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WO2019031637A1 - Gènes marqueurs du cancer pour le cancer non mutationnel p53, et procédé de dépistage d'agent thérapeutique - Google Patents

Gènes marqueurs du cancer pour le cancer non mutationnel p53, et procédé de dépistage d'agent thérapeutique Download PDF

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Publication number
WO2019031637A1
WO2019031637A1 PCT/KR2017/008788 KR2017008788W WO2019031637A1 WO 2019031637 A1 WO2019031637 A1 WO 2019031637A1 KR 2017008788 W KR2017008788 W KR 2017008788W WO 2019031637 A1 WO2019031637 A1 WO 2019031637A1
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cancer
gene
mdm2
independent
expression
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PCT/KR2017/008788
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English (en)
Korean (ko)
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김일철
김동민
최승현
이동주
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전남대학교산학협력단
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Priority to PCT/KR2017/008788 priority Critical patent/WO2019031637A1/fr
Publication of WO2019031637A1 publication Critical patent/WO2019031637A1/fr

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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a cancer marker gene and a therapeutic screening method for p53-non-mutated cancer, and more particularly, to a p53-independent MDM2 modulating gene or a protein expression encoded by the gene.
  • A culturing the cells expressing the p53-independent MDM2 modulating gene (p53-independent MDM2 modulating gene) with the test agent; And (b) in cells incubated with the test agent the gene expression and comparison with the test agent in the P 53- measured the expression level of MDM2 independently regulated genes and cultured cells without the test agent p53- independent MDM2 < / RTI > regulatory gene in a subject in need of such treatment.
  • the p53 protein is well known as a tumor suppressor protein encoded by the TP53 gene in humans. It modulates the expression of various sub-genes by regulating the signal that damages DNA in the cell, regulates the cell cycle, induces apoptosis, Protect against transformation into traits.
  • the p53-expressing gene was first discovered in 1979, but at that time, it was known as a carcinogenic gene because the mutated p53 gene was studied. In 1989, the p53 gene was actually found to be a cancer-suppressing gene.
  • the mutation of the p53 gene was observed in about 50% of the primary tumors, especially in the colon cancer, in about 85%, and the P53 gene was found to be closely related to the cell carcinogenesis. In particular, in relation to the carcinogenesis process of the tumor, it inhibits the progress of the tumor, and it is found to be involved in the cell cycle and cell death of the tumor cell, which is a very important gene related with the development and progression of cancer.
  • TP53 mutations occur in almost all types of cancer from 10% (eg, in hematopoietic malignant tumors) to nearly 100% (eg, in ovarian carcinomas with high severity) (Noa Rivl in et al., Mutations in the p53 Tumor Suppressor Gene, Genes Cancer. 2011 Apr; 2 (4): 466474.). Nonetheless, a substantial number of cancer types have been reported that have a substantially lower P53 mutation rate, i.e., no p53 mutations. Blood 82 (1993) reported that a large number of p53 mutations were not found in the leukemia cell line (M.
  • leukemia represents a type of cancer that does not involve a significant number of p53 mutations, and although there are few direct p53 mutations in such leukemias, defects in the p53 pathway due to changes in upstream or downstream modulators Has been suggested.
  • Leukemia is an overexpression of MDM2, an important negative regulator of p53.
  • p53 is a highly unstable protein in unstressed cells, with a half-life of 5-30 min and a very low level in normal cells (normal unstressed cells) due to the continuous degradation mediated by MDM2 Lt; / RTI > MDM2 gene is increased tumor formation potential of all i when cells become over-expression was, encrypts the estimated transcription factor (putat ive ion transcript factor).
  • MDM2 is primarily an E3 ligase (E3 Hgase) that promotes the degradation of p53 through the ubiquitin-dependent pathway in the nuclear and cytoplasmic 26S proteasome.
  • E3 Hgase E3 ligase
  • Mdm2 expression is induced by wi ld type p53 act ivity, EMBO J. 12 (1993) 461e468.).
  • the two molecules are self-regulating for the purpose of maintaining low p53 levels in cells when stress is absent It is associated through a negative feedback loop (autoregulatory negative feedback loop).
  • MDM2 is also known to be involved in cell cycle regulation, differentiation, cell fate determination, DNA repair, basal transcription and other processes And has a function independent of p53.
  • the expression of MDM2 during development is tissue-specific and independent of p53 in other organs.
  • MDM2 High levels of MDM2 expression are observed in human tumor cell lines with little or no functional p53 (functional p53). Furthermore, MDM2 appears to contribute to the altered phenotype in the absence of wild-type p53 (G. Ganguli, B. Wasylyk, p53- independent functions of ⁇ 2, Mol. Cancer Res. 1 (2003) 1027el035.). Although such facts have been reported in the art, the specific mechanisms involved in regulating expression of MDM2 independently of p53 in virtually p53 non-mutated cancers are not clearly known.
  • the present inventors identified genes regulating p53-independent overexpression of MDM2 overexpression, and confirmed the specific effect of genes that impose phenotypes favoring tumor survival among them.
  • p53-non-mutant cancer p53- non mutant cancer for the screening of therapeutic agents.
  • an object of the present invention is to provide a method for producing a p53-independent MDM2 modulating gene comprising: (a) culturing a cell expressing a p53-independent MDM2 modulating gene (p53-independent MDM2 modulating gene) And (b) measuring the expression level of the p53-independent MDM2 regulatory gene in the cells cultured with the test agent, and comparing the level of gene expression in the cultured cells without the test agent, wherein the test agent is a p53-independent MDM2 regulated
  • a method for screening a therapeutic agent against p53-non mutated cancer and a screened compound by the method which includes a step of confirming whether or not the expression of the gene is suppressed.
  • Another object of the present invention is to provide a p53-independent mutant strain comprising an inhibitor of expression of a p53-independent MDM2 regulatory gene or a protein inhibitor expressed by the gene as an active ingredient And to provide a pharmaceutical composition for the prevention or treatment of cancer (p53-non mutant cancer). It is another object of the present invention to provide a p53-non-mutated cancer diagnostic composition comprising a p53-independent MDM2 modulating gene (p53-independent MDM2 modulating gene) or an agent for measuring the protein expression level encoded by the gene, and a kit for diagnosing p53-non-malignant cancer.
  • the present invention provides a method for producing a p53-independent MDM2 modulating gene comprising the steps of: (a) culturing a cell expressing a p53-independent MDM2 modulating gene (p53-independent MDM2 modulating gene) And (b) measuring the expression level of the p53-independent MDM2 regulated gene in the cells cultured with the test agent, and comparing the gene expression profile in the cells cultured without the test agent, wherein the test agent is a p53-independent MDM2 regulated
  • a method for screening a therapeutic agent for p53-non mutated cancer and a compound screened by the method which comprises the step of confirming whether or not the expression of the gene is suppressed.
  • the present invention provides a p53-non mutant cancer (hereinafter referred to as " p53-non mutant ") gene comprising an expression inhibitor of p53- independent M) M2 regulatory gene or a protein inhibitor expressed by said gene as an active ingredient cancer of the present invention.
  • &quot p53-non mutant cancer
  • compositions for the prophylaxis or treatment of p53-non mutated cancer comprising an inhibitor of expression of a p53-independent MDM2 regulatory gene or a protein inhibitor expressed by said gene.
  • a p53-independent mutant cancer which is essentially constituted by an inhibitor of p53-independent MDM2 regulatory gene or a protein inhibitor expressed by said gene a pharmaceutical composition for preventing or treating mutant cancer.
  • the invention is an object to an effective amount of a composition comprising a protein inhibitors expressed by the gene or expression inhibitors of the P 53- independent adjustment MDM2 gene effectiveness minutes in need (P53-non mutant cancer), which comprises administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • the effective amount of a composition consisting of a p53-independent MDM2 regulatory gene expression inhibitor or a protein inhibitor expressed by said gene is administered to an individual in need thereof, wherein the p53-non mutant cancer ). ≪ / RTI >
  • the present invention provides a p53-independent MDM2 modulating gene or a p53-independent mutation comprising a preparation for measuring a protein expression level encoded by the gene
  • a composition for cancer diagnosis and a p53-non-mutated cancer diagnostic kit comprising the same are provided.
  • the present invention provides p53-independent MDM2 regulatory gene (p53-independent MDM2 modulat ing) from a sample collected from a subject to provide information necessary for diagnosis of p53 non- gene) or a method for qualitatively or quantitatively analyzing the expression level of the protein encoded by the gene.
  • p53-independent MDM2 modulat ing p53-independent MDM2 modulat ing
  • a diagnostic agent for p53 non- (P53-independent MDM2 modulating gene) for the manufacture of a p53-independent MDM2 modulating gene, or a preparation for measuring the protein expression level encoded by said gene.
  • the present invention provides a method of measuring p53-independent MDM2 modulating gene (p53-independent MDM2 modulating gene) in a sample of a subject or a protein expression level Provides a method for diagnosing p53-non-malignant cancers.
  • p53-independent MDM2 modulating gene p53-independent MDM2 modulating gene
  • a sample of a subject or a protein expression level Provides a method for diagnosing p53-non-malignant cancers.
  • protein is used interchangeably with “polypeptide ide” or “pept ide” and refers to a polymer of amino acid residues, as is commonly found in natural state proteins.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides in the form of single- or double-stranded.
  • MDM2 which is often overexpressed in p53 non-mutated cancers, is p53 nul 1 cells and that genes such as those listed in [Table 1] of this specification upregulate MDM2 p53- These genes And the effect on tumor cell viability related phenotype was confirmed. Therefore,
  • the present invention provides a screening method for a therapeutic agent for p53-non mutated cancer, comprising the step of confirming whether or not the expression of the gene is suppressed.
  • This 'p53-independent MDM2 modulation' has been previously shown to be associated with MDM2 being expressed by the p53-dependent pathway, as compared to other pathways in which MDM2 is highly related to the p53 gene the expression of which is regulated by the pathway or by other pathways independent of the p53 gene.
  • the 'control' means both up-regulating and down-regulating, and it may preferably mean up-regulation in the present invention.
  • Said 'modulation' is meant to include both regulation of the promoter level, RNA transcription level or protein translation level of the gene of interest.
  • the ' ⁇ 53-independent MDM2 regulatory gene' preferably means a ⁇ 53-independent MDM2 up-regulating gene, and includes all the up-regulation of the expression level of MDM2 gene, MDM2 mRNA level or MDM2 protein level, These specific genes are as described in [Table 1] of this specification.
  • the present invention is characterized in that at least one gene selected from the genes listed in [Table 1] is used, and two or more genes selected from the gene group can be used to search for the simultaneous expression suppression effect of the gene .
  • the ' ⁇ 53-independent MDM2 regulatory gene' may be a gene that performs up-regulation at the MDM2 mRNA level or the MDM2 protein level.
  • And may be one or more selected from the group of lure.
  • the p53-non mutational cancer refers to a type of cancer showing a dysfunction, defect, or inactivation of the p53 pathway without mutation in the p53 tumor suppressor gene.
  • the p53 mutation rate is usually 50 (Leukemia, 5%), lymphoma (19.1%), hematopoietic malignancy (10-12.7%), and lymphoma
  • cervical cancer 5.8%
  • sarcoma 5%
  • testicular cancer 51
  • malignant melanoma 5%
  • endocrine tumor breast cancer (25%)
  • bladder cancer 26.4%)
  • brain cancer (14.6%)
  • prostate cancer (17.5%
  • uterine cancer cancer 27%)
  • stomach cancer (32%) pancreas cancer nocar, 32.6%), skin cancer (35%), lung cancer (38.6%), larynx cancer (40.4%), head and neck cancer (head and neck cancer, 40.6%), esophageal cancer 43.1%), colorectal cancer (43.2%) or ovarian cancer (47.8%).
  • the p53 mutation rate according to specific cancer types can be found in the following references; Magali Olivier et al. , TP53 Mutations in Human Cancers: Origins, Consequences, and Clinical Use, Cold Spring Harb Perspect Biol. 2010 Jan; 2 (1): a001008.
  • ' 'Arnold J. Levine, Mutations in the p53 Tumor Suppressor Gene / Import ant Milestones at the Various Steps of Tumor i genes is Genes Cancer. 2011 Apr; 2 (4): 466474.
  • the ' ⁇ 53-non-mutant cancer' of the present invention is preferably a cancer having no mutation in the cancer suppressor gene ⁇ 53, and indicates a type of cancer in which MDM2 is up-regulated.
  • the most preferable cancer is acute myelogenous leukemia (acute myeloid leukemia).
  • the term "agent” or “test agent” refers to any substance, molecule, element, compound, Combinations. But are not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like. It may also be a natural product, a synthetic compound or a chemical compound or a combination of two or more substances.
  • the agents, substances and compounds may be used interchangeably.
  • the substance selected through the method of the present invention may be selected from the group consisting of, for example, a methionine amino acid derivative, an siRNA, an shRNA, a miRNA, a ribozyme, a DNAzyme, an enzyme nucleic acid (PNA), an antisense oligonucleotide, Peptides, natural extracts, chemicals, and the like.
  • PNA enzyme nucleic acid
  • Test agents that can be screened or identified by the methods of the invention include but are not limited to polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatics, Compounds, benzodiazepines, oligomeric N-substituted glycines, oligocarbamates, saccharides, fatty acids, purines, pyrimidines or derivatives thereof, structural analogs Or combinations thereof. Some test agents may be synthetic and other test agents may be natural. The test agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. A combinatorial library can be produced with a variety of compounds that can be synthesized step-by-step.
  • Test agent may be a naturally occurring protein or a fragment thereof. Such test agents can be obtained from natural sources, such as cell or tissue lysates.
  • Polypeptide preparation Can be obtained, for example, by conventional methods or from commercially available cDNA libraries.
  • the test agent may be a peptide, for example, a peptide having about 5-30 amino acids, preferably about 5-20 amino acids, more preferably about 15 amino acids.
  • the peptide may be a naturally occurring protein, a random peptide or a cleavage of a " biased " random peptide.
  • the test agent may also be " nucleic acid ".
  • the nucleic acid test agent may be a naturally occurring nucleic acid, a random nucleic acid, or a " biased " random nucleic acid. For example, cuts of prokaryotic or eukaryotic genomes can be used similar to those described above.
  • the test agent may also be a small molecule (e.g., a molecule having a molecular weight of about 1,000 or less).
  • a high throughput assay can preferably be applied to the method for screening the modulating agent of small molecules.
  • a combination library of small molecule test agents as described above can be easily applied to the screening method of the present invention.
  • Many assays are useful in the screening (Shultz, Bioorg. Med. Chem. Lett., 8: 2409-2414, 1998; Weller, Mol. Drivers., 3: 61-70, 1997; Fernandes, Curr. Opin Chem. Biol., 2: 597-603, 1998; and Sittampalam, Curr. Opin. Chem. Biol., 1: 384-91, 1997).
  • Test agents screened in the methods of the present invention can be prepared based on construction studies on P 53 -independent MDM2 regulatory genes or mRNAs, proteins or analogues thereof expressed thereby. This structural study enables the identification of test agents that are likely to bind to the gene, mRNA or protein.
  • the three-dimensional structure of the target protein can be studied in a number of ways, such as crystal structure and molecular modeling. Methods for studying protein structures using X-ray crystallography are well known in the literature: Physical Bio-Chemistry, Van Holde, KE (Prentice-Hall 1, New Jersey 1971), pp. 221-239 , and Physical Chemistry with Applied Physics, D. Eisengerg & D.
  • culturing the cells expressing the " ⁇ 53-independent MDM2 regulatory gene with a test agent &quot may be performed using the ⁇ 53-independent MDM2 regulatory gene itself, an expression product thereof (mRNA or protein expression) And the process of contacting the test agent with the test agent.
  • the contacting method of the test agent may be, for example, by treating the test agent outside the cell membrane in vitro and introducing it into the cell, or by introducing the test agent into the cell by the known standard recombinant DNA and molecular cloning techniques But the present invention is not limited thereto.
  • the expression " expression " in the specification means the generation of a protein or a nucleic acid in a cell, including both transcription and translation of a specific nucleotide sequence caused by a promoter
  • Independent MDM2 regulatory gene in the step (a) is a cell in which the expression of the < 53 > independent MDM2 regulatory gene described above occurs, Transfected cells constructed to retain the above-described < 53 > -independent MDM2 regulatory gene through molecular techniques such as cell or molecular cloning, which have naturally, but not exclusively, the aforementioned ⁇ 53-independent MDM2 regulatory genes
  • the type of the cells is not particularly limited, but may be preferably eukaryotic cells.
  • a 'cell expressing the ⁇ 53-independent MDM2 regulatory gene' may be overexpressed in a normal cell at a level above the level at which the gene (the ⁇ 53-independent MDM2 regulatory gene described above) is expressed. That is, it may be a cell that overexpresses a < 53 > -independent MDM2 regulatory gene at a normal level or higher.
  • step (b) whether or not the test agent inhibits mRNA transcription of the ⁇ 53-independent MDM2 regulatory gene, or suppresses translation of the mRNA after transcription , Whether it causes structural alteration or degradation of the translated protein, whether the test agent binds to the translated protein and becomes a substantive functional state, and so on.
  • the expression of the p53-independent MDM2 regulatory gene is substantially absent or the overexpressed p53-independent MDM2 regulatory gene is expressed at the level of the normal individual without disease, the MDM2 and if the state is expressed at a level of normal individuals the test agent can be determined by inhibiting the expression of p53- regulated genes MDM2 independent, as it MDM2 expression inhibitor (more preferably, over-expressing MDM2 inhibitor) P ratio 53- It can be judged as a therapeutic agent for mutant cancer.
  • the MDM2 expression inhibitor (more preferably, the expression inhibitor of MDM2) is inhibited when the expression of the p53-independent MDM2 regulatory gene is suppressed
  • MDM2 and an expression inhibitor as a therapeutic agent for p53-non-mutated cancer.
  • the screening method for the p53-non mutated cancer of the present invention can be performed by measuring the expression level of the p53-independent MDM2 regulatory gene, wherein the 'gene expression level measurement' refers to measurement of the expression level of a gene of interest (P53-independent MDM2 regulatory gene), which includes both mRNA expression level measurement and protein expression level measurement (protein amount change measurement).
  • the 'measurement of mRNA expression level' refers to measuring the amount of mRNA by measuring the presence and expression level of the target gene in the target cell. Measurement of mRNA expression levels can be performed through a variety of methods known in the art. For example, mRNA expression levels can be determined by RTPCRCSambrook et al., Molecular Cloning.
  • each gene promoter of the present invention can be monitored by using a DNA chip, an expression quantification kit using a reporter system, or the like, , ≪ / RTI > but not limited to, by monitoring MDM2 expression.
  • RNA is isolated from cells treated with the test substance, and first strand cDNA is prepared using oligo dT primer and reverse transcriptase. Then, the first strand cDNA is used as a template, and the PCR reaction is performed using the target gene-specific primer set. Then, the PCR amplification product is electrophoresed, and the band formed is analyzed to measure the change in the expression level of the target gene.
  • the 'measurement of protein expression level' is a process for confirming the presence and the degree of expression of a protein expressed from a target gene in a target cell, and for example, using an antibody that specifically binds to the protein of the target gene, The amount could be ascertained, but is not limited to this. Specifically, measurement of protein expression levels can be performed through various immunoassay methods known in the art.
  • changes in the amount of the target protein can be determined by radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition analysis,
  • ELISA enzyme-linked immunosorbent assay
  • Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, Complement Fixation Assay, Fluorescence Actuated Cell Sorter (FACS), Western blot analysis, A protein chip, and the like may be used, but the present invention is not limited thereto.
  • the screening method comprising steps (a) and (b) above may further comprise the steps of:
  • step (c) inspecting whether the test agent confirmed to inhibit the expression of the p53-independent MDM2 regulatory gene in step (b) is administered to an animal having p53-non-mutated cancer to show therapeutic effect.
  • the animal is a non-human animal.
  • the term " therapeutic effect " includes a part or all of the effect of alleviating or ameliorating p53-non-mutated cancer and its symptoms, and the effect of inhibiting the progress of p53-non-mutated cancer.
  • the present invention also relates to a pharmaceutical composition for the prevention or treatment of p53-non mutated cancer comprising an inhibitor of expression of a p53-independent MDM2 regulatory gene or a protein inhibitor expressed by said gene as an active ingredient Provide all.
  • a pharmaceutical composition for the prevention or treatment of p53-non mutated cancer comprising an inhibitor of expression of a p53-independent MDM2 regulatory gene or a protein inhibitor expressed by said gene as an active ingredient
  • the meanings and specific types of the ' ⁇ 53-independent MDM2 regulatory gene' and ' ⁇ 53-non-mutant cancer' are as described above.
  • the 'expression inhibitor of the ⁇ 53-independent MDM2 regulatory gene' may be selected from the group consisting of antisense RNA, siRNA and miRNA, which are not limited to the ⁇ 53-independent MDM2 regulatory gene, but are specific to the ⁇ 53-independent MDM2 regulatory gene.
  • the "specific" means the ability to suppress only the target gene without affecting other genes in the cell.
  • the 'antisense RNA' is complementary to the first transcript or mRNA of the target gene and is complementary to the first transcript or mRNA and prevents the processing, transport and / or translation of the first transcript or mRNA, Speak body.
  • the antisense RNA may be complementary to a portion of a particular gene transcript in a 5 'noncoding sequence, a 3' noncoding sequence, an intron, or a coding sequence.
  • the antisense RNA used herein may include a ribozyme sequence region that increases the efficacy of antisense RNAs that block gene expression.
  • Ribozyme &quot refers to catalytic RNA and includes sequence specific endoribonuclease.
  • the 'target gene' is a p53-independent MDM2 regulatory gene for the purpose of the present invention, and the specific types thereof are as described above.
  • RNA transcript' refers to a transcription-based product catalyzed by an RNA polymerase of the DNA sequence.
  • RNA transcript When the RNA transcript is a complete complementary copy of the DNA sequence, it may be an RNA sequence derived from the first transcript or from post-transcription processing of the first transcript.
  • mRNA Messenger RA
  • siRNA means a double-stranded RNA capable of inducing RNA interference (RNA interference) through cleavage of mRNA of a target gene, and a sense RNA strand having a sequence homologous to the mRNA of the target gene And antisense RNA strands with complementary sequences.
  • siRNA is provided as an efficient gene knockdown method or gene therapy method because it can inhibit the expression of a target gene.
  • the siRNA is not limited to a pair of double-stranded RNA portions that are paired with each other, but a mismatch (the corresponding base is not complementary), a bulge (no base mutually opposing one of the chains) May be included.
  • the siRNA end-structure can be blunt or cohesive.
  • the sticky end structure has both a 3-terminal protruding structure and a 5-terminal protruding structure, and the number of protruding bases is not limited.
  • the siRNA can be added to a protruding portion of one end in a range where the effect of suppressing the expression of the target gene can be maintained, for example, a low molecular RA (for example, a natural RNA molecule such as a tRNA, Molecules).
  • a low molecular RA for example, a natural RNA molecule such as a tRNA, Molecules.
  • the siRNA terminal structure does not need to have a cleavage structure on both sides, and one terminal region of the double-stranded RNA may be a stem loop structure connected by linker RNA.
  • the length of the linker is not particularly limited as long as it does not interfere with the pairing of the stem portions.
  • the siRNA used in the present invention itself may be a complete form having polynucleotide pairing, that is, a form that is introduced into a cell through two transformation processes in which siRNA is directly synthesized in a test tube, one single-stranded oligonucleotide of the nucleotide fragment that its backward (reverse) the treasure may be derived from a single-stranded polynucleotides are separated by spacer type, for example is manufactured such that the siRNA expressed in cells derived siRNA expression vector or PCR- And the introduced siRNA expression cassette is introduced into the cell through a transformation or infecting process.
  • miRNA means a short non-coding RNA derived from an endogenous gene, which acts as a post-transcriptional regulator of gene expression. miRNAs serve as post-transcriptional regulators of gene expression by base pairing with the mRNA of the target gene.
  • the nucleotide sequence and the length of the miRNA of the present invention are not particularly limited as long as the miRNA has an activity of suppressing the expression of the above-described p53-independent MDM2 regulatory gene.
  • the antisense RNA, siRNA and miRNA for the p53-independent MDM2 regulatory gene of the present invention can be introduced into cells through known methods.
  • the antisense RNA, siRNA and miRNA of the present invention may be contained in a vector and provided to an individual.
  • a polynucleotide encoding any one selected from the group consisting of the antisense RNA, siRNA, and miRNA operably linked thereto, in the form of a recombinant expression vector may be contained in a vector and provided to an individual.
  • a promoter and a polynucleotide encoding any one selected from the group consisting of the antisense RNA, siRNA, and miRNA operably linked thereto, in the form of a recombinant expression vector.
  • the expression vector includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector and a virus vector.
  • Suitable expression vectors can include expression regulatory elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers, and the like, and can be prepared in various ways depending on the purpose. Said combination vectors can be introduced into host cells using methods known in the art.
  • the inhibitor may also be an antibody specific for a protein expressed by a p53-independent MDM2 regulatory gene.
  • the term "specific " or " specific " as used herein refers to the specificity of an antibody capable of binding to only one antigen within a cell, and also refers to a specific epitope among a plurality of epitopes, It is also used in specific cases.
  • the antigen is a protein expressed by a p53-independent MDM2 regulatory gene for the purpose of the present invention.
  • the term " antibody " refers to a specific protein molecule directed against an antigenic site.
  • an antibody refers to an antibody that specifically recognizes a protein expressed by a p53-independent MDM2 regulatory gene, and includes both polyclonal and monoclonal antibodies.
  • Polyclonal antibodies can be produced by methods well known in the art for obtaining sera containing antibodies by injecting the above protein antigens into animals and collecting them from animals. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like. Monoclonal antibodies can be prepared by methods known in the art such as the fusion method (see Kohler and Mi lstein (1976) European Jounal of Iro 6: 511-519), recombinant DNA methods (U.S. Patent No. 4,816,567 (Clackson et al, Nature, 352: 624-628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991) .
  • An antibody of the invention comprises a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains.
  • a functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, including, but not limited to, Fab, F (ab ') 2, F (ab') 2 and Fv.
  • the antibody can be indirectly coupled (for example, covalently bonded) to a substance known as an existing anticancer drug directly or through a linker or the like.
  • the kind of the therapeutic agent that can be coupled is not particularly limited as long as it is known as an anticancer agent, but may be, for example, Paclitaxel or the like.
  • the pharmaceutical composition according to the present invention may be used alone or in combination with one or more pharmaceutically acceptable carrier, excipient or chelating agent for the expression inhibitor of the p53-independent MDM2 regulatory gene or the protein inhibitor expressed by the gene can do.
  • pharmaceutically acceptable refers to a composition that is physiologically acceptable and, when administered to humans, does not inhibit the action of the active ingredient and is normally a non-toxic composition that does not cause an allergic reaction such as gastrointestinal disorder, dizziness, .
  • the pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, sal roloid, magnesium stearate, stearic acid, and the like.
  • the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative.
  • Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and clorobutanol.
  • the pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components.
  • a lubricant e.g., a lubricant for lubricating a viscous fluid, a viscous fluid, etc.
  • a sweetening agent e.g., a sweetening agent
  • a flavoring agent emulsifying agent
  • a suspending agent etc.
  • Other pharmacological Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, East on, PA, 1995, which is incorporated herein by reference in its entirety.
  • the composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally.
  • Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI >
  • the pharmaceutical compositions of the present invention may be formulated into oral or parenteral formulations according to the route of administration as described above.
  • the compositions of the present invention may be formulated into powders, granules, tablets, pills , Tablets, capsules, solutions, gels, syrups, slurries, suspensions, and the like.
  • an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the granular product into tablets or sugar tablets.
  • suitable excipients include sugars such as lactose, dextrose, sucrose, sorbic, mannitol, zeolite, erythritol and maltitol, corn starch, wheat starch, rice starch and potato starch
  • Cells including rosin, gelatin, polyvinylpyrrolidone, and the like can be included in the cell including rosin, methylcellulose, sodium carboxymethylcellulose, rosin and hydroxypropylmethylcellulose, and the like. have.
  • crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant.
  • the pharmaceutical composition of the present invention may further comprise an anti-aging agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.
  • a preparation for parenteral administration it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, East on, PA, 1995, which is a commonly known form of pharmaceutical chemistry.
  • the total effective amount of the composition of the present invention may be administered to a patient in a single dose and may be administered by a fractorted treatment protocol administered over a prolonged period of time in a multidirectional dose .
  • the pharmaceutical composition of the present invention Depending on the severity of the disease, the amount of active ingredient can be different.
  • the preferred total dosage of the pharmaceutical composition of the present invention may be from about 0.01 to about 10,000 mg per kilogram body weight of patient per day, most preferably from 0.1 to 500 mg.
  • the dosage of the pharmaceutical composition should be determined based on various factors such as the formulation method, route of administration, treatment frequency, age, body weight, health condition, sex, severity of disease, diet and excretion rate of the patient, As a matter of fact, one of ordinary skill in the art will be able to determine the appropriate effective dose of the composition of the present invention in view of this point.
  • the pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.
  • the present invention also provides the use of a p53-independent MDM2 regulatory gene expression inhibitor or a protein inhibitor expressed by said gene for the preparation of a pharmaceutical composition for the prophylaxis or treatment of p53 non-mutated cancer do.
  • the present invention also relates to a p53-independent mutant cancer which comprises administering to a subject in need thereof an effective amount of a composition comprising, as an active ingredient, a p53-independent MDM2 regulatory gene expression inhibitor or a protein inhibitor expressed by said gene (p53-non mutant cancer).
  • the 'effective amount' of the present invention refers to an amount which, when administered to an individual, indicates an improvement, treatment, or prevention effect of p53-non-mutated cancer, wherein said 'individual' includes an animal, preferably a mammal, It may be an animal, or it may be an animal-derived cell, tissue, or organelle. The subject may be a patient requiring the effect.
  • the 'preparation or composition' of the present invention may be in the form of a food composition, a cosmetic composition, a pharmaceutical composition or the like. In the present invention, the composition may preferably mean a pharmaceutical composition, as described above.
  • the present invention relates to a composition for the diagnosis of p53-non-mutated cancer, which comprises a p53-independent DM2 modulating gene or an agent for measuring the expression level of the protein encoded by said electron and a p53 non-
  • a cancer diagnosis kit is provided.
  • the term " diagnosing &quot is intended to include determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder, Determining the prognosis of a diseased object, or therametrics (e.g., monitoring the status of an object to provide information about the therapeutic efficacy).
  • the diagnosis of the present invention may be to determine the expression level of a p53-independent MDM2 regulatory gene or a protein encoded by the gene to confirm the presence or absence of p53-non-mutated cancer.
  • a protein and a genetic database for example, Genbank or Uniprot known in the art for the protein encoded by the gene, as described above for the '? 53-independent MDM2 regulatory gene' And will be understood by those skilled in the art.
  • An agent for measuring the expression level of the protein encoded by the p53-independent MDM2 regulatory gene is not particularly limited as long as it is known in the art to be capable of measuring the expression level of the protein, but preferably p53-independent MDM2 Regulatory gene Or an antibody or antibody that specifically binds to a protein to be encoded.
  • the term " ant ibody " in the present invention means an immunoglobulin that specifically binds to an antigenic site.
  • the antibody of the present invention is an antibody that specifically binds only to a protein encoded by a p53-independent MDM2 regulatory gene, without opposing other proteins other than the protein encoded by the p53-independent MDM2 regulatory gene.
  • the antibody may be prepared by cloning a p53-independent MDM2 regulatory gene into an expression vector to obtain a protein encoded by the gene, and obtaining the antibody by injection of the obtained protein into an animal, .
  • the antibodies produced using a p53- MDM2 independent control may be those produced through the full-length protein sequence of a gene encoding, or P 53- independent control MDM2 poly 3 ⁇ 4 tide fragment containing the antigenic site of a protein coding gene You may.
  • the form of the antibody of the present invention is not particularly limited and includes a polyclonal antibody or a monoclonal antibody. In addition, if an antibody has antigen-antibody binding ability, part of the whole antibody is included in the antibody of the present invention.
  • any type of immunoglobulin antibody that specifically binds to a protein encoded by p53-independent MDM2 regulatory gene Respectively.
  • the antibodies of the present invention include special antibodies such as humanized antibodies, chimeric antibodies, and recombinant antibodies, as long as they can specifically bind to the protein encoded by the p53-independent MDM2 regulatory gene.
  • 'aptamer' in the present invention means a single strand nucleic acid (DNA, RA, or modified nucleic acid) having a stable tertiary structure as a substance capable of specifically binding with an analyte to be detected in a sample The presence of the target protein in the sample can be confirmed specifically.
  • aptamer is based on a general method of preparing aptamer, which is selective for a target protein to be identified (in the present invention, a protein encoded by a p53-independent MDM2 regulatory gene) and has a high binding capacity and determines a nucleotide sequence And then modifying the oligonucleotide to 5 'terminal or 3' terminal of the oligonucleotide with a functional group of an aptamer chip such as methoxy, -SH, -COOH, -OH, or NH2, but not limited thereto .
  • a target protein to be identified in the present invention, a protein encoded by a p53-independent MDM2 regulatory gene
  • a functional group of an aptamer chip such as methoxy, -SH, -COOH, -OH, or NH2, but not limited thereto .
  • the expression level of the p53-independent MDM2 regulatory gene is measured Significance is meant to include measuring the expression levels of transcripts derived from the p53-independent MDM2 regulatory gene, including, for example, measuring p53-independent MDM2 regulatory gene mRNA expression levels.
  • the agent for measuring the expression level of the p53-independent MDM2 regulatory gene is not limited thereto, but may preferably mean an agent for detecting mRNA expressed from the gene. Therefore, a preparation for detecting a p53-independent MDM2 regulatory gene is not particularly limited as long as it is a ligand that specifically binds or hybridizes to mRNA expressed from the gene, but may be, for example, a primer Pair) or a probe.
  • the " primer " is a nucleic acid sequence having a short free 3 'hydroxy 1 group and capable of forming a base pair with a complementary template and having a short nucleic acid sequence .
  • the primer can initiate DNA synthesis in the presence of reagents (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates for polymerization in a suitable buffer solution and temperature.
  • reagents i.e., DNA polymerase or reverse transcriptase
  • the PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.
  • the sequence of the primer does not need to have a sequence completely complementary to the sequence of a part of the template, and is stratified if it has a superficial complementarity within a range that can be reacted with the template and have a primer-specific action.
  • the primers for measuring the expression level of the p53-independent MDM2 regulatory gene do not need to have a perfectly complementary sequence to the p53-independent MDM2 regulatory sequence, and the p53-independent MDM2 regulatory Gene mRNA or p53-independent MDM2 regulatory gene is amplified to amplify a specific region of the cDNA and is complementary to the length of the p53-independent MDM2 regulatory gene to measure the amount of the mRNA.
  • the primer for the amplification reaction is a p53-independent MDM2 regulatory gene to be amplified.
  • the mRNA is complementary to a template (sense or sense) at opposite ends of the specific region of the mRNA and opposite (anti-sense) antisense Set (pair). Primers can be readily designed by those skilled in the art with reference to p53-independent MDM2 regulatory mRNA or cDNA sequences.
  • probe refers to a probe having a base pair length of several hundreds to several hundreds, which can specifically bind to mRNA or cDNA (complementary DNA) of a specific gene. Refers to a fragment of a polynucleotide such as RNA or DNA and is labeled so that the presence or expression level of mRNA or cDNA to be bound can be confirmed.
  • a probe by measuring the expression level of the p53-independent MDM2 regulatory gene mRNA by performing a sample complementary to the p53-independent MDM2 regulatory gene mRNA and a hybridizate ion, It can be used for diagnosis.
  • the conditions for selecting and modifying the probe may be appropriately selected according to techniques known in the art.
  • primer or probe of the present invention can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods.
  • primers or probes can be modified in various ways according to methods known in the art, so long as they do not interfere with the p53-independent MDM2 regulatory mRNA mRNA.
  • modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs and modifications between nucleotides such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) Or a combination of charged ligands (eg, phosphorothioate, phosphorodithioate, etc.) and fluorescent or enzymatic labeling materials.
  • uncharged linkers e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.
  • a combination of charged ligands eg, phosphorothioate, phosphorodithioate, etc.
  • fluorescent or enzymatic labeling materials e.g., fluorescent or enzymatic labeling materials.
  • an antibody, an aptamer or a p53-independent MDM2 regulatory gene mRNA which recognizes a protein encoded by a p53-independent MDM2 regulatory gene as a marker in order to measure the expression level of a p53-independent MDM2 regulatory gene,
  • an antibody, an aptamer or a p53-independent MDM2 regulatory gene mRNA which recognizes a protein encoded by a p53-independent MDM2 regulatory gene as a marker, in order to measure the expression level of a p53-independent MDM2 regulatory gene,
  • one or more other component compositions, solutions or devices suitable for the assay method in order to measure the expression level of a p53-independent MDM2 regulatory gene.
  • the kit can be administered by Western blot, ELISA, radioimmunoassay, radiation immunodiffusion, Oucheroton immunodiffusion, rocket immunoelectrophoresis, immuno staining, reverse precipitation assay, complement fixation assay, FACS or protein chip method
  • diagnostic kits characterized by the inclusion of known essential elements and associated elements required to perform the assay.
  • the kit comprises an antibody specific for a protein encoded by a p53-independent MDM2 regulatory gene.
  • the antibody is a monoclonal antibody, polyclonal antibody or recombinant antibody, which has high specificity and affinity for the target marker protein and little cross-reactivity to other proteins.
  • the kit may further comprise an antibody specific for a control protein.
  • the kit can detect the bound antibody May include reagents, such as labeled secondary antibodies, chromophores, enzymes (in the form conjointred with the antibody) and other substrates capable of binding to the substrate or antibody, and the like.
  • the kit of the present invention may include a washing solution or an eluting solution capable of removing surplus chromogenic substrate and unbound protein and retaining only the protein marker bound to the antibody.
  • the kit is not particularly limited as long as it is known in the art as an assay kit for providing a primer (primer pair) or a probe as a component.
  • the kit may include PCR (polymerase chain reaction), RNase protection Assay, nodb blotting, Southern blotting or kits for DNA microarray chips, and the like.
  • the diagnostic kit may be a diagnostic kit specifically comprising an essential element necessary to perform a polymerase antagonism.
  • the Polymerase Enzyme Kit contains each primer pair specific for the marker gene (mRNA).
  • a primer is a nucleotide having a sequence specific to the nucleotide sequence of each marker gene (mRNA), and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. It may also contain a primer specific for the nucleic acid sequence of the control gene.
  • Polymerase Enzyme Kit can be used in a test tube or other appropriate container, reaction buffer (pH and magnet concentration vary), deoxynucleotides (dNTPs), DNA polymerase (eg Taq polymerase) Enzymes, DNAse, RNAse inhibitor DEPC-water, sterile water, and the like.
  • reaction buffer pH and magnet concentration vary
  • dNTPs deoxynucleotides
  • DNA polymerase eg Taq polymerase
  • Enzymes DNAse, RNAse inhibitor DEPC-water, sterile water, and the like.
  • the present invention also provides p53-independent MDM2 modulating genes (p53-independent MDM2 modulating genes) or proteins expressed by these genes from samples collected from a subject to provide information necessary for diagnosis of p53-non-mutated cancer Provides a method of qualitative or quantitative analysis of the level.
  • the method comprises the steps of: (a) measuring the p53-independent MDM2 regulatory gene or protein expression level encoded by the gene from a sample of the subject; and
  • the term 'analysis' may mean 'measurement', and the qualitative analysis may be to measure and confirm presence or absence of a target substance, (Level of expression) or amount of a compound of the present invention.
  • analysis or measurement can be performed without limitation, including both qualitative and quantitative methods.
  • the step (a) is a step of measuring a p53-independent MDM2 regulatory gene or a protein expression level encoded by the gene in a sample provided from a subject (latent patient).
  • the sample can be used without limitation as long as it is collected from a subject to be diagnosed whether or not cancer (particularly, p53-non-mutant cancer) is diagnosed.
  • a cell or tissue obtained by biopsy The blood can be whole blood, blood serum, plasma, saliva, saliva, sputum, capsular fluid, amniotic fluid, ascites, cervix or vaginal discharge, cerebrospinal fluid, various secretions, urine, feces and the like.
  • the sample may be pretreated prior to use for detection or diagnosis.
  • the protein expression level is determined by a protein expression assay method known in the art, the measurement method is not particularly limited. For example, Western blotting, ELISA, radioimmunoassay, radioimmunoprecipitation, Ouchteroni immunodiffusion, Immunoassay, immunoassay, immunoassay, complement fixation, FACS, or protein chip methods, as described above. If the gene expression level is determined by a gene expression assay method known in the art, the measurement method is not particularly limited.
  • step (b) the expression level of the p53-independent MDM2 regulatory gene or the protein encoded by the gene of the test sample measured in step (a) is compared with that of a healthy individual, and a p53-independent MDM2 regulatory gene or a It is judged that the subject having an increased level of protein expression expressed by the gene is stuck in p53-non-mutated cancer.
  • Independent MDM2 regulatory gene of the test sample measured by the method of step (a) described above or the protein expression level of the gene encoded by the gene is measured by the same method as that of the p53- To the protein expression level that the gene codes.
  • the sample is preferably a sample obtained by the same kind or the same method between the subject and the normal person.
  • the expression level of the ⁇ 53-independent MDM2 regulatory gene or the protein encoded by the gene is increased compared to that of a normal healthy person, and can be judged to be in the case of ⁇ 53-unmutated cancer (or high risk group of ⁇ 53-non-mutant cancer).
  • the present invention also provides the use of a p53-independent MDM2 modulating gene (p53-independent MDM2 modulating gene) for the preparation of a drug for the diagnosis of p53-non-mutated cancer, or a preparation for measuring the protein expression level encoded by the gene.
  • the present invention also relates to a method for diagnosing p53-non-mutated cancer characterized by measuring the p53-independent MDM2 modulating gene (p53-independent MDM2 modulating gene) in the sample of the subject or the protein expression level encoded by the gene Lt; / RTI > Methods for diagnosing p53-non-mutated cancer include comparing a normal (phosphorus) sample with a p53-independent MDM2 regulated gene or a protein expression level encoded by the gene, -53 non-mutant cancer has occurred.
  • the subject of the present invention may be an animal, preferably a mammal, especially an animal including a human, and more preferably a human or a patient, who needs diagnosis. ⁇ Effects of the Invention ⁇
  • the screening method of the present invention can effectively screen p53-non-mutagenic cancer-specific anticancer agents.
  • the genes identified in the present invention are significantly up-regulated in the state of p53 non-mutated cancer, p53-non-mutant cancer Diagnosis is possible.
  • Figure 2 shows the production process of an MDM2 reporter based on two different MDM2 promoters (PI and P2 promoters). The top panel represents each promoter on the chromosome and the bottom panel represents the cloned region in each reporter system. The number of each region corresponds to the number in the promoter region.
  • FIG. 3 shows the combination of conditions of reverse transfection performed on HCT116 p53 + / + and HCT116 p53 - / - cells, in particular the type combination of promoter reporter transfected with the screening library in Example ⁇ 2-2 > .
  • FIG. 4 shows the results of measuring MDM2 P1 and P2 promoters and p21 promoter activity using a dual luciferase system for 368 KUGI clones. The color represents the MDM2 promoter reporter activity ratio relative to the Mock clone.
  • Figure 5 shows the log2 scale of the MDM2 promoter activity in HCT116 p53 - / - cells against the clone in Table 1 (white bars: P2 promoter activity, black bars: P1 promoter activity).
  • FIG. 6 shows the log2 scale of the MDM2 promoter activity in HCT116 p53 + / + cells against the clone in Table 1 (white bars: P2 promoter activity, black bars: P1 promoter activity).
  • FIG. 7 shows the results of RT-PCR analysis of the level of MDM2 mRNA expression by each clone in HCT116 p53 - / - cells.
  • Figure 8 shows the results of Western blotting of MDM2 protein expression levels by HCT116 p53 - / - and HCT116 p53 + / + cells, respectively.
  • 9 is a schematic diagram showing a point mutation position (underlined portion) for substantially eliminating the AP-1 binding motif in the MDM2 P2 promoter.
  • 10A shows the results of measuring the cell survival rate using an early cell proliferating counting system in which each clone was track-specific to HCT116 p53 + / + cells and irradiated with UV crosslinker (White bar, no treatment; black bar, 5 mJ / cm UV radiation 30 sec).
  • Fig. 10B shows the result of measuring cell viability by counting cells under a microscope after each clone was track-specific to LLC-PK cells and then irradiated with an UV crosslinker.
  • Wild type p53 HCT116 colon cancer cells (HCT116 + / +) and isogenic p53 knockout broth (HCT116 - / -) were cultured in 10% (v / v) fetal bovine serum (Hyclone, (GIBCO-BRL, Grand Island, NY, USA) supplemented with 100 IU / ml penicillin and 100 g / ml streptomycin (GIBCO-BRL). 2.
  • the P1 or P2 region of the MDM2 promoter was amplified by PCR polymerase chain reaction using a human breast genomic DNA library and inserted into pcDNA-luc (see FIG. 2).
  • the P1 and P2 promoters are MDM2P1F1 (5'-CTGACTCGAGGTTGGTCTTGAACTCCTG-3 ') and MDM2P1R1 (5'-
  • GACTGAATTCCCTCAAGACTCCCCAGTT-3 ' GACTGAATTCCCTCAAGACTCCCCAGTT-3 '
  • MDM2P2F1 5'-CTGACTCGAGTCTTGAGGGACCCCCGA-3'
  • MDM2P2R1 5 '
  • GACTGMTTCGCCACTGAACACAGCTG-3 ' primer set The amplified product was inserted into the XhoI / EcoRI site of pcDNA-Luc. PCR was carried out using denaturation at 94 ° C for 1 minute, using the QuikChange® Site-Directed Mutagenesis Kit (# 200518; Stratagene, La Jolla, CA, USA) to construct a P2 promoter with point mutations in the AP- annealing: 58 ° C for 30 seconds, extention: 72 ° C for 30 seconds, 30 cycles).
  • the cDNA clones used for the screening of the MDM2 expression modulators were obtained from the Korean UniGene (UGI) Library (http://kugi.kribb.re.kr/KUGI/doc/KUG-Methods.html).
  • UMI Korean UniGene
  • a high-throughput reverse-transfection of 368 gene-arrayed cDNA libraries was performed. Briefly, each well was spotted with gelatin in a 96-well plate (Corning Inc., Corning, NY, USA) containing 0.5 different cDNAs in each well.
  • Luciferase assay reagent 40 / from Assay System (E1910; Prwnega) was added to each 3 ⁇ 4 and analyzed immediately by Acquest Plate Reader (LJL Biosystems Ems, Sunnyvale, CA, USA).
  • RNA extraction and reverse transcription-PCR Total RNA was extracted from the cells with Trizol (Invitrogen) and counterstained with AMV reverse transcriptase (Promega) for 1 hour at 42 ° C according to the manufacturer's instructions. Were synthesized.
  • the cDNA was amplified by PCR using a primer set of GDH1 (5'-TGAGAACGGGAAGCTTGTCA-3 ') and GDH2 (5'-GGAAGGCCATGCCAGTGA-3') for amplification of g1cecer1 dehyde-3-phosphate dehydrogenase (GAPDH) (5'-CGTGCCMGCTTCTCTGTGAAA-3 ') and HDM2R1 (5'-CTCATCATCTTCATCTGAGAGTT-3') primers for amplification of the primers.
  • the primers were annealed at 58 ° C during the PCR.
  • the final PCR products were analyzed by 1.23 ⁇ 4> (w / v) agarose gel electrophoresis.
  • the transfected cells were recovered and lysed in lysis buffer (1% (v / v) Triton X-100, 20 mM HEPESC pH 7.9), 300 mM NaCl, 100 mM KCl, 10 mM EDTA, 10 g / ml aprotinin, 100 g / ml leupeptin, and ⁇ ⁇ M PMSF). After quantification, 20 ⁇ g / ml total protein lysate was electrophoresed on acrylamide gel and transferred to Trans-Blot ® membrane (162-0145; Bio-Rad Laboratories, Hercules, CA, USA).
  • lysis buffer 1% (v / v) Triton X-100, 20 mM HEPESC pH 7.9
  • 300 mM NaCl 100 mM KCl
  • 10 mM EDTA 10 g / ml aprotinin
  • 100 g / ml leupeptin 100 g
  • the membranes were immunoblotted with antibodies and developed with an enhanced chemiluminescence detection system (Thermo Scientific, Rockford, I I, USA).
  • the antibodies used were mouse anti-human MDM2 monoclonal anti-body (K0165-3; Medical and Biological Laboratories, Nagoya, Japan), mouse anti-p53 monoclonal anti body (sc-126; Santa Cruz Biotechnology, Inc Santa Cruz Biotechnology), and mouse anti-ib-act in monoclonal anti-body (sc-8432; Santa Cruz Biotechnology).
  • Cell proliferation was assayed using Cell Counting-8 (Dojindo Laboratories) and 96-well microplates. After the addition of CCK-8 solut ion, the absorbance of the transfected cells in each well was measured at 450 nm using a microplate reader. The reference wavelength was 600 nm.
  • MDM2 and p53 expression were confirmed in patients with acute myelogenous leukemia (AML).
  • AML acute myelogenous leukemia
  • Real-time PCR real-time PCR was performed to monitor mRNA levels of MDM2 and p53 in 17 AML patients.
  • MDM2 mRNA levels were found to be higher in the 17 patients with ALM compared to av_NR (Normal sample) in 8 patients (1, 2, 5, 7, 9, 10, 12, 14; 47% 1). High MDM2 mRNA levels were identified in 5 out of the 8 patients (1, 2, 7, 9, 12; 62.5%) regardless of p53 mRNA expression (see FIG.
  • MDM2 has p53-independent (independent) functions in cell cycle regulation, differentiation, cell fate determination, DNA repair, basal transcription and other processes. It is assumed that p53-independent MDM2 expression (p53-independent MDM2 expression) occurs by altering the MDM2 upstream regulator in leukemia.
  • MDM2 expression profile is similar to the expression profile of a gene 'A', a 'co-express ion' link (l ink) is generated.
  • l ink a 'co-express ion' link
  • Estimated co-expression associations by the nul l hypothesis were evaluated using the t-distribution with n-2 degrees of freedom (where n is the number of samples). The P-value was obtained by a permutation-based test on a subset of the data. The large deviations from the hypothesis that there is no correlation between the MDM2 gene and the gene 'A' are of significant relevance. Similar concurrent expression patterns between MDM2 and gene 'A' were also found in other data sets to confirm simultaneous expression.
  • MDM2 had 1089 links that were searched for more than 2 (stringency of 2), i.e., two or more data sets. A total of 746 and 343 genes were positively or negatively associated with MDM2 mRNA levels, respectively. Of the 1089 MDM2-coexpressed genes, a total of 560 clones were matched on the human KUGI cDNA libary (http: // kugi. Kribb.re.kr /).
  • 368 clones (368 ful l-length clones) having full-length sequences were inserted into a mammalian expression vector (pCMV-SP0RT6, pCNS or pME8S-FL3) regulated by the CMV promoter or the SV40 promoter.
  • the clones were reverse transfected with a reporter vector and used for screening tests.
  • MDM2 has a P1 promoter with RAR / RXR and E2F binding sites and a P2 promoter with AP-1, p53, and c-myc binding sites. Since the activity of the MDM2 P2 promoter is known to be much stronger than the MDM2 P1 promoter, generally isolated P1 and P2 promoter reporters were cloned and used to study promoter activity. Therefore, in order to evaluate the promoter activity including the P53-dependent method, two promoter reporters used for screening were separated (see Fig. 2) and inserted into pGL3-Basic plasmid.
  • FIG. 3 schematically shows the combination of the conditions of the reverse transmission.
  • MDM2 P1 and P2 promoter activity and p21 promoter activity were measured by dual luci ferase (Promega) and a luminometer.
  • MDM2 is also the subject of p53-mediated transcription (p53-mediated transcription), although p53 is a substrate for E2 lysase.
  • Hierarchical association (Hierarchical inkage l) was HCT116 p53 - p21 and illustrates the fact that very different profile our emitter activity of MDM2 in the P1 and P2 promoter activity in cells HCT116 p53 + / + cells, '- /.
  • clones with elevated MDM2 P1 and P2 promoter activity and reduced p21 promoter activity have been identified as candidates for the p53-independent MDM2 transcription initiator ivator, Clones of the bark as described in Table 1 were screened as p53-independent MDM2 transcriptional activation factors.
  • the MDM2 promoter activity in HCT116 p53 - / - cells and HCT116 p53 + / + cells was determined as a clone of the bark (see Figures 5 and 6). As shown in FIG. 5, 25 clones in HCT116 p53 - / - cells showed enhanced MDM2 P1 promoter activity.
  • SFRS2, L0C51035, ELAVL1, NTRK2, GNA15, or SERPINA3 enhanced MDM2 protein expression in HCT116 p53 - / - cells (see FIG. 8).
  • expression of SFRS2, L0C51035, EVAVL1, MAGEA12, GNA15, SERPINA3, NTRK2, EIF5A, PRCC, or KNG1 increased MDM2 transcript levels without significant changes in P53 protein levels in HCT116 p53 + / + cells.
  • MDM2 P2 promoter reporter with mutation in API binding motif was constructed. As shown in FIG. 9, point mutations were generated in the MDM2 P2 promoter to make the state substantially the same as that of AP-1 binding site removed. The effect on the mutant P2 promoter was confirmed against the clone of Ganoderma lucidum. To distinguish the relative intensities of each promoter, the fold change was converted to log2 value after dividing the promoter activity of the mutant P2 promoter of the wi ld type P2 promoter (see Table 1).
  • Table 1 below shows the expression of AP-1 independent MDM2 expression by monitoring the ratio of the activity of the p2 promoter and the wi ld type P2 promoter from which the AP-1 binding site was removed.
  • p53 will be a major regulator as compared to FIG.
  • three clones of NTRK2, GNA15 or SFRS2 showed activation of the P2 promoter without the AP-1 binding site.
  • the clones have an MDM2 transcriptional activation mechanism in a manner independent of p53 and AP-1.
  • HCT116 p53 + / + cells and LLC-PK cells were transfected with the above claws and UV crosslinkers were used to evaluate the effect of increased MDM2 expression on the cell viability by the selected clones in the previous examples After radiation, cell viability was measured using a cell proliferating counting system.
  • Cells transfected with L0C51035, MAGEA12, SFRS2, ELAVLl, PRCC, SERPINA3, GNA15, or NTRK2 showed improved cell growth (see FIG. 10A).
  • EIF5A or KNG1 did not affect cell growth (see Fig. 10A). This tendency was similar in p53-deficient cells (data not shown).
  • the present invention relates to a method for screening cancer marker genes and therapeutic agents against p53-non-mutated cancer, and more particularly, (A) a p53-independent MDM2 modulating gene (p53-independent MDM2 modulating gene) or a p53-independent MDM2 modulating gene (p53-independent MDM2 modulating gene) Lt; / RTI > with a test agent; And (b) determining the level of expression of the p53-independent MDM2 regulatory gene in cells incubated with the test agent and comparing the level of expression of the p53-independent MDM2 regulatory gene with the level of gene expression in the cultured cells without the test agent, (P53-non mutant cancer), comprising the step of determining whether or not the expression of the regulatory gene is suppressed.
  • the present inventors Unlike the mutation found in the cancer suppressor gene P53, which is present in most cancers, the present inventors have found that, based on the first regulated p53-independent upstream regulatory genes (see Table 1) that regulate MDM2 overexpression
  • the screening method of the present invention can effectively screen p53-non-mutant cancer-specific anticancer drugs.
  • an expression inhibitor of a p53-independent MDM2 regulatory gene or a protein inhibitor expressed by the gene can be used as a pharmaceutical composition for the prevention or treatment of p53-non-malignant cancer, Since the genes identified in the inventions (see Table 1) are significantly up-regulated in the p53 non-mutated cancer state, the diagnosis of p53-non-mutated cancer It is highly likely to be used industrially in the field of pharmaceutical and in vitro diagnostic industries.

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Abstract

La présente invention concerne des gènes marqueurs du cancer pour le cancer non mutationnel p53, et un procédé de dépistage d'agent thérapeutique. Bien que des mutations du gène p53 suppresseur de tumeur ont été trouvées jusqu'à présent dans la plupart des cancers, le procédé de dépistage selon la présente invention, basé sur des gènes régulateurs amont p53, pour réguler indépendamment la surexpression de MDM2, qui a clairement été étudié pour la première fois par le présent demandeur, permet de dépister efficacement des agents anticancéreux spécifiques du cancer non mutationnels p53. De plus, les gènes étudiés dans la présente invention (voir tableau 1) sont régulés de manière significative dans un état de cancer non mutationnel p53, ce qui permet de diagnostiquer l'apparition d'un cancer non mutationnel p53 alors que le diagnostic du cancer classique a été dépendant de l'existence de mutations de p53.
PCT/KR2017/008788 2017-08-11 2017-08-11 Gènes marqueurs du cancer pour le cancer non mutationnel p53, et procédé de dépistage d'agent thérapeutique WO2019031637A1 (fr)

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CN115161396A (zh) * 2021-09-24 2022-10-11 四川大学华西第二医院 Ppip5k2及其复合物在调控卵巢癌进展中的应用
WO2023133275A1 (fr) * 2022-01-07 2023-07-13 Sanford Burnham Prebys Medical Discovery Institute Inhibition de la glutaryl-coa déshydrogénase pour le traitement du mélanome
CN116421615A (zh) * 2023-02-10 2023-07-14 暨南大学 Xrcc5基因抑制剂在制备治疗t-all药物中的应用

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Publication number Priority date Publication date Assignee Title
CN115161396A (zh) * 2021-09-24 2022-10-11 四川大学华西第二医院 Ppip5k2及其复合物在调控卵巢癌进展中的应用
WO2023133275A1 (fr) * 2022-01-07 2023-07-13 Sanford Burnham Prebys Medical Discovery Institute Inhibition de la glutaryl-coa déshydrogénase pour le traitement du mélanome
CN116421615A (zh) * 2023-02-10 2023-07-14 暨南大学 Xrcc5基因抑制剂在制备治疗t-all药物中的应用

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