lung adenocarcinoma cells that has at least 70% homology with the polynucleotide of a) or b).
La presente invención se refiere además a inhibidores de la sobreexpresión del gen de RhoB para su uso como medicamento. Dicho uso como medicamento o método de tratamiento comprende la administración a sujetos con NSCLC o a sujetos susceptibles de desarrollar NSCLC incluyendo adenocarcinoma de pulmón, carcinoma de células escamosas, o carcinoma de células grandes de una cantidad eficaz de un inhibidor de la sobreexpresión del gen de RhoB para combatir las afecciones asociadas con NSCLC, en particular para evitar el desarrollo posterior a metástasis ósea, o la reaparición o reincidencia de actividad neoplásica.The present invention further relates to inhibitors of RhoB gene overexpression for use as a medicament. Such use as a medicament or method of treatment comprises administration to subjects with NSCLC or to subjects susceptible to developing NSCLC including lung adenocarcinoma, squamous cell carcinoma, or large cell carcinoma of an effective amount of an RhoB gene overexpression inhibitor. to combat the conditions associated with NSCLC, in particular to prevent the development after bone metastasis, or the recurrence or recurrence of neoplastic activity.
La presente invención también se refiere al uso de los inhibidores de la sobreexpresión del gen de RhoB o cualquier subgrupo de los mismos en la fabricación de un medicamento para el tratamiento de NSCLC incluyendo adenocarcinoma de pulmón, carcinoma de células escamosas, o carcinoma de células grandes, en particular para evitar el desarrollo posterior en metástasis ósea, o la reaparición o reincidencia de actividad neoplásica. Dicho de otra manera, la presente invención proporciona inhibidores de la sobreexpresión del gen de RhoB para su uso en el tratamiento de NSCLC incluyendo adenocarcinoma de pulmón, carcinoma de células escamosas, o carcinoma de células grandes, en particular para evitar el desarrollo posterior a metástasis ósea, o la reaparición o reincidencia de actividad neoplásica.The present invention also relates to the use of RhoB gene overexpression inhibitors or any subgroup thereof in the manufacture of a medicament for the treatment of NSCLC including lung adenocarcinoma, squamous cell carcinoma, or large cell carcinoma. , in particular to prevent further development in bone metastases, or the recurrence or recurrence of neoplastic activity. In other words, the present invention provides inhibitors of RhoB gene overexpression for use in the treatment of NSCLC including adenocarcinoma. of lung, squamous cell carcinoma, or large cell carcinoma, in particular to prevent the development after bone metastasis, or the recurrence or recurrence of neoplastic activity.
El término "tratar", tal como se utiliza en la presente invención, se refiere a invertir, aliviar o inhibir el progreso del trastorno o afección al que se aplica dicho término, o uno o más síntomas de dichos trastornos o afección. El término "tratamiento" se refiere al hecho de tratar, lo cual se ha definido anteriormente.The term "treat", as used in the present invention, refers to reversing, alleviating or inhibiting the progress of the disorder or condition to which said term applies, or one or more symptoms of said disorders or condition. The term "treatment" refers to the act of treating, which has been defined above.
El término "cantidad terapéuticamente eficaz" o "cantidad eficaz" tal como se utilizan en la presente invención, significan la cantidad de compuesto o componente o agente farmacéutico activo que obtiene la respuesta biológica o médica en un tejido, sistema, animal o humano que se está buscando, a la luz de la presente invención, por un investigador, veterinario, doctor u otro profesional clínico, que incluye la mejora de los síntomas de la enfermedad en tratamiento.The term "therapeutically effective amount" or "effective amount" as used in the present invention means the amount of compound or component or active pharmaceutical agent that obtains the biological or medical response in a tissue, system, animal or human that is It is seeking, in the light of the present invention, a researcher, veterinarian, doctor or other clinical professional, which includes the improvement of the symptoms of the disease being treated.
Dado que la presente invención incluye combinaciones que comprenden dos o más agentes, la "cantidad terapéuticamente eficaz" es aquella cantidad de los agentes tomados en conjunto, de manera que el efecto combinado proporciona la respuesta biológica o médica deseada .Since the present invention includes combinations comprising two or more agents, the "therapeutically effective amount" is that amount of the agents taken together, so that the combined effect provides the desired biological or medical response.
El término "inhibidor" pretende incluir antagonistas y, se refiere a una molécula que directa o indirectamente reduce la sobreexpresión del gen de RhoB, o la actividad biológica de un polipéptido o proteína RhoB. Los inhibidores de la sobreexpresión del gen de RhoB pueden incluir anticuerpos, ácidos nucleicos, u otras moléculas. En una realización, los inhibidores de la sobreexpresión del gen de RhoB se pueden seleccionar entre: a) un anticuerpo o fragmento del mismo que se une específicamente a una proteína RhoB, o un polipéptido que comprende una región de unión a RhoB; b) un péptido mimético del polipéptido Rhob; c) un ácido nucleico inhibidor del gen de RhoB seleccionado entre un oligonucleótido antisentido, un ribozima, y un agente ARNi seleccionado entre ARNds y ARNsh; o d) una combinación que comprende lovastatina y ácido zoledrónico, y/o sales farmacéuticamente aceptables de la misma.The term "inhibitor" is intended to include antagonists and, refers to a molecule that directly or indirectly reduces the overexpression of the RhoB gene, or the biological activity of a RhoB polypeptide or protein. RhoB gene overexpression inhibitors may include antibodies, nucleic acids, or other molecules. In one embodiment, RhoB gene overexpression inhibitors may be selected from: a) an antibody or fragment thereof that specifically binds to a RhoB protein, or a polypeptide comprising a RhoB binding region; b) a Rhob polypeptide mimetic peptide; c) a RhoB gene inhibitor nucleic acid selected from an antisense oligonucleotide, a ribozyme, and an RNAi agent selected from RNAds and shsh; od) a combination comprising lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof.
La molécula de anticuerpo que se une específicamente a una proteína RhoB puede ser monoclonal, policlonal, quimérico, humanizado o biespecífico, tal como se describe anteriormente en la presente invención.The antibody molecule that specifically binds to a RhoB protein can be monoclonal, polyclonal, chimeric, humanized or bispecific, as described above in the present invention.
El término "fragmento de anticuerpo" se refiere más específicamente a estos fragmentos y/o polipéptidos de tipo inmunoglobulina que no comprenden una inmunoglobulina completa .The term "antibody fragment" refers more specifically to these immunoglobulin-like fragments and / or polypeptides that do not comprise a complete immunoglobulin.
Un "polipéptido que comprende una región de unión específica a RhoB" significa un polipéptido que incorpora una o más regiones de unión que se unen específicamente a la proteína RhoB de la SEC ID NO. 2. Un tipo clásico de región de unión específica a antígeno es una región determinante de complementariedad (CDR) hallada en una inmunoglobulina. Las CDRs son secuencias de aminoácidos cortas que se unen específicamente al antígeno en cuestión y proporcionan la base de la selectividad de unión del polipéptido en el que reside. Las CDRs se identifican habitualmente a partir de inmunoglobulinas, pero se pueden generar mediante otros medios.A "polypeptide comprising a RhoB specific binding region" means a polypeptide that incorporates one or more binding regions that specifically bind to the RhoB protein of SEQ ID NO. 2. A classic type of antigen specific binding region is a complementarity determining region (CDR) found in an immunoglobulin. CDRs are short amino acid sequences that specifically bind to the antigen in question and provide the basis for the binding selectivity of the polypeptide in which it resides. CDRs are usually identified from immunoglobulins, but can be generate by other means.
Las regiones de unión especifica a antigeno también incluyen secuencias de aminoácidos relativamente cortas que se unen a un antigeno, incluso si estas secuencias no derivan de CDRs. WO2004/044011, incorporada en la presente invención por referencia, proporciona un ejemplo de cómo dichas regiones de unión especifica a antigeno (que no son CDRs) se pueden identificar y desarrollar. Existen otros procedimientos conocidos por los expertos en la materia. Una vez desarrollada, la región de unión especifica a RhoB se puede utilizar en una amplia variedad de estructuras y armazones conocidas y futuribles, incluyendo cualquier isotipo de inmunoglobulina o fragmento de la misma, y otros polipéptidos de estructura o armazón que no son inmunoglobulinas para proporcionar la especificidad de unión a antigeno. Todos estos polipéptidos se consideran en la presente invención como "polipéptidos que comprenden una región de unión especifica a RhoB".Antigen specific binding regions also include relatively short amino acid sequences that bind to an antigen, even if these sequences are not derived from CDRs. WO2004 / 044011, incorporated in the present invention by reference, provides an example of how said antigen specific binding regions (which are not CDRs) can be identified and developed. There are other procedures known to those skilled in the art. Once developed, the RhoB-specific binding region can be used in a wide variety of known and futuristic structures and frameworks, including any immunoglobulin isotype or fragment thereof, and other structure or framework polypeptides that are not immunoglobulins to provide the specificity of antigen binding. All these polypeptides are considered in the present invention as "polypeptides comprising a specific RhoB binding region".
Un "peptido mimético" es un agente no peptidico derivado de manera sintética creados en base al conocimiento de los residuos críticos del polipéptido de un sujeto que puede mimetizar la función normal del polipéptido. Los miméticos de péptido pueden romper la unión de un polipéptido a su receptor o a otras proteínas y, de este modo, interferir con la función normal de un polipéptido. Por ejemplo, un mimético de RhoB interferiría con la función normal de RhoB.A "mimetic peptide" is a synthetically derived non-peptide agent created based on knowledge of the critical residues of the polypeptide of a subject that can mimic the normal function of the polypeptide. Peptide mimetics can break the binding of a polypeptide to its receptor or other proteins and, thus, interfere with the normal function of a polypeptide. For example, a RhoB mimetic would interfere with normal RhoB function.
"Secuencia de ácidos nucleicos", tal como se utiliza en la presente invención, se refiere a un oligonucleótido, nucleótido o polinucleótido, y fragmentos o partes de los mismos, cuyos componentes poliméricos pueden ser ADN, ARN, nucleótidos modificados, miméticos de nucleótidos, o combinaciones de los mismos; y pueden ser de origen genómico o sintético, y pueden ser cadena simple o doble, y representan la cadena de sentido o antisentido."Nucleic acid sequence", as used in the present invention, refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or parts thereof, whose polymeric components may be DNA, RNA, modified nucleotides, nucleotide mimetics, or combinations thereof; and they can be of genomic or synthetic origin, and they can be single or double strands, and represent the sense or antisense chain.
El término "antisentido" tal como se utiliza en la presente invención, se refiere a secuencias de nucleótidos que son complementarias a una secuencia de ADN o ARN especifica, habitualmente un ARNm diana. Se unen a ARNm mediante el emparejamiento de bases de Watson-Crick, e inhiben su mensaje diana a través de la degradación mediante ribonucleasa (RNasa) H o mediante interferencia con la maquinaria de traducción. Los oligonucleótidos antisentido se pueden estabilizar opcionalmente con fosforotionatos, metilfosfonatos, nanoparticulas de polialquilcianoacrilato, y modificaciones en 2' de los azúcares. El término "cadena antisentido" se utiliza en referencia a una cadena de ácido nucleico que es complementaria a la "cadena de sentido". Las moléculas antisentido se pueden producir mediante cualquier procedimiento, incluyendo la síntesis mediante unión del gen de interés en orientación inversa a un promotor viral que permite la síntesis de una cadena complementaria. La designación "negativa" se utiliza algunas veces en referencia a la cadena antisentido, y "positiva" se utiliza algunas veces en referencia a la cadena de sentido .The term "antisense" as used in the present invention refers to nucleotide sequences that are complementary to a specific DNA or RNA sequence, usually a target mRNA. They bind to mRNA by pairing Watson-Crick bases, and inhibit their target message through degradation by ribonuclease (RNase) H or by interference with the translation machinery. The antisense oligonucleotides can optionally be stabilized with phosphorothionates, methylphosphonates, polyalkylcyanoacrylate nanoparticles, and 2 'modifications of the sugars. The term "antisense chain" is used in reference to a nucleic acid chain that is complementary to the "sense chain". Antisense molecules can be produced by any method, including synthesis by binding the gene of interest in reverse orientation to a viral promoter that allows the synthesis of a complementary chain. The designation "negative" is sometimes used in reference to the antisense chain, and "positive" is sometimes used in reference to the sense chain.
Tal como se utiliza en la presente invención, el término "ribozima" significa una molécula de ARN que tiene una actividad enzimática que es capaz de dividir o empalmar otras moléculas de ARN separadas en una secuencia de bases de nucleótidos de una manera específica. En referencia a una molécula de ARN catalítica o enzimática se entiende una molécula de ARN que tiene complementariedad en una región de unión a sustrato con el ARNm de RhoB, y también tiene actividad enzimática que es activa para dividir y/o empalmar ese ARNm, alterándolo de esta maneraAs used in the present invention, the term "ribozyme" means an RNA molecule that has an enzymatic activity that is capable of dividing or splicing other separate RNA molecules into a nucleotide base sequence in a specific manner. Referring to a catalytic or enzymatic RNA molecule is meant an RNA molecule that has complementarity in a substrate binding region with RhoB mRNA, and also has enzymatic activity that is active to divide and / or splice that mRNA, thereby altering it
El término "agente ARNi" tal como se utiliza en la presente invención, se refiere a compuestos y composiciones que pueden actuar a través de un mecanismo de interferencia de ARN (véase, para una referencia general, He y Hannon, (2004) Nat . Genet . 5:522-532) . Habitualmente se utilizan agentes ARNi tales como ARN de corta interferencia (ARNsi), ARN de doble cadena (ARNds), ARN en horquilla corta (ARNsh) , también denominado algunas veces como ARN en horquilla pequeña, otros están en desarrollo. Cuando se introducen en una célula o se sintetizan en una célula, los agentes ARNi se incorporan en un complejo macromolecular que utiliza cadenas del agente ARNi para marcar y dividir cadenas de ARN que contienen la secuencia complementaria (o sustancialmente complementaria) .The term "RNAi agent" as used in the present invention, refers to compounds and compositions that can act through an RNA interference mechanism (see, for general reference, He and Hannon, (2004) Nat. Genet. 5: 522-532). Usually RNAi agents are used such as short interference RNA (siRNA), double stranded RNA (RNAs), short hairpin RNA (shRNA), also sometimes referred to as small hairpin RNA, others are in development. When introduced into a cell or synthesized in a cell, RNAi agents are incorporated into a macromolecular complex that uses chains of the RNAi agent to label and divide RNA chains that contain the complementary (or substantially complementary) sequence.
Tal como se contempla en la presente invención, se diseñan oligonucleótidos antisentido, ADN de triple hélice, aptámeros de ARN, agentes ARNi tales como ARNsi, ARNds, y ARNsh, ribozimas y ARN de cadena simple para inhibir la expresión de RhoB, de manera que se diseña la secuencia de nucleótidos elegida de la molécula inhibidora para provocar la inhibición de la síntesis endógena de proteína RhoB.As contemplated in the present invention, antisense oligonucleotides, triple helix DNA, RNA aptamers, RNAi agents such as siRNA, RNAds, and shRNAs, ribozymes and single stranded RNA are designed to inhibit RhoB expression, so that the nucleotide sequence chosen from the inhibitor molecule is designed to cause the inhibition of endogenous RhoB protein synthesis.
Por ejemplo, en base a la presente descripción, se puede utilizar el conocimiento de la secuencia de nucleótidos de RhoB para diseñar una molécula de ARNsi que inhiba la expresión de RhoB sin demasiada experimentación. De manera similar, los ribozimas se pueden sintetizar para reconocer secuencias de nucleótidos específicas de una proteína de interés y dividirla (Cech. J. Amer . Med Assn. 260:3030 (1988)) . Las técnicas para el diseño de dichas moléculas para su uso en la inhibición dirigida de la expresión génica son bien conocidas por los expertos en la materia.For example, based on the present description, knowledge of the RhoB nucleotide sequence can be used to design an siRNA molecule that inhibits RhoB expression without too much experimentation. Similarly, ribozymes can be synthesized to recognize specific nucleotide sequences of a protein from interest and divide it (Cech. J. Amer. Med Assn. 260: 3030 (1988)). Techniques for the design of such molecules for use in the directed inhibition of gene expression are well known to those skilled in the art.
En una realización de la presente invención, el inhibidor de la sobreexpresión del gen de RhoB es una molécula ARNsh que tiene por los menos un 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, o 100% de homología con la SEC ID NO. 3, tal como se proporciona a continuación.In one embodiment of the present invention, the RhoB gene overexpression inhibitor is an RNAsh molecule that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99%, or 100% homology with SEQ ID NO. 3, as provided below.
Figure imgf000059_0001
Figure imgf000059_0001
Los oligonucleótidos antisentido, ribozimas, y agentes ARNi se pueden preparar mediante cualquier procedimiento conocido en la técnica para la síntesis de moléculas de ácidos nucleicos. Se incluyen técnicas para la síntesis química, tal como síntesis química de fosforamidita en fase sólida. Alternativamente, los oligonucleótidos antisentido, ribozimas, y agentes ARNi se pueden generar mediante transcripción in vitro de secuencias de ADN que codifican la molécula pertinente. Dichas secuencias de ADN se pueden incorporar en una amplia variedad de vectores con promotores de ARN polimerasa adecuados.Antisense oligonucleotides, ribozymes, and RNAi agents can be prepared by any method known in the art for the synthesis of nucleic acid molecules. Techniques for chemical synthesis, such as chemical synthesis of solid phase phosphoramidite, are included. Alternatively, antisense oligonucleotides, ribozymes, and RNAi agents can be generated by in vitro transcription of DNA sequences encoding the relevant molecule. Such DNA sequences can be incorporated into a wide variety of vectors with suitable RNA polymerase promoters.
Los oligonucleótidos antisentido, ribozimas, y agentes ARNi se pueden liberar a células mediante aplicación directa, tal como transfección o formuladas en liposomas. Las técnicas de transfección convencionales incluyen una variedad de técnicas reconocidas en el sector para introducir ácido nucleico exógeno (por ejemplo, ADN o ARN) en una célula huésped, incluyendo coprecipitación con fosfato calcico o cloruro calcico, transfección mediada por DEAE-dextrano, lipofección, (micro) inyección, o electroporación.Antisense oligonucleotides, ribozymes, and RNAi agents can be released to cells by direct application, such as transfection or formulated in liposomes. Conventional transfection techniques include a variety of techniques recognized in the sector for introducing exogenous nucleic acid (eg, DNA or RNA) into a host cell, including coprecipitation with calcium phosphate or calcium chloride, DEAE-dextran-mediated transfection, lipofection, (micro) injection, or electroporation
Los liposomas se pueden preparar utilizando técnicas convencionales incluyendo sonicación, diálisis con quelatos, homogeneización, infusión de disolvente acoplado con extrusión, extrusión por congelación-descongelación, microemulsificación, y otros. Los reactivos utilizados para reticular un liposoma u otro agente que contenga lipidos con oligonucleótidos antisentido, ribozimas, y agentes ARNi comprenden un derivado fosfolipido para anclarse a un extremo de la reticulación en la capa lipidica y un grupo reactivo en el otro extremo para proporcionar un punto de unión a la biomolécula diana. También se pueden utilizar liposomas polimerizados o liposomas recubiertos con polímero. Dichos polímeros pueden estabilizar el liposoma, reducir su depuración del cuerpo, y/o reducir su inmunogenicidad. El liposoma se puede cargar con un residuo funcional tal como un agente de diagnóstico o terapéutico durante o después de su formación. El agente puede estar contenido en el núcleo acuoso del liposoma o se puede incorporar o unir a su membrana circundante.Liposomes can be prepared using conventional techniques including sonication, chelation dialysis, homogenization, solvent infusion coupled with extrusion, freeze-thaw extrusion, microemulsification, and others. Reagents used to crosslink a liposome or other agent containing lipids with antisense oligonucleotides, ribozymes, and RNAi agents comprise a phospholipid derivative to anchor at one end of the cross-linking in the lipid layer and a reactive group at the other end to provide a point binding to the target biomolecule. Polymerized liposomes or polymer coated liposomes can also be used. Such polymers can stabilize the liposome, reduce its clearance from the body, and / or reduce its immunogenicity. The liposome can be loaded with a functional residue such as a diagnostic or therapeutic agent during or after its formation. The agent may be contained in the aqueous nucleus of the liposome or it may be incorporated or bound to its surrounding membrane.
Alternativamente, la liberación de oligonucleótidos antisentido, ribozimas, y agentes ARNi se realiza a través de una estrategia de terapia génica en la que los vectores, incluyendo plásmidos, cósmidos, bacteriófagos, o vectores virales, particularmente vectores retrovirales, lentivirales, o adenovirales, se liberan a una célula o sujeto; o células que dirigen la expresión de las moléculas, por ejemplo, células en las que se ha introducido un vector que dirige la expresión de la molécula, se administran al sujeto. Los vectores que dirigen la síntesis in vivo de oligonucleótidos antisentido, ribozimas, y agentes ARNi se pueden introducir en lineas celulares, células o tejidos de manera constitutiva o inducible.Alternatively, the release of antisense oligonucleotides, ribozymes, and RNAi agents is performed through a gene therapy strategy in which vectors, including plasmids, cosmids, bacteriophages, or viral vectors, particularly retroviral, lentiviral, or adenoviral vectors, are they release a cell or subject; or cells that direct the expression of the molecules, for example, cells in which a vector that directs the expression of the molecule has been introduced, are administered to the subject. Vectors that direct the in vivo synthesis of antisense oligonucleotides, ribozymes, and RNAi agents can be introduce into cell lines, cells or tissues constitutively or inducibly.
Una molécula "vector" es una molécula de ácido nucleico en la que se puede insertar ácido nucleico heterólogo que entonces se puede introducir en una célula huésped apropiada. Los vectores tienen preferiblemente uno o más orígenes de replicación, y uno o más sitios en los que se puede insertar el ADN recombinante . Los vectores frecuentemente tienen medios adecuados mediante los cuales se pueden seleccionar las células con vectores de aquellas sin vectores, por ejemplo, que codifican genes de resistencia fármacos.A "vector" molecule is a nucleic acid molecule into which heterologous nucleic acid can be inserted that can then be introduced into an appropriate host cell. The vectors preferably have one or more origins of replication, and one or more sites where the recombinant DNA can be inserted. Vectors often have suitable means by which cells with vectors can be selected from those without vectors, for example, which encode drug resistance genes.
Una "célula huésped", tal como se utiliza en la presente invención, se refiere a una célula procariota o eucariota que contiene ADN heterólogo que ha sido introducido en la célula mediante cualquier medio, por ejemplo, electroporación, precipitación con fosfato de calcio, microinyección, transformación, infección viral, y similares .A "host cell," as used in the present invention, refers to a prokaryotic or eukaryotic cell that contains heterologous DNA that has been introduced into the cell by any means, for example, electroporation, calcium phosphate precipitation, microinjection. , transformation, viral infection, and the like.
"Heterólogo" tal como se utiliza en la presente invención significa "de origen natural diferente" o representa un estado no natural. Por ejemplo, si se transforma una célula huésped con un ADN o gen derivado de otro organismo, particularmente de otra especie, ese gen es heterólogo con respecto a la célula huésped y también con respecto a los descendientes de la célula huésped que portan ese gen. De forma similar, heteróloga se refiere a una secuencia de nucleótidos derivada de e insertada en el mismo tipo de células natural original, pero que está presente en un estado no natural, por ejemplo un número de copias diferentes, o bajo el control de diferentes elementos reguladores. Los protocolos adicionales de terapia génica pueden implicar aislar una población de células, por ejemplo, células madre o células del sistema inmune de un sujeto, opcionalmente expandir las células en cultivo de tejido, y administrar un vector de terapia génica a las células in vitro. A continuación, las células se pueden devolver al sujeto. Opcionalmente, se pueden seleccionar in vitro células que expresan el oligonucleótido antisentido, ribozima, o agente ARNi deseados, antes de introducirlas en el sujeto. Alternativamente, se puede utilizar una población de células, que pueden ser células de una linea celular o de un individuo que no es el sujeto. Los procedimientos de aislamiento de células madre, células del sistema inmune, etc, de un sujeto son bien conocidos en la técnica. Dichos procedimientos se utilizan, por ejemplo, para el trasplante de médula ósea, trasplante de células madre de sangre periférica, etc., en individuos que está realizando quimioterapia."Heterologous" as used in the present invention means "of different natural origin" or represents an unnatural state. For example, if a host cell is transformed with a DNA or gene derived from another organism, particularly from another species, that gene is heterologous with respect to the host cell and also with respect to the descendants of the host cell carrying that gene. Similarly, heterologous refers to a nucleotide sequence derived from and inserted into the same original natural cell type, but which is present in an unnatural state, for example a number of different copies, or under the control of different elements. regulators Additional gene therapy protocols may involve isolating a population of cells, for example, stem cells or cells of a subject's immune system, optionally expanding the cells in tissue culture, and administering a gene therapy vector to the cells in vitro. Next, the cells can be returned to the subject. Optionally, cells expressing the desired antisense oligonucleotide, ribozyme, or RNAi agent may be selected in vitro before being introduced into the subject. Alternatively, a population of cells can be used, which can be cells of a cell line or an individual that is not the subject. The isolation procedures of stem cells, immune system cells, etc., of a subject are well known in the art. Such procedures are used, for example, for bone marrow transplantation, peripheral blood stem cell transplantation, etc., in individuals undergoing chemotherapy.
En una realización adicional de la presente invención, el inhibidor de la sobreexpresión de gen de RhoB es una combinación que comprende lovastatina y ácido zoledrónico, y/o sales farmacéuticamente aceptables de los mismos.In a further embodiment of the present invention, the RhoB gene overexpression inhibitor is a combination comprising lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof.
El término "y/o" se refiere a un "o" no exclusivo, es decir, "A y/o B" incluye "A y B", asi como "A o B". Como tal, la expresión "lovastatina y ácido zoledrónico, y/o sales farmacéuticamente aceptables de los mismos", se refiere a las siguientes combinaciones: a) lovastatina y ácido zoledrónico; b) una sal farmacéuticamente aceptable de lovastatina y ácido zoledrónico; c) lovastatina y una sal farmacéuticamente aceptable de ácido zoledrónico; y d) una sal farmacéuticamente aceptable de lovastatina y una sal farmacéuticamente aceptable de ácido zoledrónico .The term "and / or" refers to a "or" non-exclusive, that is, "A and / or B" includes "A and B", as well as "A or B". As such, the term "lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof," refers to the following combinations: a) lovastatin and zoledronic acid; b) a pharmaceutically acceptable salt of lovastatin and zoledronic acid; c) lovastatin and a pharmaceutically acceptable salt of zoledronic acid; Y d) a pharmaceutically acceptable salt of lovastatin and a pharmaceutically acceptable salt of zoledronic acid.
La lovastatina es un producto comercial que puede ser proporcionado por las compañías TCI Europe NV, Sigma Aldrich, entre otras. El número de registro CAS de lovastatina es 75330-75-5, y su fórmula estructural es:Lovastatin is a commercial product that can be provided by the companies TCI Europe NV, Sigma Aldrich, among others. The CAS registry number of lovastatin is 75330-75-5, and its structural formula is:
Figure imgf000063_0001
Figure imgf000063_0001
Acido zoledrónico, al que también se hace referencia como zoledronato, es un producto comercial que puede ser proporcionado por las compañías Interchim Ambinter, ACICZoledronic acid, which is also referred to as zoledronate, is a commercial product that can be provided by Interchim Ambinter, ACIC
Figure imgf000063_0002
Figure imgf000063_0002
El término "farmacéuticamente aceptable" significa que un compuesto o combinación de compuestos deben ser compatibles con los otros ingredientes de una formulación, y no ser prejudiciales para el paciente. Para el uso terapéutico, las sales de lovastatina o ácido zoledrónico son aquellas en las que el contraión es farmacéuticamente aceptable .The term "pharmaceutically acceptable" means that a compound or combination of compounds must be compatible with the other ingredients of a formulation, and not be prejudicial for the patient. For therapeutic use, the salts of lovastatin or zoledronic acid are those in which the counterion is pharmaceutically acceptable.
Las sales de adición de ácido farmacéuticamente aceptables tal como se han mencionado anterior pretenden comprender las formas de sales de adición de ácidos no tóxicos terapéuticamente activas que la lovastatina y el ácido zoledrónico son cada uno capaces de formar. Las sales de adición de ácido farmacéuticamente aceptables se pueden obtener de manera adecuada mediante el tratamiento de la forma básica de lovastatina y el ácido zoledrónico con dicho ácido apropiado. Los ácidos apropiados comprenden, por ejemplo, ácidos inorgánicos tales como hidrácidos, por ejemplo ácido clorhídrico o bromhídrico, sulfúrico, nítrico, fosfórico y ácidos similares; u ácidos orgánicos tales como, por ejemplo, acético, propanoico, hidroxiacético, láctico, pirúvico, oxálico (es decir, etanodioico) , malónico, succínico (es decir, ácido butanodioico) , maleico, fumárico, málico (es decir, ácido hidroxibutanodioico) , tartárico, cítrico, metanosulfónico, etanosulfónico, bencenosulfónico, p-toluenosulfónico, ciclámico, salicílico, p-aminosalicílico, pamoico y ácidos similares. A la inversa, dichas formas de sales se pueden convertir mediante el tratamiento con una base apropiada en la forma de base libre.Pharmaceutically acceptable acid addition salts as mentioned above are intended to comprise the forms of therapeutically active non-toxic acid addition salts that lovastatin and zoledronic acid are each capable of forming. Pharmaceutically acceptable acid addition salts can be suitably obtained by treating the basic form of lovastatin and zoledronic acid with said appropriate acid. Suitable acids comprise, for example, inorganic acids such as hydracids, for example hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and similar acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e. hydroxybutanedioic acid) , tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and similar acids. Conversely, said salt forms can be converted by treatment with an appropriate base in the free base form.
En una realización de la presente invención, la combinación que comprende lovastatina y ácido zoledrónico, y/o sales farmacéuticamente aceptables de las mismas se pueden administrar de manera simultánea, por separado o de una manera secuencial.In an embodiment of the present invention, the combination comprising lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof can be administered simultaneously, separately or sequentially.
Como tal, la presente invención se refiere además a una combinación de formulaciones farmacéuticas separadas en forma de kit. Por ejemplo, un kit puede comprender dos formulaciones farmacéuticas separadas que comprenden: 1) lovastatina o sales farmacéuticamente aceptables de la misma; y 2) ácido zoledrónico o sales farmacéuticamente aceptables del mismo. El kit puede comprender también un recipiente para las formulaciones separadas, tal como una botella compartimentada o un recipiente de papel de aluminio compartimentado . Ejemplos adicionales de recipientes incluyen jeringas, cajas, bolsas y similares. Habitualmente, un kit comprende instrucciones para la administración de los componentes separados. La forma kit es particularmente ventajosa cuando los componentes separados se administran preferiblemente en formas de dosificación diferentes (por ejemplo, oral y parenteral), se administran a diferentes intervalos de dosificación, o cuando se desea el ajuste de los componentes individuales de la combinación por profesional clínico.As such, the present invention further relates to a combination of separate pharmaceutical formulations in kit form. For example, a kit may comprise two separate pharmaceutical formulations comprising: 1) lovastatin or pharmaceutically acceptable salts thereof; and 2) zoledronic acid or pharmaceutically acceptable salts thereof. The kit may also comprise a container for separate formulations, such as a compartmentalized bottle or a compartmentalized aluminum foil container. Additional examples of containers include syringes, boxes, bags and the like. Usually, a kit comprises instructions for the administration of the separate components. The kit form is particularly advantageous when the separated components are preferably administered in different dosage forms (eg, oral and parenteral), are administered at different dosage intervals, or when adjustment of the individual components of the combination is desired by professional. clinical.
En general, se contempla que se puede utilizar una cantidad diaria eficaz de ácido zoledrónico de aproximadamente 0,5 hasta aproximadamente 2000 mg. Una cantidad diaria eficaz de lovastatina puede variar desde aproximadamente 5 mg hasta aproximadamente 400 mg.In general, it is contemplated that an effective daily amount of zoledronic acid of about 0.5 to about 2000 mg can be used. An effective daily amount of lovastatin can vary from about 5 mg to about 400 mg.
La dosis exacta y la frecuencia de administración de los inhibidores de la sobreexpresión del gen de RhoB dependen del compuesto concreto o la combinación utilizada, la afección concreta en tratamiento, la gravedad de la afección en tratamiento, la edad, el peso, el sexo, la extensión del trastorno y la condición física general del paciente concreto, así como otra medicación que el individuo puede estar tomando, tal como es bien sabido por los expertos en la materia. Además, es evidente que dicha cantidad diaria eficaz puede disminuir o aumentar dependiendo de la respuesta del sujeto tratado y/o dependiendo de la evaluación del médico que prescribe los compuestos de la presente invención. Las variaciones de la cantidad diaria eficaz mencionada anteriormente sirven por tanto únicamente como guias.The exact dose and frequency of administration of RhoB gene overexpression inhibitors depend on the particular compound or combination used, the specific condition being treated, the severity of the condition being treated, age, weight, sex, the extent of the disorder and the general physical condition of the specific patient, as well as other medication that the individual may be taking, as is well known by those skilled in the art. Furthermore, it is evident that said effective daily amount may decrease or increase depending on the response of the treated subject and / or depending on the evaluation of the prescribing physician of the compounds of the present invention. The variations of the effective daily amount mentioned above therefore serve only as guides.
Los siguientes ejemplos no limitativos ayudan a ilustrar los principios de la invención.The following non-limiting examples help to illustrate the principles of the invention.
EjemplosExamples
Animales y cultivos celulares. Se adquirieron ratones desnudos atimicos de cuatro semanas de vida de Harían (Barcelona, España) y se mantuvieron en condiciones especificas libres de patógenos. La linea celular de adenocarcinoma A549 fueron obtenidas del Dr. Gazdar. Todas las lineas celulares se mantuvieron en medio RPMI 1640 suplementado con un 10% de suero bovino fetal.Animals and cell cultures. Atomic nude mice four weeks old from Harían (Barcelona, Spain) were acquired and kept in specific pathogen-free conditions. The A549 adenocarcinoma cell line was obtained from Dr. Gazdar. All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum.
Aislamiento de ARN, síntesis de ADNc e hibridación con microarraysRNA isolation, cDNA synthesis and hybridization with microarrays
Se extrajo ARN utilizando Reactivo TRIzol (LifeRNA was extracted using TRIzol Reagent (Life
Technologies) de tres placas de cultivo de 10 cm por línea celular y se purificó utilizando el kit RNeasy (Qiagen, Hilden, Alemania) . La pureza y calidad se evaluó utilizando nanochips RNA 6000 (Agilent Technologies, Alemania) . A continuación, se utilizó el ADN para sintetizar sondas complementarias (ADNc) utilizando el kit de síntesis de ADNc de un ciclo (Affimetrix) . Posteriormente, se sintetizó ARNc y se marcó con ADNc como plantilla utilizando el kit de marcado IVT (Affimetrix) , y se purificó mediante GeneChip Sample Cleanup Module (Affimetrix) . Los ARNc marcados se fragmentaron y se hibridaron a microarrays de oligonucleótidos de alta densidad de GeneChip de Affimetrix Human Genome U133 2.0 según los protocolos descritos en Gene Expression Analysis Technical Manual (http://www.affimetrix.com) . Se hibridaron 3 microarrays con 3 réplicas biológicas independientes por línea celular de 3 líneas diferentes (denominadas MlMl, M1M3, y M1M4) . Para cada línea celular, se utilizó un grupo de tres extracciones de ARNm independientes para hibridar cada microarray de oligonucleótidos de alta densidad humanos de GeneChip. Como controles se utilizaron 6 líneas de células tumorales parentales: 3 transíectadas con el vector de luciferasa y 3 no transíectadas . En total, se híbrido un conjunto de 15 muestras: 6 controles y 9 líneas metastáticas .Technologies) of three 10 cm culture plates per cell line and was purified using the RNeasy kit (Qiagen, Hilden, Germany). Purity and quality was evaluated using RNA 6000 nanochips (Agilent Technologies, Germany). Next, the DNA was used to synthesize complementary probes (cDNA) using the one-cycle cDNA synthesis kit (Affimetrix). Subsequently, cRNA was synthesized and labeled with cDNA as a template using the IVT labeling kit (Affimetrix), and purified by GeneChip Sample Cleanup Module (Affimetrix). The labeled cRNAs were fragmented and hybridized to high density oligonucleotide microarrays of Affimetrix Human Genome U133 2.0 GeneChip according to the protocols described in Gene Expression Analysis Technical Manual (http://www.affimetrix.com). 3 microarrays were hybridized with 3 independent biological replicates per cell line of 3 different lines (called MlMl, M1M3, and M1M4). For each cell line, a group of three independent mRNA extractions was used to hybridize each GeneChip human high density oligonucleotide microarray. As controls, 6 lines of parental tumor cells were used: 3 transcribed with the luciferase vector and 3 untranslated. In total, a set of 15 samples was hybridized: 6 controls and 9 metastatic lines.
PCR cuantitativa a tiempo realQuantitative real-time PCR
Se aisló el ARN total del 70-80% de cultivos confluentes utilizando Trizol RNA (2 μg) y DNasa I, se trató y transcribió de forma inversa utilizando la transcriptasa inversa Superscript II y cebadores oligo(dT) . Se realizó un Assay-on-Demand™ (Applied Biosystems) utilizando un sistema de detección de secuencias Gene Amp 7300 (Applied Biosystems) para RHOB según las recomendaciones del fabricante. El valor promedio de ciclo umbral (Ct) de las muestras por triplicado se utilizó para calcular la expresión génica. Los productos de PCR se normalizaron hasta los niveles de β-actina. El experimento se repitió tres veces con resultados idénticos.Total RNA from 70-80% of confluent cultures was isolated using Trizol RNA (2 μg) and DNase I, treated and transcribed in reverse using Superscript II reverse transcriptase and oligo (dT) primers. An Assay-on-Demand ™ (Applied Biosystems) was performed using a Gene Amp 7300 (Applied Biosystems) sequence detection system for RHOB according to the manufacturer's recommendations. The average threshold cycle value (Ct) of the triplicate samples was used to calculate gene expression. The PCR products were normalized to β-actin levels. The experiment was repeated three times with identical results.
Análisis de los datos de microarray: normalización, cálculo de la señal, expresión diferencial significativa y agrupación de los perfiles de muestra/genAnalysis of microarray data: normalization, signal calculation, significant differential expression and grouping of sample / gene profiles
El análisis de los datos de microarrays se realizó utilizando la estrategia y procedimientos descritos en la bibliografía (Castellano et al. 2007) . En resumen, se utilizó el algoritmo de RMA para la corrección de la base, la normalización intra- e intermicroarrays y el cálculo de la señal de expresión (2-4) . Una vez calculada la señal de expresión absoluta para cada gen (es decir, el valor de la señal para cada conjunto de sondas) en cada microarray, se aplicó un procedimiento (denominado SAM) (Tusher et al. 2001) para calcular la expresión diferencial significativa y encontrar los conjuntos de sondas para genes que caracterizan las muestras altamente metastáticas . El procedimiento utiliza permutaciones para proporcionar una inferencia estadística robusta de los genes más significativos y proporciona valores de p ajustados a tests múltiples utilizando FDR (Benjamini et al. 1995) . El test principal se realizó mediante el contraste de 4 muestras de control frente a 9 muestras altamente metastáticas. Sin embargo, con 3 líneas metastáticas diferentes (MlMl, M1M3, MM1M4) realizadas por triplicado, se realizaron análisis SAM múltiples para encontrar los genes que mostraban una expresión diferencial significativa que también eran estables en diferentes tests SAM. Se utilizó un corte de FDR < 0,20 para todos los cálculos de expresión diferencial. Se aplicaron todos estos procedimientos utilizando las herramientas estadísticas "R" y Bioconductor . Tras la identificación del conjunto de sondas para genes expresados de manera diferencial, se analizó la correspondiente matriz de valores de expresión para todas las hibridaciones de microarrays utilizando el algoritmo de agrupación "hclust" implementado en "R". Este algoritmo realiza un análisis de agrupación jerárquica con unión completa para buscar similitudes entre los conjuntos de sondas basados en sus valores de expresión en los diferentes microarrays de chips analizados. El algoritmo clasifica los conjuntos de sondas en grupos correlacionados que presentan perfiles de expresión o firmas de expresión similares. Esta estrategia global permitió la selección de genes claves significativos con perfiles de expresión similares.The analysis of microarray data was performed using the strategy and procedures described in the literature (Castellano et al. 2007). In summary, the RMA algorithm was used for base correction, intra- and intermicroarray normalization and expression signal calculation (2-4). Once the absolute expression signal was calculated for each gene (that is, the value of the signal for each set of probes) in each microarray, a procedure (called SAM) was applied (Tusher et al. 2001) to calculate the significant differential expression and find the sets of probes for genes that characterize highly metastatic samples. The procedure uses permutations to provide a robust statistical inference of the most significant genes and provides p-values adjusted to multiple tests using FDR (Benjamini et al. 1995). The main test was performed by contrasting 4 control samples against 9 highly metastatic samples. However, with 3 different metastatic lines (MlMl, M1M3, MM1M4) performed in triplicate, multiple SAM analyzes were performed to find the genes that showed a significant differential expression that were also stable in different SAM tests. A cut of FDR <0.20 was used for all differential expression calculations. All these procedures were applied using the statistical tools "R" and Bioconductor. After identifying the set of probes for differentially expressed genes, the corresponding array of expression values for all microarray hybridizations was analyzed using the "hclust" clustering algorithm implemented in "R". This algorithm performs a hierarchical clustering analysis with complete binding to look for similarities between the sets of probes based on their expression values in the different microarray of chips analyzed. The algorithm classifies probe sets into correlated groups that have similar expression profiles or expression signatures. This global strategy allowed the selection of significant key genes with similar expression profiles.
Ensayos de metástasis: i.c. se realizó tal como se ha descrito anteriormente (González et al. 2007) . Creación de imágenes por bioluminescencia y análisis. Se anestesiaron ratones y se les inyectó intraperitonealmente 1,5 mg de D-Luciferina en 100 μl de PBS. La imagen se completó a los 2 min exactamente para cada grupo de ratones con un sistema Xenogen IVIS acoplado un software de adquisición y análisis Living Image (Xenogen Inc.) . El flujo de fotones se calculó para cada ratón mediante la utilización de una región circular de interés para cada pata trasera. Los valores base (de ratón inyectado con luciferina sin células tumorales) se restaron de cada medición .Metastasis trials: ic was performed as described previously (González et al. 2007). Bioluminescence imaging and analysis. Mice were anesthetized and 1.5 mg of D-Luciferin in 100 µl of PBS was injected intraperitoneally. The image was completed at 2 min exactly for each group of mice with an Xenogen IVIS system coupled with a Living Image acquisition and analysis software (Xenogen Inc.). The photon flow was calculated for each mouse by using a circular region of interest for each hind leg. The base values (of mouse injected with luciferin without tumor cells) were subtracted from each measurement.
Ensayo de proliferaciónProliferation test
Se evaluó la proliferación celular mediante un ensayo MTT según las recomendaciones del fabricante (Roche Diagnostics GmbH, Mannheim, Alemania) . Los experimentos se repitieron tres veces y los datos se representaron como la media de los pocilios por sextuplicado ± SEM.Cell proliferation was evaluated by an MTT assay according to the manufacturer's recommendations (Roche Diagnostics GmbH, Mannheim, Germany). The experiments were repeated three times and the data were represented as the mean of the wells per sextuplicate ± SEM.
Análisis radiográfico y tomográfico microcomputerizado (μCT)Microcomputerized radiographic and tomographic analysis (μCT)
Se realizó una radiografía por rayos X bajo anestesia, en posición de cubito supino sobre una película radiográfica sensible (MIN-R, Eastman Kodak) . Los ratones se expusieron a radiación X a 20 kV durante 20 s utilizando un instrumento Faxitron (modelo MX-20; Faxitron, Buffalo Grove, IL, USA) . El área de metástasis osteolítica se evaluó con un sistema de análisis de imagen computerizada, AnalySIS® (sistema de obtención de imágenes suave GmbH, Münster, Alemania) . Se capturaron imágenes escaneadas en una película de rayos X de alta resolución con doble aumento a 1.200 ppi utilizando un scanner Epson Expression 1680 Pro (Long Beach, CA, USA) . La cuantificación del área metastática se realizó dos veces después de la calibración por dos observadores independientes. Para las curvas de Kaplan-Meier la metástasis se valoró como el tiempo transcurrido hasta la primera aparición de signos de caquexia y dificultad para caminar.An X-ray was performed under anesthesia, in a supine cube position on a sensitive radiographic film (MIN-R, Eastman Kodak). Mice were exposed to X radiation at 20 kV for 20 s using a Faxitron instrument (model MX-20; Faxitron, Buffalo Grove, IL, USA). The area of osteolytic metastasis was evaluated with a computerized image analysis system, AnalySIS® (soft imaging system GmbH, Münster, Germany). Scanned images were captured on a high resolution X-ray film with double magnification at 1,200 ppi using an Epson Expression 1680 Pro scanner (Long Beach, CA, USA). Quantification of the metastatic area was performed twice after calibration by two independent observers. For the curves of Kaplan-Meier metastasis was assessed as the time elapsed until the first appearance of signs of cachexia and difficulty walking.
Se analizaron todas las articulaciones femorotibial mediante un sistema μCT (micro CAT ™II, SiemensAll femorotibial joints were analyzed using a μCT system (micro CAT ™ II, Siemens
Preclinical Solutions, Knoxville, TN, USA) a 75,0 kVp yPreclinical Solutions, Knoxville, TN, USA) at 75.0 kVp and
250,0 uA. Los escaneados se realizaron a una resolución de250.0 uA. The scans were performed at a resolution of
10 μm. SE reconstruyeron imágenes 2D CT utilizando un procedimiento de proyección de retro-convolución estándar con un filtro Shepp-Logan . Las imágenes se almacenaron en grupos de 3D con un tamaño de vóxel de 19 μm x 19 μm x 23 μm.10 μm 2D CT images were reconstructed using a standard retro-convolution projection procedure with a Shepp-Logan filter. The images were stored in 3D groups with a voxel size of 19 μm x 19 μm x 23 μm.
Construcciones "knock-down" Se obtuvieron construcciones lentivirales para ARNsh de RhoB humana de Sigma (St. Louis, MO, USA) . Para obtener partículas virales, se transíectaron células de empaquetamiento con 8 μg de ADN para cada tipo de fusión mediante el procedimiento con fosfato clasicote según las recomendaciones del fabricante. Se utilizó el vector pLKO "scramble" como control (que contiene una secuencia shRHOB en la que se han cambiado aleatoriamente las bases nucleotídicas) . Dos días después de la transíección, los sobrenadantes se centrifugaron durante 10 minutos a 600 g y se filtraron a través de un filtro de acetato de celulosa de 0,45 μm de poro. Para la infección por transducción, se sembraron A549 a IxIO5 células y se incubaron durante toda la noche con sobrenadantes virales en presencia de 4 μg/ml de polibreno (Sigma) . Cuarenta y ocho horas después de la infección, se incubaron poblaciones de células en medio que contenía el antibiótico apropiado durante dos semanas adicionales. Los grupos resistentes a antibiótico se expandieron y se congelaron en cada pasaje celular. Aislamiento de colonias derivadas de una única célula (SCC) de huesos largosKnock-down constructions Lentiviral constructions were obtained for human RhoB shRNA from Sigma (St. Louis, MO, USA). To obtain viral particles, packaging cells were transiected with 8 μg of DNA for each type of fusion using the classic phosphate procedure according to the manufacturer's recommendations. The pLKO "scramble" vector was used as a control (containing a shRHOB sequence in which the nucleotide bases have been randomly changed). Two days after the transection, the supernatants were centrifuged for 10 minutes at 600 g and filtered through a 0.45 μm pore cellulose acetate filter. For transduction infection, A549 was seeded into IxIO 5 cells and incubated overnight with viral supernatants in the presence of 4 µg / ml polybrene (Sigma). Forty-eight hours after infection, cell populations were incubated in medium containing the appropriate antibiotic for two additional weeks. Antibiotic resistant groups expanded and frozen in each cell passage. Isolation of colonies derived from a single cell (SCC) from long bones
Se aislaron células metastáticas de la médula ósea de las extremidades inferiores. Los ratones se sacrificaron según los protocolos aprobados del Comité Local de Animales (Universidad de Navarra, protocolo 003-04) . Se extirparon los huesos largos, y se lavaron de todos los tejidos blandos. Se liberaron las células de la médula mediante "flushing", introduciendo 5-10 mi de medio CC-MEM que contenia 2 x penicilina/estreptomicina con una aguja de calibre 27 en la epífisis distal a través del compartimento de la médula ósea. Las agrupaciones de células se desagregaron pasando el medio que contenía las células a través de la jeringa con aguja del calibre 27 G. Las células se sembraron en placas de 10 ó 15 cm expandidas durante 5 días en un medio que contenía 0,4 mg/ml de G-418. Este procedimiento se realizó por separado para cada fémur y tibia de 7 ratones por grupo. Se contaron las SCC bajo la luz de un microscopio después de tinción con violeta cristal.Metastatic cells from the bone marrow were isolated from the lower extremities. Mice were sacrificed according to the approved protocols of the Local Animal Committee (University of Navarra, protocol 003-04). Long bones were removed, and washed from all soft tissues. The marrow cells were released by flushing, introducing 5-10 ml of CC-MEM medium containing 2 x penicillin / streptomycin with a 27 gauge needle into the distal epiphysis through the bone marrow compartment. The cell clusters were disaggregated by passing the medium containing the cells through the 27G needle syringe. The cells were seeded in expanded 10 or 15 cm plates for 5 days in a medium containing 0.4 mg / ml of G-418. This procedure was performed separately for each femur and tibia of 7 mice per group. SCCs were counted under the light of a microscope after staining with crystal violet.
Análisis histológicoHistological analysis
Se extirparon las patas traseras, se extrajeron cuidadosamente tejidos blandos del fémur, se fijaron durante 24 h en formalina tamponada neutra al 10%, se deshidrataron y se descalcificaron en solución Osteosoft®.The hind legs were removed, soft tissues of the femur were carefully removed, fixed for 24 h in 10% neutral buffered formalin, dehydrated and decalcified in Osteosoft® solution.
Después de la completa deshidratación con alcohol, los huesos se incluyeron en parafina y se tiñeron secciones deAfter complete dehydration with alcohol, the bones were included in paraffin and sections of stained
5 μm con H&E.5 μm with H&E.
Expresión de RhoB y valor de predicción en pacientes con adenocarcinomaRhoB expression and predictive value in patients with adenocarcinoma
PacientesPatients
Para este análisis, se seleccionaron 35 individuos que se separaron en dos grupos seleccionados en base al grupo de Enfermedad Progresiva (PD) comparado con un grupo Sin Enfermedad (DF) después de la cirugía. El grupo PD incluía pacientes con un historial de un cáncer de pulmón primario completamente reseccionado que reincidió (reaparición o metástasis) después de un periodo sin enfermedad de 6 meses después de la cirugía. Los pacientes DF fueron el resto de individuos con cáncer de pulmón primario completamente reseccionado sin signos de la enfermedad. Los pacientes que realizaron cualquier tratamiento, ya sea radioterapia o quimioterapia, después de la cirugía se excluyeron del análisis. El protocolo de estudio fue aprobado por el Comité Ético Local. Para cada paciente se obtuvo un consentimiento y cuestionarios por escrito (antecedentes y seguimiento) . Los criterios de inclusión fueron: histología (NSCLC: adenocarcinoma) , y ausencia de cáncer en los 5 años previos a la intervención quirúrgica del cáncer de pulmón, excepto el melanoma o el cáncer de piel. El diagnóstico histológico se determinó según la clasificación WHO. El estadiaje patológico de los tumores se realizó según el sistema internacional para el cáncer de pulmón. El periodo de seguimiento continuó hasta el 31 de octubre del 2007. El tiempo de reincidencia se calculó desde la intervención quirúrgica hasta la fecha de reincidencia .For this analysis, 35 individuals were selected who separated into two selected groups based on the group of Progressive Disease (PD) compared to a No Disease (DF) group after surgery. The PD group included patients with a history of a completely resected primary lung cancer that relapsed (recurrence or metastasis) after a disease-free period of 6 months after surgery. DF patients were the rest of individuals with completely resected primary lung cancer with no signs of the disease. Patients who performed any treatment, whether radiotherapy or chemotherapy, after surgery were excluded from the analysis. The study protocol was approved by the Local Ethical Committee. Written consent and questionnaires (background and follow-up) were obtained for each patient. Inclusion criteria were: histology (NSCLC: adenocarcinoma), and absence of cancer in the 5 years prior to the lung cancer surgery, except melanoma or skin cancer. The histological diagnosis was determined according to the WHO classification. The pathological staging of the tumors was performed according to the international system for lung cancer. The follow-up period continued until October 31, 2007. The recidivism time was calculated from the surgical intervention until the recidivism date.
Immunohistoquímica (IHC)Immunohistochemistry (IHC)
Se utilizaron secciones de tejido fijados a formalina y embebidos en parafina para los procedimientos immunohistoquímicos . La parafina se extrajo de los tejidos y las secciones se hidrataron a través de una serie graduada de etanol. La actividad de peroxidasa endógena se detuvo con hidrógeno peroxidasa al 3% durante 10 minutos. La extracción de antígeno por microondas se llevó a cabo con tampón EDTA (mM, pH 8) durante 2 x 15 min. Los sitios de unión no específica se bloquearon con suero normal de cabra al 5% en TBS-Tween (tampón de lavado, Dako) durante 30 minutos. Las secciones se incubaron con anticuerpo policlonal de conejo anti-RhoB (sc-180, Santa Cruz Biotechnology, Inc) durante toda la noche a 40C. La dilución de trabajo fue: 1:75. Después de enjuagar con TBS, las secciones se incubaron con el complejo policlonal Envision (Dako) . La actividad peroxidasa se demostró mediante diaminobencidina . Finalmente, las secciones se lavaron en agua, se contrastaron ligeramente con hematoxilina, se deshidrataron y se montaron en DPX. Los tejidos que expresaban diferentes niveles de RhoB se incluyeron en cada banda inmunohistoquimica para unificar la posible discordancia de intensidad.Sections fixed to formalin and embedded in paraffin were used for immunohistochemical procedures. The paraffin was extracted from the tissues and the sections were hydrated through a graduated series of ethanol. Endogenous peroxidase activity was stopped with 3% hydrogen peroxidase for 10 minutes. Microwave antigen extraction was carried out with EDTA buffer (mM, pH 8) for 2 x 15 min. Non-specific binding sites were blocked with normal serum of 5% goat in TBS-Tween (wash buffer, Dako) for 30 minutes. Sections were incubated with rabbit anti-RhoB polyclonal antibody (sc-180, Santa Cruz Biotechnology, Inc) overnight at 4 0 C. The working dilution was: 1:75. After rinsing with TBS, the sections were incubated with the Envision polyclonal complex (Dako). The peroxidase activity was demonstrated by diaminobenzidine. Finally, the sections were washed in water, slightly contrasted with hematoxylin, dehydrated and mounted on DPX. Tissues expressing different levels of RhoB were included in each immunohistochemical band to unify the possible intensity mismatch.
Evaluación de la inmunotinciónImmunostaining Evaluation
Dos observadores de manera independiente y a ciegas evaluaron la extensión e intensidad de la tinción en las muestras. Para RhoB, la extensión se puntuó como el porcentaje de células positivas (0-100%) y la intensidad de tinción se evaluó en comparación con un control positivo externo conocido (mareaje 1+, ligero; 2+, moderado y 3+, intenso) . Las lecturas independientes discordantes se resolvieron mediante la revisión simultánea por ambos observadores.Two observers independently and blindly evaluated the extent and intensity of staining in the samples. For RhoB, the extension was scored as the percentage of positive cells (0-100%) and the intensity of staining was evaluated compared to a known external positive control (tidal 1+, light; 2+, moderate and 3+, intense ). Discordant independent readings were resolved by simultaneous review by both observers.
Análisis estadístico. Se utilizó el test de log-rank para calcular la significancia estadística (valor de p) de las diferencias observadas entre las curvas de Kaplan-Meier . Para estudiar las diferencias en las velocidades de proliferación, crecimiento del tumor, se analizaron las diferencias en el área metastática mediante el test de Kruskall-Wallis con el test de comparación múltiple de Dunn . Los valores se expresaron como la media ± SEM y la significancia estadística se definió como p < 0,05 (*), p < 0,01 (**), p < 0,001 (***) . Métodos complementarios proporcionan una información adicional.Statistic analysis. The log-rank test was used to calculate the statistical significance (p-value) of the differences observed between the Kaplan-Meier curves. To study the differences in proliferation rates, tumor growth, differences in the metastatic area were analyzed using the Kruskall-Wallis test with the Dunn multiple comparison test. The values were expressed as the mean ± SEM and the statistical significance was defined as p <0.05 (*), p <0.01 (**), p <0.001 (***). Complementary methods They provide additional information.
ReferenciasReferences
Mazieres et al. (2004) Loss of RhoB Expression in Human Lung Cáncer Progression. Clinical Cáncer Research 10: 2742-2750.Mazieres et al. (2004) Loss of RhoB Expression in Human Lung Cancer Progression. Clinical Cancer Research 10: 2742-2750.
Sato et al. (2007) . RhoB is frequently downregulated in non-small-cell lung cáncer and resides in the 2p24 homozygous deletion región of a lung cáncer cell line. Int J Cáncer. 120 (3 ) : 543-51.Sato et al. (2007). RhoB is frequently downregulated in non-small-cell lung cancer and resides in the 2p24 homozygous deletion region of a lung cancer cell line. Int J Cancer. 120 (3): 543-51.
Li et al. (2008) . Lung Cáncer. 2008 Feb; 59 (2 ) : 180-91.Li et al. (2008). Lung Cancer 2008 Feb; 59 (2): 180-91.
Turner et al. (2008) . Effects of lovastatin on Rho isoform expression, activity, and association with guanine nucleotide dissociation inhibidores. Biochemical Pharmacology, 75, 405-413.Turner et al. (2008). Effects of lovastatin on Rho isoform expression, activity, and association with guanine nucleotide dissociation inhibitors. Biochemical Pharmacology, 75, 405-413.
Hawk et al. (1996) . Inhibition of lung tumor cell growth in vitro and mouse lung tumor formation by lovastatin. Cáncer Letters, 109(1,2), 217-222.Hawk et al. (nineteen ninety six) . Inhibition of lung tumor cell growth in vitro and mouse lung tumor formation by lovastatin. Cancer Letters, 109 (1,2), 217-222.
Castellano E, De Las Rivas J, Guerrero C, Santos E. Transcriptional networks of knockout cell lines identify functional specificities of H-Ras and N-Ras : significant involvement of N-Ras in biotic and defense responses. Oncogene 2007; 26 : 917-33.Castellano E, De Las Rivas J, Guerrero C, Santos E. Transcriptional networks of knockout cell lines identify functional specificities of H-Ras and N-Ras: significant involvement of N-Ras in biotic and defense responses. Oncogene 2007; 26: 917-33.
Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Nati Acad Sci U S A 2001 ; 98 : 5116-21.Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Nati Acad Sci U S A 2001; 98: 5116-21.
Benjamini Y, Hochberg Y. Controlling the false discovery rate: A practical and powerful approach to múltiple testing. J RoyStat Soc 1995; (Ser B) 57:289-300.Benjamini Y, Hochberg Y. Controlling the false discovery rate: A practical and powerful approach to multiple testing. J RoyStat Soc 1995; (Ser B) 57: 289-300.
González I, Vicent S, de Álava E, Lecanda F. EWS/FLI-1 oncoprotein subtypes impose different requirements for transíormation and metastatic activity in a murine model. J Mol Med 2007;85:1015-29.González I, Vicent S, of Álava E, Lecanda F. EWS / FLI-1 oncoprotein subtypes impose different requirements for transíormation and metastatic activity in a murine model. J Mol Med 2007; 85: 1015-29.
Yin JJ, Selander K, Chirgwin JM, et al. TGF-beta signaling blockade inhibits PTHrP secretion by breast cáncer cells and bone metastases development. J Clin Invest 1999;103:197-206 Yin JJ, Selander K, Chirgwin JM, et al. TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. J Clin Invest 1999; 103: 197-206

Claims

REIVINDICACIONES
1. Procedimiento de pronóstico de la evolución de la enfermedad en un sujeto con adenocarcinoma de pulmón que comprende : a) la medición de la expresión del gen de RhoB; o b) la medición del número de copias del gen de RhoB; en una muestra de dicho sujeto con adenocarcinoma de pulmón, caracterizado porque la detección de la sobreexpresión o el aumento en el número de copias del gen de RhoB en relación con la expresión o el número de copias del mismo gen en una muestra de control es indicativo del riesgo de metástasis, o reaparición o reincidencia de actividad neoplásica.1. Method of prognosis of the evolution of the disease in a subject with lung adenocarcinoma comprising: a) the measurement of RhoB gene expression; or b) measuring the number of copies of the RhoB gene; in a sample of said subject with lung adenocarcinoma, characterized in that the detection of overexpression or the increase in the number of copies of the RhoB gene in relation to the expression or the number of copies of the same gene in a control sample is indicative of the risk of metastasis, or recurrence or recurrence of neoplastic activity.
2. Procedimiento según la reivindicación 1, en el que el pronóstico de la evolución de la enfermedad comprende la determinación de un resultado clínico seleccionado del grupo que consiste en velocidad de respuesta (RR) , respuesta completa (CR) , respuesta parcial (PR) , enfermedad estable (SD) , tiempo hasta la progresión (TTP) , supervivencia global (OS) , y beneficio clínico, que comprende respuesta completa (CR) , respuesta parcial (PR) , y enfermedad estable (SD) .2. The method according to claim 1, wherein the prognosis of the disease evolution comprises the determination of a clinical result selected from the group consisting of response speed (RR), complete response (CR), partial response (PR) , stable disease (SD), time to progression (TTP), overall survival (OS), and clinical benefit, which includes complete response (CR), partial response (PR), and stable disease (SD).
3. Procedimiento según cualquiera de las reivindicaciones 1-2, en el que la medición de la expresión del gen de RhoB se determina a nivel de proteína, ARNm o ADNc.3. A method according to any one of claims 1-2, wherein the measurement of RhoB gene expression is determined at the level of protein, mRNA or cDNA.
4. Uso de un gen de RhoB, o una región del mismo, como marcador del pronóstico de metástasis, o reaparición o reincidencia de actividad neoplásica en un sujeto con adenocarcinoma de pulmón.4. Use of a RhoB gene, or a region thereof, as a marker of the prognosis of metastasis, or recurrence or recurrence of neoplastic activity in a subject with lung adenocarcinoma.
5. Procedimiento para el screening de fármacos activos contra adenocarcinoma de pulmón, comprendiendo dicho procedimiento : c) la medición de la expresión del gen de RhoB, o la medición del número de copias del gen de RhoB, en una muestra con células de adenocarcinoma de pulmón en presencia de un fármaco, o la medición de la afinidad de unión entre un fármaco y un polipéptido RhoB; d) la comparación de la expresión o números de copias medidas del gen de RhoB, o la afinidad de unión de la etapa a) a una muestra de control; caracterizado porque una expresión o número de copias reducido del gen de RhoB, o una mayor afinidad de unión en la etapa a) , en relación con la expresión o el número de copias del mismo gen o la afinidad de unión del mismo polipéptido en una muestra de control es indicativa de la actividad del fármaco contra el adenocarcinoma de pulmón.5. Procedure for screening active drugs against lung adenocarcinoma, said procedure comprising: c) measuring the expression of the RhoB gene, or measuring the number of copies of the RhoB gene, in a sample with lung adenocarcinoma cells in the presence of a drug, or the measurement of binding affinity between a drug and a RhoB polypeptide; d) comparison of the expression or measured copy numbers of the RhoB gene, or the binding affinity of step a) to a control sample; characterized in that a reduced expression or copy number of the RhoB gene, or a higher binding affinity in step a), in relation to the expression or the number of copies of the same gene or the binding affinity of the same polypeptide in a sample Control is indicative of the activity of the drug against lung adenocarcinoma.
6. Procedimiento para determinar la eficacia de una pauta de tratamiento terapéutico en un sujeto con adenocarcinoma de pulmón, comprendiendo dicho procedimiento : a) la medición de la expresión del gen de RhoB, o la medición del número de copias del gen de RhoB, en una muestra con células de adenocarcinoma de pulmón de dicho sujeto, generando de este modo un nivel inicial; b) la medición de la expresión del gen de RhoB, o la medición del número de copias del gen de RhoB, en una segunda muestra con células de adenocarcinoma de pulmón del mismo sujeto en un momento posterior a la administración de la pauta de tratamiento, obteniendo de este modo un nivel de prueba; y c) la comparación de los niveles inicial y de prueba; caracterizado porque una expresión o número de copias reducido del gen de RhoB en el nivel de prueba en relación con el nivel inicial es indicativo de que la pauta de tratamiento es eficaz en el sujeto.6. Procedure for determining the efficacy of a therapeutic treatment regimen in a subject with lung adenocarcinoma, said procedure comprising: a) measuring the expression of the RhoB gene, or measuring the number of copies of the RhoB gene, in a sample with lung adenocarcinoma cells of said subject, thereby generating an initial level; b) the measurement of the RhoB gene expression, or the measurement of the copy number of the RhoB gene, in a second sample with lung adenocarcinoma cells of the same subject at a time after the administration of the treatment regimen, thus obtaining a test level; and c) comparison of initial and test levels; characterized in that an expression or number of copies Reduced RhoB gene at the test level in relation to the initial level is indicative that the treatment regimen is effective in the subject.
7. Uso de una molécula de nucleótido o anticuerpo que interacciona con ADN, ARN, proteina de RhoB, o un fragmento de los mismos, para el pronóstico de la evolución de la enfermedad en un sujeto con adenocarcinoma de pulmón, para el screening de fármacos activos contra el adenocarcinoma de pulmón, o para determinar la eficacia de una pauta de tratamiento terapéutico en un sujeto con adenocarcinoma de pulmón.7. Use of a nucleotide molecule or antibody that interacts with DNA, RNA, RhoB protein, or a fragment thereof, for the prognosis of disease progression in a subject with lung adenocarcinoma, for drug screening active against lung adenocarcinoma, or to determine the efficacy of a therapeutic treatment regimen in a subject with lung adenocarcinoma.
8. Uso según la reivindicación 7, en el que la molécula de nucleótido es un nucleótido que tiene por lo menos un8. Use according to claim 7, wherein the nucleotide molecule is a nucleotide having at least one
70% de homología con la SEC ID NO. 4-6.70% homology with SEQ ID NO. 4-6.
9. Uso según la reivindicación 7, en el que la molécula de anticuerpo es un anticuerpo que tiene por lo menos un 70% de homología con RhoB (119) .9. Use according to claim 7, wherein the antibody molecule is an antibody that has at least 70% homology to RhoB (119).
10. Kit para el pronóstico de la evolución de la enfermedad en un sujeto con adenocarcinoma de pulmón que comprende reactivos para la medición de la expresión o el número de copias del gen de RhoB (119) .10. Kit for the prognosis of the evolution of the disease in a subject with lung adenocarcinoma comprising reagents for measuring the expression or copy number of the RhoB gene (119).
11. Kit según la reivindicación 10, que comprende además reactivos para realizar un análisis por microarray.11. Kit according to claim 10, further comprising reagents for microarray analysis.
12. Kit según cualquiera de las reivindicaciones 10 u 11, en el que dicho kit comprende un nucleótido que tiene por lo menos un 70% de homología con la SEC ID NO. 4-6, o una molécula de anticuerpo que tiene por lo menos un 70% de homología con RhoB (119) . 12. Kit according to any of claims 10 or 11, wherein said kit comprises a nucleotide having at least 70% homology with SEQ ID NO. 4-6, or an antibody molecule that has at least 70% homology to RhoB (119).
13. Amplicón del gen de RhoB aislado, en el que el amplicón comprende más de una copia de un polinucleótido seleccionado entre: a) un polinucleótido que codifica el polipéptido establecido en la SEC ID NO. 2 ; b) un polinucleótido establecido en la SEC ID NO. 1 ; c) un polinucleótido que tiene por lo menos un 70% de homología con el polinucleótido de a) o b) ; y d) un polinucleótido que se sobreexpresa en células de adenocarcinoma de pulmón que tiene por lo menos un 70% de homología con el polinucleótido de a) o b) .13. Amplicon of the isolated RhoB gene, wherein the amplicon comprises more than one copy of a polynucleotide selected from: a) a polynucleotide encoding the polypeptide set forth in SEQ ID NO. 2 ; b) a polynucleotide established in SEQ ID NO. one ; c) a polynucleotide that has at least 70% homology with the polynucleotide of a) or b); and d) a polynucleotide that is overexpressed in lung adenocarcinoma cells that has at least 70% homology to the polynucleotide of a) or b).
14. Inhibidores de la sobreexpresión del gen de RhoB para su uso como medicamento.14. RhoB gene overexpression inhibitors for use as a medicine.
15. Inhibidores de la sobreexpresión del gen de RhoB según la reivindicación 14, donde los inhibidores se seleccionan entre: a) un anticuerpo o fragmento del mismo que se une específicamente a una proteína RhoB, o un polipéptido que comprende una región de unión a RhoB; b) un péptido mimético del polipéptido RhoB; c) un ácido nucleico inhibidor del gen de RhoB seleccionado entre un oligonucleótido antisentido, un ribozima, y un agente ARNi seleccionado entre15. RhoB gene overexpression inhibitors according to claim 14, wherein the inhibitors are selected from: a) an antibody or fragment thereof that specifically binds to a RhoB protein, or a polypeptide comprising a RhoB binding region ; b) a RhoB polypeptide mimetic peptide; c) a RhoB gene inhibitor nucleic acid selected from an antisense oligonucleotide, a ribozyme, and an RNAi agent selected from
ARNds, ARNsi y ARNsh; o d) una combinación que comprende lovastatina y ácido zoledrónico, y/o sales farmacéuticamente aceptables de la misma.RNAds, siRNAs and siRNAs; or d) a combination comprising lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof.
16. Inhibidores de la sobreexpresión del gen de RhoB según la reivindicación 15, donde el inhibidor es una molécula de ARNsh que tiene por lo menos un 70% de homología con la SEC ID No. 3.16. RhoB gene overexpression inhibitors according to claim 15, wherein the inhibitor is a shRNA molecule that has at least 70% of homology with SEQ ID No. 3.
17. Inhibidor de la sobreexpresión del gen de RhoB según la reivindicación 16, en el que el ARNsh está en forma de un oligonucléotido o vector.17. RhoB gene overexpression inhibitor according to claim 16, wherein the shRNA is in the form of an oligonucleotide or vector.
18. Inhibidor de la sobreexpresión del gen de RhoB según la reivindicación 17, en el que el vector es un plásmido, cósmido, bacteriófago, o un virus.18. The RhoB gene overexpression inhibitor according to claim 17, wherein the vector is a plasmid, cosmid, bacteriophage, or a virus.
19. Inhibidores de la sobreexpresión del gen de RhoB según cualquiera de las reivindicaciones 14-18 para su uso en el tratamiento de cáncer de pulmón no microcítico (NSCLC) .19. RhoB gene overexpression inhibitors according to any of claims 14-18 for use in the treatment of non-small cell lung cancer (NSCLC).
20. Uso de inhibidores de la sobreexpresión del gen de RhoB según cualquiera de las reivindicaciones 14-18 para la fabricación de un medicamento destinado al tratamiento de NSCLC.20. Use of RhoB gene overexpression inhibitors according to any of claims 14-18 for the manufacture of a medicament for the treatment of NSCLC.
21. Método de tratamiento de NSCLC en un sujeto, que comprende la administración a dicho sujeto de una cantidad eficaz de un inhibidor de la sobreexpresión del gen de RhoB según cualquiera de las reivindicaciones 14-18.21. Method of treatment of NSCLC in a subject, comprising administering to said subject an effective amount of a RhoB gene overexpression inhibitor according to any of claims 14-18.
22. Inhibidores según cualquiera de las reivindicaciones 14-15 ó 19, uso según la reivindicación 20, o método según la reivindicación 21, en los que el inhibidor es una combinación que comprende lovastatina y ácido zoledrónico, y/o sales farmacéuticamente aceptables de los mismos, y dicha combinación es para la administración simultánea, separada o secuencial.22. Inhibitors according to any of claims 14-15 or 19, use according to claim 20, or method according to claim 21, wherein the inhibitor is a combination comprising lovastatin and zoledronic acid, and / or pharmaceutically acceptable salts thereof. themselves, and said combination is for simultaneous, separate or sequential administration.
23. Inhibidores según la reivindicación 19, uso según la reivindicación 20, o método según la reivindicación 21, donde el NSCLC se selecciona entre adenocarcinoma de pulmón, carcinoma de células escamosas y carcinoma de células grandes. 23. Inhibitors according to claim 19, use according to the claim 20, or method according to claim 21, wherein the NSCLC is selected from lung adenocarcinoma, squamous cell carcinoma and large cell carcinoma.
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