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WO2008007807A1 - Biomolécules de diagnostic - Google Patents

Biomolécules de diagnostic Download PDF

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Publication number
WO2008007807A1
WO2008007807A1 PCT/JP2007/064288 JP2007064288W WO2008007807A1 WO 2008007807 A1 WO2008007807 A1 WO 2008007807A1 JP 2007064288 W JP2007064288 W JP 2007064288W WO 2008007807 A1 WO2008007807 A1 WO 2008007807A1
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WIPO (PCT)
Prior art keywords
pa2g4
protein
gene
expression
subject
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PCT/JP2007/064288
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English (en)
Inventor
Osamu Nishimura
Tetsuo Ichikawa
Keli Ou
Patrick Tan
Kumaresan Ganesan
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Shimadzu Corporation
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Application filed by Shimadzu Corporation filed Critical Shimadzu Corporation
Priority to JP2009501060A priority Critical patent/JP5229214B2/ja
Publication of WO2008007807A1 publication Critical patent/WO2008007807A1/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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/57407—Specifically defined cancers
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to biological molecules or biomolecules.
  • biomarkers More in particular, the present invention relates to biomolecules used for diagnosis or prognostic evaluation of a disease or to monitor efficacy of a disease treatment.
  • Breast cancer is the second leading cause of cancer deaths in women today (after lung cancer) and is one of the most common cancers among women.
  • the World Health Organization estimates that more than 1.2 million people worldwide each year will be diagnosed with breast cancer.
  • Breast cancer is usually first detected as a palpable lump in the breast and/or by unusual presentation of the breast such as dimpling, puckering, bulging of the skin, a nipple that has changed position or an inverted nipple, redness, soreness, rash, swelling, or asymmetry of the breasts.
  • Detection by physical examination may be followed by mammography by x- ray or ultrasound.
  • mammography is fraught with false positive results due to cysts, calcifications and benign lumps such as fibroadenomas. Consequently, further diagnoses based on cytology on tissue samples obtained from biopsies are usually necessary. Interpretation of cytological samples requires highly trained professionals.
  • the present invention addresses the problems above and provides methods for detecting and/or treating a proliferative cell disorder such as cancer.
  • the disorder is cancer, particularly breast cancer.
  • the present invention provides: [1] a method of detecting and/or quantitating the presence of, predisposition to, and/or severity of, a proliferative cell disorder in a subject, the method comprising:
  • control may be, for example, is at least one subject not diagnosed with a cell proliferative disorder.
  • control may be at least one subject having a low level of expression of PA2G4 gene and/or protein.
  • the control may also be an average value in expression obtained from a selected population.
  • the gene, RNA and/or mutation thereof may be human PA2G4 gene, RNA and/or mutation thereof, and the PA2G4 protein, derivative, mutant and/or fragment may be human PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the detecting may be by labelling the PA2G4 protein, derivative, mutant and/or fragment thereof, with at least one sulfenyl halide agent followed by 2-dimensional gel electrophoresis and mass spectrometry.
  • the sulfenyl halide agent may be 2- nitrobenzenesulfenyl chloride (NBS).
  • a method of treating a proliferative cell disorder in a subject comprising varying the expression of a PA2G4 gene, RNA, and/or mutation thereof.
  • the PA2G4 gene may have a sequence as given in SEQ ID NO:1.
  • the varying may be obtained by administering nucleic acid hybridisable to all or part of the PA2G4 gene, RNA and/or mutation thereof.
  • the hybridisable nucleic acid may be DNA or RNA.
  • the hybridisable nucleic acid may be siRNA.
  • the varying may be obtained by administering a compound hybridisable to all or part of the PA2G4 gene,
  • a method of treating a proliferative cell disorder in a subject comprising varying the expression of a PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the method may comprise varying the expression of the PA2G4 protein having the amino acid sequence of SEQ ID NO:2.
  • the varying may be obtained by administering a polypeptide binding to all or part of the amino acid sequence of the PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the binding polypeptide may be an antibody.
  • the varying may be obtained by administering a compound hybridisable to all or part of the amino acid sequence of the PA2G4 protein.
  • the varying of the expression may comprise reducing the expression of the PA2G4 gene, RNA and/or mutation thereof; and/or PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the analysing step may comprise separating the protein(s) based on a first characteristic, optionally separating the protein(s) based on a second characteristic, ionising the protein(s) and detecting and identifying the separated protein(s).
  • the protein and/or fragment thereof may be a PA2G4 protein and/or fragment thereof.
  • the PA2G4 protein has the sequence of SEQ ID NO:2.
  • the agent may be a sulfenyl halide.
  • the agent may be 2-nitrobenzenesulfenyl chloride (NBS).
  • NBS 2-nitrobenzenesulfenyl chloride
  • the amino acid residue may be tryptophan.
  • the separating steps may be based on at least one characteristic selected from the group consisting of mass, charge, functional group and isoelectric point.
  • the separating steps may be steps for SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two- dimensional gel electrophoresis (2DE), high performance liquid chromatography (HPLC) and/or free flow electrophoresis (FFE).
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • 2DE two- dimensional gel electrophoresis
  • HPLC high performance liquid chromatography
  • FFE free flow electrophoresis
  • the ionising may be selected from the group consisting of electrospray ionising (ESI), matrix assisted laser desorption/ionisation (MALDI), surface enhanced laser desorption/ionisation (SELDI), nanospray ionisation, atmospheric pressure chemical ionisation (APCI), chemical ionisation (Cl), electron impact (El), fast atom bombardment (FAB), field desorption / field ionisation (FD/FI) and thermospray ionisation (TSP).
  • ESI electrospray ionising
  • MALDI matrix assisted laser desorption/ionisation
  • SELDI surface enhanced laser desorption/ionisation
  • nanospray ionisation nanospray ionisation
  • APCI atmospheric pressure chemical ionisation
  • Cl chemical ionisation
  • El electron impact
  • FAB fast atom bombardment
  • FD/FI field desorption / field ionisation
  • TSP thermospray ionisation
  • the analysing step may be by tandem mass spectrometry.
  • the method tandem mass spectrometry may use quadrupole time-of-flight geometry.
  • a method of selecting at least one candidate for a clinical trial, experimentation and/or diagnostic test comprising: (a) providing at least one sample from at least one subject;
  • control may be at least one subject not diagnosed with a cell proliferative disorder.
  • control may be a subject not diagnosed with a cell proliferative disorder.
  • the control may also be at least one subject having a low or reduced level of expression of PA2G4 gene and/or protein.
  • the control may also be an average value in expression obtained from a selected population.
  • the proliferative cell disorder may be a cancer.
  • the proliferative cell disorder may be breast cancer.
  • the subject may be a mammal.
  • the subject may be a human being.
  • a diagnostic and/or prognostic kit for the diagnosis and/or prognostic evaluation of a cell proliferative disorder in a subject, the diagnostic and/or prognostic kit comprising at least one molecule or compound hybridisable to a PA2G4 gene, RNA and/or mutation thereof; and/or binding to a PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the molecule may be a nucleic acid sequence hybridisable to the PA2G4 gene, RNA and/or mutation thereof.
  • the molecule may be a siRNA hybridisable to the PA2G4 gene transcript.
  • the molecule may be a protein binding to the PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the molecule may be an antibody binding to the PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the kit may further comprise packaging and information pertaining to the use of the compound for purposes such as diagnosis and/or prognostic evaluation of disease states or treatment conditions.
  • the proliferative cell disorder may be a cancer.
  • the proliferative cell disorder may be breast cancer.
  • the subject may be a mammal; in particular, a human being.
  • FIG. 1 (A) Modification of tryptophan residues with 2-nitrobenzensulfenyl chloride (NBS). * represents either 12 C (light) or 13 C (heavy). (B) Schematic representation of the NBS/2DE/MS protein identification/quantitation strategy. In this study, the selection of spots for excision was based on a prior analysis using conventional 2DE gels (see Results for details). However, it is also clearly feasible to excise all the observable spots on the NBS gel for subsequent analysis, if a more global proteomic survey is required.
  • Figure 2. (A) MALDI-TOF spectrum of tryptic peptides of chicken ovalbumin.
  • Figures 4 2DE map of NBS-labeled protein spots that were excised and identified by MS analysis. Fifty-eight protein spots were positively identified by peptide mass fingerprinting from 88 spots with differential fluorescence stain intensity as determined by 2DE analysis. Among them, 44 protein spots were detected with NBS-labeled peptide pairs, which were quantified by calculating 13 C/ 12 C peptide volume ratios.
  • FIG. 7 Mass spectra of tryptic peptides of Spot-21 (CK19).
  • the peptide sequencing mass spectra of the matched (m/z 1222.75) and unmatched peptide (m/z 1419.85) are shown with their y-ions.
  • the matched peptide 1222.75 (TKFETEQALR) (SEQ ID NO:56) was found from CK19, while the unmatched peptide m/z 1419.85 (LEGLTDEINFLR) (SEQ ID NO:57) was from CK8.
  • FIG. 8 Two isoforms of the heat shock 60 kDa protein (CH60JHUMAN) with different isoelectric points (Spots 7 and 8).
  • CH60JHUMAN heat shock 60 kDa protein
  • spots 7 and 8 different isoelectric points.
  • A Based on the NBS labeling quantitation, this protein was up-regulated on Spot 8 but down- regulated on Spot 7.
  • B The ratios of fluorescence labeling of 2DE spots were concordant with the NBS labeling ratios.
  • FIG. 10 Validation of NBS quantification by immunoblotting.
  • Two representative proteins GRP78 and CK19 which were found to change upon TAM treatment, were validated by immunoblotting with the protein specific antibodies.
  • GRP78 is up-regulated in TAM treated MCF-7 cells.
  • B (Top) lmmunoblot revealing the down-regulation of PA2G4 protein at 24 and 48 h after TAM treatment. Blot with ⁇ -Actin antibody is shown for the equal loading of total cell lysates.
  • (Below) RT-PCR amplification of PA2G4 and ⁇ - actin transcripts from oligo-dT primed cDNA shows the relative down- regulation of PA2G4 mRNA upon TAM treatment.
  • FIG. 1 2DE images of MCF-7 cells treated with TAM (A) or vehicle (B) for 48 hr, and the NBS-labeled combined TAM or vehicle treated cells (C). All the images show very similar protein patterns. The results of qualitative and quantitative image analyses are presented in Figure 12.
  • Figure 12 PDQuest image analysis results comparing the 2DE images of TAM treated or vehicle treated (untreated) MCF-7 cells. Triplicate gels were run for each treatment.
  • A Match rates for the two sets of gels was ca. 85- 95% among the ca. 340 visible gel spots;
  • B Numbers of differentially displayed protein spots at qualitative and quantitative levels.
  • Figure 13 (A) Observation result of IHC staining for PA2G4 on normal tissue with inlet showing enlarged part of the normal tissue, and (B) observation result of IHC staining for PA2G4 on tumor tissue, with inlet showing enlarged part of the tumor tissue.
  • FIG 14 Western Blotting on fractionated protein extracts (i.e. the cytosolic protein extract (i), membrane/ organelle protein extract (ii) and nucleic protein extract (Ni)) to show the localization of PA2G4.
  • fractionated protein extracts i.e. the cytosolic protein extract (i), membrane/ organelle protein extract (ii) and nucleic protein extract (Ni)
  • FIG. 15 Validation of PA2G4: (A) Western Blotting on PA2G4 in Cancer sample and Normal sample using Anti-actin as a reference, (B) the integrated value in absorbance unit of the band of PA2G4 respectively in the Cancer sample and Normal sample found in (A). Both in (A) and (B), ER+ represents Cancer sample and TNT represents Normal sample, and in (B) the vertical axis represents an integrated value in absorbance unit of the band.
  • Biomarker any material from a biological source.
  • the material may comprise biomolecules, cells, tissue and/or fluid.
  • the material may be obtained from cell culture, an animal, mammalian or human subject.
  • Biomolecule - A biological molecule such as protein acid, nucleic acid (DNA and/or RNA), lipid, carbohydrate, and their derivatives.
  • Biomarker - Biomarkers are biochemical or molecular molecules (proteins, polypeptides, carbohydrates, lipids, etc, and their derivatives) associated with the presence and severity of specific disease states or different treatment conditions. Under the present invention, a protein or protein derivative may be used as a biomarker. Biomarkers are detectable and measurable by a variety of methods.
  • Biomarker To identify a biomarker, it is usually necessary to detect a difference or change in the expression or abundance of a biological molecule and identifying the particular molecule.
  • Expression or abundance of a biomarker - the expression of a biomarker may be determined from the presence or abundance of its gene, gene transcript, and gene product.
  • the terms “gene” and “gene transcript” include RNA sequences complementary to the gene and cDNA sequences obtained by reverse transcription of the gene transcript. The terms also include the wild type gene, variations and mutations of the gene and gene transcript wherein the variation or mutation share substantial identity with the gene or gene transcript.
  • gene product includes wild type gene product, variations, fragments or derivatives thereof.
  • Substantial identity means that the variants of the gene or gene product retain sufficient identity for them to be detectable by methods and probes used for the wild type gene or gene product and retain the same functions as the wild type gene or gene products.
  • the determination may be qualitative such as whether the biomarker is expressed, or the determination may be quantitative, or the determination may be semi-quantitative by any method known in the art such as by microarray technology or polymerase chain reaction. Departure (increase or decrease) from normal levels in the undiseased state can indicate a disease state or predisposition to a disease state.
  • the overexpression or increased abundance of a protein may be indicative of a disease state, the severity of the disease state and hence the prognosis for the subject in which the biomarker was determined, and/or it might be an indicator of susceptibility to a disease state.
  • the difference in expression and abundance may be determined between different biomarkers or between the same biomarker under different conditions or time points.
  • the expression of the biomarker(s) may then be compared and correlated to a reference value or to other values obtained at different time points or between different biomarkers to determine the correlation with the presence, severity of a disease state, to determine the efficacy of a treatment, or to determine a prognosis of the disease outcome for the subject.
  • Reference values may be determined from a statistically significant number of subjects suffering from or not suffering from.
  • the expression of the biomarker(s) may also be used in conjunction with other suitable diagnostic or prognostic markers, biomarkers or indices to obtain a higher level of confidence.
  • a biomarker is said to be "over expressed” when compared to controls, it is meant that the expression of that biomarker is at an abundance or level that is statistically significantly more than that naturally expressed by at least one wild type or non-mutant control subject not diagnosed with the disease state or condition.
  • a biomarker is "under expressed"
  • the expression of that biomarker is statistically significantly less than that naturally expressed by at least one wild type or non-mutant control subject not diagnosed with the disease state or condition.
  • Control A reference subject, experiment or value by which values obtained in tests can be compared against.
  • Control values or ranges s usually represent the "normal" state so that a statistically difference or deviation of the control values or ranges represents an abnormal or disease state.
  • a subject or candidate - to determine the suitability of a subject or candidate for a clinical trial, experimentation, diagnostic and/or other tests by measuring at least one characteristic of the subject or candidate. The value representing the characteristic is then compared to a reference or control value or range of values, and the result is used to determine if the subject or candidate is suitable for the trial, experimentation, diagnostic and/or other test.
  • a person skilled in the art will know how to select subjects or candidates based on their amenability to a particular treatment, or their susceptibility to a particular challenge or disease.
  • Cell proliferation disorder a condition in which there is abnormal growth of cells.
  • Cancer A malignant and uncontrolled growth of cells in one part of the body that can spread to other parts of the body.
  • Nucleic acid - “Nucleic acids” are linear polymers of nucleotides, linked by 3 1 , 5' phosphodiester linkages.
  • the sugar group is deoxyribose and the bases of the nucleotides adenine, guanine, thymine and cytosine.
  • RNA or ribonucleic acid has ribose as the sugar and uracil replaces thymine.
  • An "isolated nucleic acid” is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid.
  • the term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein.
  • PCR polymerase chain reaction
  • the invention also features an isolated nucleic acid at least 50% (including any percentage between 50% and 100%, e.g., 85%, 95%, or 100%) identical to SEQ ID NO: 1 , which can be used, e.g., for detection of the human PA2G4 gene in a subject, or screening of therapeutic compounds for treating a cell proliferation-associated disorder.
  • Gene product - the gene product refers to a gene product other than a protein.
  • the gene product may be a length of RNA with a biological functional other than a gene transcript coded for by a particular gene.
  • Gene transcript - the messenger RNA sequence coded for by a gene.
  • Protein - A biological molecule composed of one or more chains of amino acids in a specific order, coded for by a particular gene.
  • any mutation in the gene may be reflected in a mutant protein being expressed.
  • a mutant protein that has substantially the same biological functions and activities of the non-mutant protein is considered to be the same as the non-mutant protein.
  • Proteins may have derivatives such as isoforms.
  • a protein isoform is a version of a protein with some small differences, usually a splice variant or the product of some post-translational modification.
  • a protein also encompasses fragments that are sufficiently large enough for the protein to be detected, identified and/or quantified by the method(s) used.
  • a protein complex is a mixture of different proteins.
  • Proteome The set of proteins expressed by a cell or organ at a particular time and under specific conditions. Accordingly, proteomic analysis is the study of the full set of proteins encoded by a genome.
  • Percentage Identity The "percent identity" of two nucleic acid sequences is determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the XBLAST programs of Altschul et al. (1990). BLAST nucleic acid searches are performed with the XBLAST program. Where gaps exist between two sequences, Gapped BLAST is utilised as described in Altschul et al. (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST) are used. See the World Wide Web address http://ncbi.nih.gov. The percentage identity of other sequences such as protein sequences may similarly be determined.
  • Antibody - An immunoglobulin protein produced by B-lymphocytes of the immune system that binds to a specific antigen molecule.
  • the term includes monoclonal antibodies, polyclonal antibodies as well as fragments thereof, such as Fab, F(ab')2, and Fv fragments.
  • An "effective amount" is an amount of the composition that is capable of producing a medically desirable result, e.g., as described above, in a treated subject. This method can be performed alone or in conjunction with other drugs or therapy. Treating includes reduction of the biomarker to a level found in the non-disease state.
  • Hybridizing, hybridisable - a biomolecule such as a sequence of nucleic acids may hybridize to another sequence of nucleic acids if there are sufficient stretches of complementary nucleotides between them.
  • one protein such as an antibody or fragment thereof may hybridize to another protein if there are sufficient complementary structures between these two biomolecules.
  • Diagnose, diagnosis - To diagnose a disease is to is to determine the nature or the identity of a disease.
  • a diagnosis may be accompanied by a determination as to the severity of the disease.
  • Prognosticate, prognosis, - To prognosticate is to predict the outcome or prognosis of a disease, such as to give a chance of survival based on observations and results of clinical tests.
  • Predisposition The likelihood of being diagnosed with, or susceptibility to a particular disease.
  • Blood derivative Blood is made up of a cellular and a liquid component.
  • a blood derivative is any of one or more of the blood components less than whole blood.
  • the present invention addresses the problems above and provides methods for detecting and/or treating a proliferative cell disorder such as cancer.
  • the disorder is cancer, particularly breast cancer.
  • the present invention provides a method of detecting and/or quantitating the presence of, predisposition to, and/or severity of, a proliferative cell disorder in a subject, the method comprising:
  • the control may be, for example, is at least one subject not diagnosed with a cell proliferative disorder.
  • the control may also be a subject having a low level of expression of PA2G4 gene and/or protein.
  • the control may also be an average value in expression obtained from a selected population.
  • the gene, RNA and/or mutation thereof may be human PA2G4 gene, RNA and/or mutation thereof, and the PA2G4 protein, derivative, mutant and/or fragment may be human PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the detecting may be by labelling the PA2G4 protein, derivative, mutant and/or fragment thereof, with at least one sulfenyl halide agent followed by 2- dimensional gel electrophoresis and mass spectrometry.
  • the sulfenyl halide agent may be 2-nitrobenzenesulfenyl chloride (NBS).
  • NBS 2-nitrobenzenesulfenyl chloride
  • the PA2G4 gene may have a sequence as given in SEQ ID NO:1.
  • the varying may be obtained by administering nucleic acid hybridisable to all or part of the PA2G4 gene, RNA and/or mutation thereof.
  • the hybridisable nucleic acid may be DNA or RNA.
  • the hybridisable nucleic acid may be siRNA.
  • the varying may be obtained by administering a compound hybridisable to all or part of the PA2G4 gene, RNA and/or mutation thereof.
  • a method of treating a proliferative cell disorder in a subject comprising varying the expression of a PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the method may comprise varying the expression of the PA2G4 protein having the amino acid sequence of SEQ ID NO:2.
  • the varying may be obtained by administering a polypeptide binding to all or part of the amino acid sequence of the PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the binding polypeptide may be an antibody.
  • the varying may be obtained by administering a compound hybridisable to all or part of the amino acid sequence of the PA2G4 protein.
  • the varying of the expression may comprise reducing the expression of the PA2G4 gene, RNA and/or mutation thereof; and/or PA2G4 protein, derivative, mutant and/or fragment thereof.
  • a method of screening for at least one drug that causes a variation in expression of a at least one protein and/or fragment thereof comprising: administering at least one drug to at least one subject; obtaining at least one protein from the subject; labelling at least one amino acid residue of the protein(s) with at least one agent; and analysing the protein(s) by mass spectrometry, thereby identifying the at least one protein and/or fragment thereof, and determining whether a variation of expression has occurred.
  • the analysing step may comprise separating the protein(s) based on a first characteristic, optionally separating the protein(s) based on a second characteristic, ionising the protein(s) and detecting and identifying the separated protein(s).
  • the protein and/or fragment thereof may be a PA2G4 protein and/or fragment thereof.
  • the PA2G4 protein has the sequence of SEQ ID NO:2.
  • the agent may be a sulfenyl halide.
  • the agent may be 2-nitrobenzenesulfenyl chloride (NBS).
  • NBS 2-nitrobenzenesulfenyl chloride
  • the amino acid residue may be tryptophan.
  • the separating steps may be based on at least one characteristic selected from the group consisting of mass, charge, functional group and isoelectric point.
  • the separating steps may be steps for SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2DE), high performance liquid chromatography (HPLC) and/or free flow electrophoresis (FFE).
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • 2DE two-dimensional gel electrophoresis
  • HPLC high performance liquid chromatography
  • FFE free flow electrophoresis
  • the ionising may be selected from the group consisting of electrospray ionisation (ESI), matrix assisted laser desorption/ionisation (MALDI), surface enhanced laser desorption/ionisation (SELDI), nanospray ionisation, atmospheric pressure chemical ionisation (APCI), chemical ionisation (Cl), electron impact (El), fast atom bombardment (FAB), field desorption / field ionisation (FD/FI) and thermospray ionisation (TSP).
  • the analysing step may be by tandem mass spectrometry.
  • the method tandem mass spectrometry may use quadrupole time-of-flight geometry.
  • a method of selecting at least one candidate for clinical trial(s), experimentation and/or diagnostic test comprising:
  • diagnostic and/or prognostic kit for the diagnosis and/or prognostic evaluation of a cell proliferative disorder in a subject, the diagnostic and/or prognostic kit comprising at least one molecule or compound hybridisable to a PA2G4 gene, RNA and/or mutation thereof; and/or binding to a PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the molecule may be a nucleic acid sequence hybridisable to the PA2G4 gene, RNA and/or mutation thereof.
  • the molecule may be a siRNA hybridisable to the PA2G4 gene transcript.
  • the molecule may be a protein binding to the PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the molecule may be an antibody binding to the PA2G4 protein, derivative, mutant and/or fragment thereof.
  • the proliferative cell disorder may be a cancer.
  • the proliferative cell disorder may be breast cancer.
  • the subject may be a mammal; in particular, a human being.
  • the present application describes the novel use of a gene, gene transcript, RNA gene product and/or mutation thereof, and/or protein, derivative, mutant and/or fragment thereof, for the diagnosis of breast cancer, for determining a prognosis and for the monitoring of the efficacy of a treatment for breast cancer.
  • MS mass spectrometry
  • PA2G4 proliferation associated gene 2G4
  • Mass spectrometry may be used to analyse molecules.
  • molecules of interest are ionised, separated in an analyzer based on their m/z ratios and then detected by a detector. The results may be displayed in the form of a m/z spectrum.
  • the MS system usually includes a data analysis sub-system for the analysis of the data.
  • lonisation of the molecules may be achieved by many methods, including: Atmospheric Pressure Chemical lonisation (APCI), Thermospray lonisation (TSP), Chemical lonisation (Cl), Electron Impact (El), Electrospray lonisation (ESI), Fast Atom Bombardment (FAB), Field Desorption / Field lonisation (FD/FI), Surface Enhanced Laser Desorption lonisation (SELDI) and Matrix Assisted Laser Desorption lonisation (MALDI).
  • APCI Atmospheric Pressure Chemical lonisation
  • TSP Thermospray lonisation
  • Cl Chemical lonisation
  • El Electron Impact
  • EI Electrospray lonisation
  • FAB Fast Atom Bombardment
  • FD/FI Field Desorption / Field lonisation
  • SELDI Surface Enhanced Laser Desorption lonisation
  • MALDI Matrix Assisted Laser Desorption lonisation
  • the analyser may utilize different principles or geometries for the analysis of the ions, including quadrupoles, magnetic sectors, and both Fourier transform, quadrupole ion traps and time-of-flight (TOF) geometries.
  • Two or more analysers may be arranged in tandem.
  • Commonly used tandem geometries include quadrupole-quadrupole, magnetic sector-quadrupole, and quadrupole-time-of-flight geometries.
  • MS a person skilled in the art will be able to select different ionisation, analysis and detection methods to best study a biomolecule of interest such as proteins.
  • NBS labeling involves the binding of light and heavy NBS moieties to tryptophan residues, resulting in a 6 Da mass difference between light and heavy NBS labeled tryptophan peptides.
  • TAM tamoxifen
  • CK19, GRP78, and PA2G4 Three proteins, CK19, GRP78, and PA2G4, were also validated as bona-fide TAM-responsive proteins in MCF-7 cells by Western blotting, and further shown that in primary breast cancers, PA2G4 expression behaves as a novel prognostic factor in two independent patient cohorts.
  • PA2G4 Proliferation Associated 2G4
  • the gene sequence is given as SEQ ID NO: 1 and the gene product (protein) sequence is given in SEQ ID NO: 2.
  • PA2G4 (Ebp-1) belongs to a large family of cell cycle regulatory proteins or replication proteins that maintain the cell cycle activities of proliferating cells. It appears to be a proliferation-dependent gene that probably encodes a nuclear DNA-binding protein involved in the control of cell replication.
  • PA2G4/Ebp-1 has been shown to participate in the differentiation of human ErbB receptor-positive breast and prostate cancer cells, and is capable of inducing anti-proliferative effects by virtue of its interactions with the retinoblastoma (Rb) tumor suppressor protein (Rao et al., 2002). This protein is found in both mammals and humans.
  • Rb retinoblastoma
  • E2 mitogenic effects have been attributed in large part to its ability to increase the expression of key cell cycle regulatory genes.
  • NBS/2DE/MS we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity.
  • TAM tamoxifen
  • PA2G4 has been identified as a reliable biomarker for breast cancer, its gene, gene transcript, RNA gene product, gene derivative, gene mutation, protein, protein derivative, protein mutant, and protein fragment may be used accordingly for this purpose. Any method known in the art may be used to qualitatively detect or quantitatively measure the presence of PA2G4.
  • a nucleic acid of the invention may be expressed in vitro by DNA transfer into a suitable host cell by methods known in the art.
  • the nucleic acid may be inserted into a recombinant expression vector.
  • a variety of host-expression vector systems may be utilised to express a nucleic acid of the invention. These include; but are not limited to, micro-organisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors; yeast transformed with recombinant yeast expression vectors; and human cell lines infected with recombinant virus or plasmid expression vectors. Isolation and purification of recombinant polypeptides, or fragments thereof, can be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
  • the invention also features methods for determining prognosis for, diagnosing, treating, and identifying therapeutic compounds for treating a cell proliferation-associated disorder.
  • a prognostic method of the present invention involves comparing expression of the biomarker in samples from patients with reference values obtained from one or more patients with known disease outcomes, or from known benign or malignant tissues or cells. Thereafter, it will be possible to determine the prognoses of these patients.
  • a diagnostic method of the invention involves comparing one or more of the PA2G4, GRP78 and CK19 genomic DNA levels in a sample prepared from a subject (i.e., an animal or a human) with that in a sample prepared from a normal subject, i.e., a subject who does not suffer from or at risk for developing a cell proliferation-associated disorder.
  • a higher PA2G4 genomic DNA level indicates that the subject is suffering from, or at risk for, a cell proliferation-associated disorder.
  • This methods can be used on their own or in conjunction with other procedures to diagnose a cell proliferation- associated disorder in appropriate subjects and to provide a prognosis as to the outcome of the disease.
  • Amplification of a gene locus can be detected by a variety of methods known in the art. For example, the copy number of a gene locus can be determined and compared by PCR amplification of genomic DNA prepared from a test sample and a control sample. Amplification of a gene locus can also be identified by Southern blot analysis. Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location and an amount of the DNA sequence present in the chromosome.
  • FISH Fluorescence in situ hybridization
  • the invention also provides a method for identifying compounds (e.g., proteins, peptides, peptidomimetics, peptoids, antibodies, or small molecules) that decrease the PA2G4 genomic DNA level, modulate the expression, decrease the level of PA2G4 or PA2G4 mRNA or protein, or decrease the level of PA2G4 interaction with other molecules recognizing it.
  • the resulting identified compounds can then be used, e.g., for preventing and treating a cell proliferation-associated disorder.
  • the candidate compounds of the invention may be obtained using any of the numerous approaches in combinatorial library methods known in the art.
  • libraries include: peptide libraries, peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non- peptide backbone that is resistant to enzymatic degradation); spatially addressable parallel solid phase or solution phase libraries; synthetic libraries obtained by deconvolution or affinity chromatography selection; and the "one-bead one-compound” libraries. See, e.g., Lam (1997). Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: Gallop et al. (1994).
  • Libraries of compounds may be presented in solution (e.g., Houghten, 1992), or on beads (Lam, 1991), chips (Fodor, 1993), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No. 5,223,409), plasmids (Cull et al, 1992), or phages (U.S. Pat. No. 5,223,409).
  • the cell can contain amplified PA2G4 genomic DNA (e.g., a cancer cell), naturally express PA2G4, be modified to express a recombinant nucleic acid, for example, having a PA2G4 gene fused to a heterologous promoter. If the level of PA2G4 genomic DNA is changed due to the candidate compound; that is, either lower in the presence of the candidate compound than in its absence, or if the expression of the marker nucleotide sequence in the presence of the candidate compound differs from that in the absence of the candidate compound, the candidate compound is identified as being useful for preventing and treating a cell proliferation-associated disorder.
  • PA2G4 genomic DNA e.g., a cancer cell
  • PA2G4 genomic DNA e.g., a cancer cell
  • PA2G4, GRP78 and CK19 genomic DNA levels can be determined by methods described above and any other methods well known in the art.
  • the expression of the marker nucleotide sequence, PA2G4, GRP78 and CK19 can be determined either at the mRNA level or at the protein level.
  • Methods of measuring mRNA levels in a tissue or a body fluid sample are known in the art.
  • cells can be lysed and the levels of mRNA in the lysates or in RNA purified or semi-purified from the lysates can be determined by any of a variety of methods including; without limitation, hybridization assays using detectably labeled DNA or RNA probes and quantitative or semi-quantitative RT-PCR methodologies using appropriate oligonucleotide primers.
  • quantitative or semi- quantitative in situ hybridization assays can be carried out using, for example, tissue sections or non-lysed cell suspensions, and detectably (e.g., fluorescently or enzyme) labeled DNA or RNA probes.
  • detectably e.g., fluorescently or enzyme
  • Additional methods for quantifying mRNA include RNA protection assay (RPA) and serial analysis of gene expression (SAGE).
  • NBS/2DE/MS other methods of measuring protein levels in a tissue or body fluid sample are also known in the art.
  • Many such methods employ antibodies (e.g., monoclonal or polyclonal antibodies) that bind specifically to the target protein.
  • the antibody itself or a secondary antibody that binds to it can be detectably labeled.
  • binding with detectably labeled avidin can be used to detect its presence.
  • Combinations of these approaches can be used to enhance the sensitivity of the methodologies.
  • Some of these protein-measuring assays can be applied to lysates of cells, and others can be applied to histological sections or non-lysed cell suspensions, e.g., immunohistological methods or fluorescence flow cytometry. Methods of measuring the amount of label will be depend on the nature of the label and are well known in the art.
  • Appropriate labels include, without limitation, radionuclides (e.g., 125 I, 131 I 1 35 S, 3 H, or 32 P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or beta- galactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot.TM. nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, California).
  • Other applicable assays include quantitative immunoprecipitation or complement fixation assays.
  • the level of PA2G4 can be determined by any method known in the art, e.g., by in vitro binding assay or by using a yeast hybrid system.
  • the binding domains of PA2G4 can be identified, e.g., by mutagenesis, and used in the screening assays.
  • This invention further provides a method for preventing and treating a cell proliferation-associated disorder.
  • Subjects to be treated can be identified; for example, by determining the PA2G4 genomic DNA, DNA transcript or DNA product level in a sample prepared from a subject by methods described above. If the PA2G4 genomic DNA level is higher in the sample from the subject than that in a sample (control) from a normal subject, the subject is a candidate for treatment with an effective amount of compound that decreases the PA2G4 genomic DNA, DNA transcript or DNA product level.
  • a therapeutic composition e.g., a composition containing a compound identified as described above
  • the compound will be suspended in a pharmaceutically- acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecal ⁇ , intraperitoneal ⁇ , intrarectally, intravaginally intranasally, intragastrically, intratracheally, or intrapulmonarily.
  • a pharmaceutically- acceptable carrier e.g., physiological saline
  • intravenous infusion e.g., physiological saline
  • the compound can be delivered directly to the cancer tissue.
  • the dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the subject's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100.0 mg/kg. Wide variations in the needed dosage are to be expected in view of the variety of compounds available and the different efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
  • a suitable delivery vehicle e.g., polymeric microparticles or implantable devices
  • liposomes prepared by standard methods.
  • the vectors can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies.
  • tissue-specific antibodies one can prepare a molecular conjugate composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces.
  • Poly-L-lysine binds to a ligand that can bind to a receptor on target cells (Cristiano et al, 1995).
  • tissue specific targeting can be achieved by the use of tissue-specific transcriptional regulatory elements (TRE) which are known in the art.
  • TRE tissue-specific transcriptional regulatory elements
  • nucleic acid sequence encoding an anti-sense PA2G4 RNA is operatively linked to a promoter or enhancer-promoter combination.
  • Enhancers provide expression specificity in terms of time, location, and level. Unlike a promoter, an enhancer is able to function when located at variable distances from the transcription initiation site, provided a promoter is present. An enhancer can also be located downstream of the transcription initiation site.
  • RNA interference This is a technique for post- transcriptional gene silencing ("PTGS"), in which target gene activity is specifically abolished with cognate double-stranded RNA (“dsRNA").
  • dsRNA double-stranded RNA
  • dsRNA of about 18-26 nucleotides, in particular, 20-24 nucleotides, more particularly 21-23 nucleotides.
  • a 21- nucleotide sequence homologous to the target gene is introduced into the cell and a sequence specific reduction in gene activity is observed.
  • RNA interference provides a mechanism of gene silencing at the mRNA level. It offers an efficient and broadly applicable approach for gene knock-out as well as for therapeutic purposes.
  • Sequences capable of inhibiting gene expression by RNA interference may be of any desired length.
  • the sequence can have at least 15, 20, 25, or more consecutive nucleotides.
  • the sequence may be dsRNA or any other type of polynucleotide, provided if the sequence may form a functional silencing complex to degrade the target mRNA transcript.
  • the sequence consists of or comprises a short interfering RNA (siRNA).
  • siRNA may be, for example, dsRNA having 19- 25 nucleotides.
  • siRNAs may be produced endogenously by degradation of longer dsRNA molecules by an RNase Ill-related nuclease called Dicer.
  • siRNAs may also be introduced into a cell exogenously or by transcription of an expression construct. Once formed, the siRNAs and protein components assemble into endoribonuclease-containing complexes known as RNA- induced silencing complexes (RISCs).
  • RISCs RNA- induced silencing complexes
  • ATP-generated unwinding of the siRNA activates the RISCs, which in turn targets the complementary mRNA transcript by Watson-Crick base pairing, thereby cleaving and destroying the mRNA. Cleavage of the mRNA takes place near the middle of the region bound by the siRNA strand. This sequence-specific mRNA degradation results in gene silencing.
  • Polynucleotides may be administered in a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are biologically compatible vehicles that are suitable for administration to an animal or a human, e.g., physiological saline or liposomes.
  • a preferred dosage for administration of polynucleotide is from approximately 10 6 to 10 12 copies of the polynucleotide molecule. This dose may be repeatedly administered, as needed. Routes of administration may be any of those listed above.
  • Antibodies (monoclonal or polyclonal) to PA2G4 may be used to reduce the level of PA2G4 protein, or to decrease the level of PA2G4 in a subject.
  • the term "antibody” includes intact molecules as well as fragments thereof, such as Fab, F(ab') 2 , and Fv which are capable of binding to an epitopic determinant present in the PA2G4 protein.
  • Methods of making monoclonal and polyclonal antibodies and fragments thereof are known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
  • Other compounds that may be used to inhibit PA2G4 expression include a peptide or polypeptide containing an amino acid sequence at the C-terminus of the PA2G4 protein.
  • the invention provides a method of developing a procedure for treating a cell proliferation-associated disorder by providing a plurality of subjects suffering from a cell proliferation-associated disorder (e.g., subjects having amplified PA2G4 genomic DNA or PA2G4 protein expression); administering to each subject an effective amount of an PA2G4 inhibitor followed by an effective amount of a nucleotide or nucleoside analogue (e.g., gemcitabine), each at a unique time point; and selecting an optimal time point at which the cell proliferation-associated disorder is inhibited to the greatest extent. Once the optimal time point has been identified, the procedure may be used to treat a cell proliferation-associated disorder in appropriate subjects.
  • a cell proliferation-associated disorder e.g., subjects having amplified PA2G4 genomic DNA or PA2G4 protein expression
  • a nucleotide or nucleoside analogue e.g., gemcitabine
  • MCF-7 breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Tamoxifen (TAM), chicken ovalbumin, streptomycin, L-glutamine, penicillin, 4-hydroxycinnamic acid (CHCA), 3- hydroxy-4-nitrobenzoic acid (HNBA) 1 2,5-dihydroxybenzoic acid (DHB) 1 protease inhibitor cocktail, phenyl methyl sulfonyl fluoride (PMSF), sodium deoxychlate and sodium orthovanadate were from Sigma (St Louis, MO, USA).
  • the sulfenyl halide agent and 2-nitrobenzenesulfenyl chloride were from Shimadzu Biotech (Kyoto, Japan).
  • Tris (2- carboxyethyl) phosphine (TCEP) hydrochloride was from Pierce (Rockford, IL, USA).
  • Deep Purple fluorescent dye, dithiotritol (DTT) and horseradish peroxidase conjugated secondary antibodies were from GE HealthCare (Uppsala, Sweden).
  • Ethylene diamine tetra acetic acid disodium salt dihydrate (EDTA) and polyvinylidene difluoride membrane (PVDF) were from Bio-Rad (Hercules, CA, USA).
  • C18 ZipTips were from Millipore (Bedford, MA, USA). Sequencing grade modified trypsin was from Promega (Madison, Wl, USA). Dulbecco modified Eagle medium 5 (DMEM) was from Gibco (Grand Island, NY, USA). Dextran charcoal-stripped fetal bovine serum (FBS) was from HyClone Laboratories (Pittsburg, PA, USA). Anti-vinculin and anti- PA2G4 antibodies were from Upstate Biotechnology (VA, USA). Anti- cytokeratin 19 and anti-GRP78 antibodies were from Santa Cruz Biotechnology (Santa Cruz Inc, CA, USA). Trizol and Reverse transcriptase enzymes were from Invitrogen (CA, USA). Oligonucleotide primers used in cDNA synthesis and RT-PCR were synthesized from Sigma Proligo (Sigma, Singapore).
  • Example 2 Cell Culture and TAM Treatment of MCF-7 Cells
  • MCF-7 breast cancer cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 U/mL streptomycin, and 2 mM L-glutamine. Before TAM treatment, cells were washed in PBS and maintained in phenol red free DMEM with 5 % Dextran charcoal-stripped FBS for 24 hrs. Subsequently, cells were treated with 10 ⁇ M TAM at 40-50% confluence and harvested at 24 and 48 hrs. As control, sister cultures were treated with an equivalent volume of the vehicle (0.1 % ethanol).
  • TAM-treated and control MCF-7 cells were lysed in 2DE lysis buffer (7M urea, 2M thiourea, 4% CHAPS, 1mM PMSF, 50 ⁇ g/mL Dnase I 1 50 ⁇ g/mL Rnase I and protease inhibitor cocktail) and the extracted proteins buffer exchanged into 8M urea and 5mM EDTA using a 2D-sample clean up kit (Bio-Rad).
  • the sample was dried with a vacuum concentrator and resuspended in 50 ⁇ L of 5OmM Tris buffer (pH 8.5) containing 0.1% SDS and reduced with 4mM TCEP for 30 min at 37 0 C, followed by alkylation with 1OmM iodoacetetamide for 45 min at room temperature in the dark.
  • the lysate was then buffer exchanged into 2DE lysis buffer (7M urea, 2M thiourea and 4% CHAPS).
  • each IPG strip (18 cm, pH 4-7, GE
  • the IPG strips were equilibrated in two steps: (1) 15 min in 5OmM Tris-HCI, pH 8.8, 6M urea, 30% glycerol, 2% SDS, 1% DTT and a trace of bromophenol blue; (2) 15 min in a similar solution containing 2.5% iodoacetamide instead of DTT.
  • the strips were transferred onto 10% isocratic polyacrylamide gels (18cm x 20cm x 0.75mm).
  • the IPG gels were then sealed with 0.5% w/v agarose in running buffer (25mM Tris-HCI, pH8.3, 192mM glycine and 0.1% SDS).
  • the second dimensional separation was then performed using the Protean Il XL system (Bio-Rad).
  • the SDS-PAGE was carried out at 17 0 C with 8mA/gel for 1 hr followed by 24mA/gel constant current, until the dye front reached the bottom of the gel.
  • the gels were then labeled with the Deep Purple fluorescence dye according to the manufacturer's protocol (GE HealthCare).
  • the gels were digitized using a FX Molecular Imager (Bio-Rad), and image analysis performed using PDQuest 7.3 image analysis software (Bio-Rad).
  • Fluorescence labeled protein gel spots were excised and in-gel trypsin digested using the Xcise robotic system (Shimadzu Biotech, Kyoto, Japan).
  • the automatic operation involved washing the gel pieces with water twice, shrinking with 100% acetonitrile (ACN), and drying prior to digestion. Thirty microliters of trypsin (3.33ng/ ⁇ L in 5OmM ammonium bicarbonate, pH 8.5) was added to each gel plug and the digestion was performed overnight at 30 0 C with shaking. The samples were then cleaned and concentrated with C18 ZipTips.
  • peptide mixtures were eluted onto the MALDI sample plate with 1 ⁇ l_ of 50% ACN/0.5% TFA and mixed with 1 ⁇ l_ matrix containing 10mg/ml_ ⁇ -cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3-hydroxy-4-nitrobenzoic acid (HNBA) in 50% ACN/0.5% TFA prior to MS analysis.
  • CHCA methyl ⁇ -cyano-4-hydroxycinnamic acid
  • HNB 2,5-dihydroxybenzoic acid
  • HNBA 3-hydroxy-4-nitrobenzoic acid
  • MALDI-TOF MS analyses were performed using the AXIMA-CFR plus mass spectrometer (Shimadzu Corporation, Kyoto, Japan and Kratos Analytical, Manchester, UK), under the following settings : nitrogen laser (337 nm); reflectron mode; detection of positive ions.
  • the acceleration potential was set to 35 kV using a gridless-type electrode.
  • MALDI-TOF MS spectra were acquired in manual mode, from m/z 800 to 3000 and internally calibrated with two trypsin autolysis peaks (m/z 842.51 and 2211.10).
  • Peak lists from peptide mass mapping spectra were automatically extracted and submitted to an in-house Mascot server (Matrix Science, London, UK) to. search against UniProt and NCBInr databases.
  • Mascot server Mobile Network Science, London, UK
  • two modification values of 12CNBS (+153 Da) and 13CNBS (+159 Da) were added to the Mascot server as variable modifications.
  • the NBS-labeled paired peaks were manually selected from the TOF MS spectrum and the relative quantitation was achieved by calculating the volume ratio of each NBS labeled peak pairs using the Kompact software (Shimadzu/Kratos).
  • MS/MS peptide sequencing was performed using the AXIMA-QIT MALDI quadrupole ion trap time-of- flight mass spectrometer (Shimadzu Corporation, Kyoto, Japan and Kratos Analytical, Manchester, UK) equipped with a 337 nm nitrogen laser.
  • AXIMA-QIT MALDI quadrupole ion trap time-of- flight mass spectrometer (Shimadzu Corporation, Kyoto, Japan and Kratos Analytical, Manchester, UK) equipped with a 337 nm nitrogen laser.
  • One microliter of tryptic-digested sample was deposited on to the MALDI plate and mixed with 1 ⁇ L of DHB (10 mg/mL in 50% ACN/0.5% TFA).
  • the TOF spectrum was externally calibrated using fullerite pre-deposited onto the MALDI sample stage. All spectra were acquired with standard instrument settings for optimum transmission at medium and high masses. Data acquisition and processing were performed using the Kompact software (Shimadzu/Krato
  • Example 6 Statistical analysis To investigate the biological and experimental variations associated with NBS/2DE/MS, and to compare protein quantities between NBS/2DE/MS and conventional 2DE analysis, we applied a series of Pearson's correlation tests to assess the similarity of the two datasets under investigation. To determine the significance of any observed correlation value, a bootstrap permutation test was performed by scrambling the index of the two datasets being compared, and re-calculating the correlation coefficient for the randomized datasets. For the correlation tests, to determine the significance of any observed correlation value, a bootstrap permutation test was performed by scrambling the order of the proteins in the two datasets being compared, and re-calculating the correlation coefficient for the randomized datasets. This procedure was repeated 10,000 times, the frequency at which the randomized coefficients exceeded the observed value was computed, and this was reflected as an empirical p-value.
  • PA2G4-RTF CACGTGCCTTCTTCAGTGAG (SEQ ID NO:58); PA2G4-RTR: ACTCTGGAGGAGGGCCTTTA ((SEQ ID NO:59); ⁇ -actin-RTF: CGGGAAATCGTGCGTGACATT (SEQ ID NO: 60); ⁇ -actin-RTR: TGATCTCCTTCTGCATCCTGT (SEQ ID NO:61 ).
  • Example 9 Quantitative NBS/2DE/MS analyses of TAM-treated MCF-7 cells
  • the 44 identified NBS protein spots representing 23 different protein species.
  • the 13C/12C ratios were calculated based on the NBS-paired peak intensities. Three proteins were identified with more than one pair of NBS peptides. For them, the 13C/12C ratios were the average of all the paired peptide ratios of the same protein.
  • protein lysates from the exact same batch of TAM and vehicle treated MCF-7 cells were re-labelled with NBS, separated on 2DE gels, and re-quantified based on the 13C/12C ratios.
  • 88 spots were 26 excised from the NBS-labelled 2DE gels, out of which 58 protein spots were positively identified by peptide mass fingerprinting.
  • Spots 1-44 were proteins detected with NBS-labelled peptide peaks. Among them 44 protein spots were detected with NBS-labelled peptide pairs, which were quantified by calculating 13C/12C peptide volume ratio.
  • the 13C/12C ratios were calculated based on the NBS-paired peak intensities (Column 13C/12C (1)). Three proteins were identified with more than one pair of NBS peptides. For these proteins, the 13C/12C ratios were the average of all the paired peptide ratios of the same protein.
  • protein lysates from the exact same batch of TAM and vehicle treated MCF-7 cells were re-labelled with NBS, separated on 2DE gels, and re-quantified based on the 13C/12C ratios (Column 13C/12C (2)). The mean and SD values are based on combining values from the 13C/12C (1) and 13C/12C (2) columns.
  • Table 2 (below): Guided by the conventional 2DE gel image analysis, 88 spots were excised from the NBS-labeled 2DE gels. A total of 58 protein spots were positively identified by peptide mass fingerprinting (Figure 4 displays the NBS/2DE reference map with the identified protein spots), of which 44 contained NBS peptide pairs (Table 2). Some protein spots possessed more than one NBS peptide pair, indicating the presence of multiple tryptophan containing peptides in these proteins. For these proteins, the final NBS ratio was calculated by averaging the ratios of all NBS paired peptides associated with the same protein spot.
  • NBS ratio analysis 21/44 protein spots showed a >2-fold intensity alteration upon TAM treatment, and the remaining 23/44 spots changed less than 2-fold between TAM and vehicle. In comparison, for the conventional 2DE analysis, 17/44 of these spots were associated with a >2 fold difference, while the remaining 27/44 exhibited a ⁇ 2 fold difference.
  • the 44 identified NBS labeled protein spots represented 23 different protein species (Table 2), where 10 species, including K1C18JHUMAN (CK18), were represented by multiple isoforms/spots on the 2DE gel.
  • Example 11 Proteomics of PA2G4 in breast cancer cells, a) 2DE analysis of 2 breast cancer cell lines (MCF-7, HCC-38) and a CRL cell line indicated that PA2G4 was over-expressed in the ER+ cell lines but not the ER- cell lines.
  • the over-expression of PA2G4 in MCF-7 cells can be suppressed by tamoxifen treatment. It was found a 5x decrease using NBS analysis and a 3x decrease using 2DE analysis.
  • PA2G4 originally identified as an ERBB3 receptor interacting protein.
  • TAM TAM treatment.
  • FIG. 10b exposure of MCF7 cells to TAM induced a modest but noticeable down-regulation of PA2G4 protein after 24h, which persisted until 48hr.
  • the expression of PA2G4 protein is >2 fold higher in MCF-7 (ER+) than in HCC-38 (ER-) and CCD-1059sk (ER-) breast cell lines.
  • PA2G4 transcript expression demonstrated a highly significant correlation with disease-free survival in both data sets (p-values are 1.75x10-5 (GIS) and 0.001 (Oxford) respectively), with patients with high PA2G4 expressing tumors exhibiting poorer clinical outcome (Table 4A).
  • PA2G4 expression was also observed in both data sets when the univariate analysis was restricted to only those patients receiving adjuvant hormonal therapy (p values are 0.002 (GIS) and 0.008 (Oxford) respectively; Table 4a).
  • p values are 0.002 (GIS) and 0.008 (Oxford) respectively; Table 4a.
  • PA2G4 expression might behave as an independent prognostic factor in comparison to other clinical variables, such as tumor grade, patient age, and tumor size.
  • PA2G4 is a novel survival- associated breast cancer gene and is suitable for use as a prognostic biomarker.
  • Suitable antibodies against PA2G4 protein are available from commercial sources and may be used to detect and/or quantify PA2G4 in any immunological binding/hybridization reaction known in the art. For example, a
  • PA2G4 polyclonal antibody is available from the Abnova Corporation (Taiwan;
  • Product Number H00005036-A01 This is a mouse polyclonal antibody raised against a partial recombinant PA2G4 immunogen.
  • another example is an anti-Ebp1 polyclonal antibody raised from rabbit available from
  • Example 14 - Suitable recombinant expression vectors for PA2G4 A person skilled in the art will be able to select a suitable vector and insert the PA2G4 gene sequence into it for subsequent expression of the gene.
  • vectors with the PA2G4 inserted are commercially available.
  • Origene (USA) has the PA2G4 sequence in its pCMV6-XL5 vector as Product Number SC103017.
  • Example 12 - Use of PA2G4 in diagnosis discloses the differential expression of the PA2G4 gene and protein in ER+ breast cancer cells and disease patients, and the suppression effect of tamoxifen (TAM) in its expression. Based on this finding, the invention provides a method for diagnosing or prognosticating ER+ breast cancer disease in a subject, and for determining whether a subject is at increased risk of developing breast cancer disease. Furthermore, this invention provides therapeutic and prophylactic methods for treating or preventing breast caner disease using the PA2G4 gene and its corresponding gene products.
  • TAM tamoxifen
  • the anti-human PA2G4 antibody can be used to analyse (detect and measure) this protein in human samples such as serum, nipple aspirate fluid, extracellular fluids and primary tumour tissues.
  • PA2G4 gene/protein has the potential use for monitoring therapy of breast cancer.
  • siRNA for gene silencing to observe the physiological change of ER+ cells (e.g. MCF-7) after the transfection of siRNA. Then we can evaluate the role of PA2G4 gene in the ER+ cell survival and determine whether we can suppress the ER+ cancer cells by specifically inhibiting the PA2G4 gene.
  • the assay can be a dose-dependant time-course response study and has particular application to serum and other body fluidic samples. The result will be able to show the feasibility of using PA2G4 to monitor therapy in breast cancer patients.
  • Molecules for the treatment of a proliferative cell disorder such as breast cancer can be prepared using PA2G4 gene sequence (SEQ ID NO:1) or PA2G4 protein sequence (SEQ ID NO:2).
  • a person skilled in the art will be able to produce nucleic acids complementary to the PA2G4 gene or mRNA such as siRNA, by any method known in the art, to inhibit or decrease the translation of the mRNA.
  • antibodies such as those cited in Example 10 based on the PA2G4 protein, can be raised by any method known in the art, to produce antibodies that bind to, and hence, inactivate the actions of the PA2G4 protein.
  • Such therapeutic molecules can be mixed with suitable excipients, vehicles or carriers and administered into the diseased tissues or cells to obtained the desired therapeutic effect. In particular, the desired therapeutic effect is to lower or decrease the expression of the PA2G4 gene and/or protein.
  • the present invention also provides a kit comprising at least one biomarker complementary or hybridisable to PA2G4 gene, gene transcript, gene product, variant or fragment thereof.
  • complementary or hybridisable biomarkers include nucleic acid probes recognizing and binding to the PA2G4 gene or gene products or fragments or variations thereof, and antibodies recognizing and binding to the PA2G4 gene product or fragments thereof.
  • the gene can be wild type or mutants thereof, and the proteins can be wild type or mutants thereof as long as sufficient homology exists to allow recognition of the gene or protein by the complementary biomarker.
  • the complementary or hybridisable biomolecules can be labeled and hence be used for prognostic evaluation (predicting the susceptibility to), or for the diagnosis of, the cell proliferative disease, or to indicate the severity of the disease state, or to monitor the efficacy of a course of treatment.
  • Example 18 Other biomarkers for breast cancer
  • CK18 neoepitope M30 Another TAM-regulated protein, CK18 neoepitope M30, which has been shown to be a useful serum marker of apoptotic epithelium in septic patients (de Graauw et al, 2005). During apoptosis, DNA fragmentation, nuclear condensation and CK18 cleavage are known events, and a neoepitope in CK18 called M30 neoantigen, becomes available at an early caspase cleavage event. The observation of TAM mediated down- regulation of CK18 show that measurement of CK18 down regulation is useful in TAM treated patients as a measure of drug responsiveness.
  • TAM is the primary hormonal therapy for breast cancer
  • CK18 is useful as a biomarker for breast cancer.
  • GRP78 is a known drug resistance effector and is also a stress response chaperon. Lung cancer and neuroblastoma patients with a positive GRP78 expression tended to show a better prognosis and improved survival (Uramoto et al., 2005).
  • the observed up-regulation of GRP78 in the TAM treated MCF-7 cells represent the immediate stress response to the drug treatment, and also could couple cellular stress to the cell death program (Rao et al., 2002; Ding et al., 2004). This is supported by accompanied up-regulation of another ER stress gene, HERP, at the mRNA level upon TAM treatment.
  • NBS/2DE/MS is a reliable approach for cellular protein quantitation than conventional 2DE approaches.
  • breast cancer biomarkers were discovered from the integrated proteomic and genomic study, and were validated using breast tissue microarrays.
  • the breast tissue arrays were provided and cross-examined by one single pathologist in order to minimize bias (Dr. Manuel Salto-Tellez: Associate Professor, Department of Pathology; Senior Research Engineer, Oncology Research Institute; Senior Consultant Pathologist, Clinical Director, Molecular Diagnostic Oncology Centre, NUH, Singapore).
  • IHC immunohistochemical staining
  • MCF7 breast cancer cells were cultured in 75ml_ flask. Upon confluence, the cells were trypsinized and spun down to >5mm thick cell pellets.
  • the cell pellets were incubated with formalin for 20 min and proceeded to paraffin embedment.
  • the stained positive control tissue slides were examined by the pathologist after optimization.
  • the optimal condition including the specific antigen retrieval method and the specific primary antibody dilution, was determined after consulting the pathologist and was applied to the breast tissue microarrays.
  • citric acid adjust pH to 6.0 with 2M NaOH (about 65mL) top up with deionized water to 5L (If over pH, add citrate acid, if below pH, add NaOH.)
  • 60ml Dako (10x) (Cat. S2369) target retrieval, top up with deionized water to 60OmL.
  • DAKO pH9.0 Buffer 60ml Dako (10x) (Cat. S2367) target retrieval, top up with deionized water to 60OmL.
  • array slides Before deparaffinization, array slides should be kept at room temperature for 60 min or heated in oven at 60 0 C for 20 min in a horizontal position. Align slides in a metal tray, 1. Immerse the slides in xylene for 5 min, repeat once in new xylene for 5 min. (Xylene is toxic.)
  • Boil 2 L of citrate buffer in a stainless pressure cooker for 10-15 min 2. Once the buffer boils, put the slides vertically into the pot and shut the lid tightly. After 2 buttons on lid are popped up, start to time 3min. (Don't allow the slides to touch the bottom of cooker directly) 3. After 3 min, remove the pot and cool in sink under running water. Pressure can be released by turning the knob. After the pressure is released, open the lid and keep the slides under running water to cool down.
  • PA2G4 IHC staining on the tumor tissue microarray showed 96.5% (i.e. 83 out of 86 tumor tissues) positive staining(brown colored). Among those positively stained tumor tissues, 40.7% (i.e. 35 out of 86) tumor tissues were of grade 3 positive staining; 36% (i.e. 31 out of 86) tumor tissues were of grade 2 positive staining; and 20% (i.e. 17 out of 86) tumor tissues were of grade 1 positive staining.
  • FIG.13(A) observation result of IHC staining for PA2G4 on normal tissue is shown in FIG.13(A), with inlet showing enlarged part of the normal tissue.
  • FIG.14 is result of western blotting on fractionated protein extracts for PA2G4. From left to right lane, they are the cytosolic protein extract (i), membrane/ organelle protein extract (ii) and nucleic protein extract (iii). As the picture shown, the protein expression for PA2G4 is at both cytoplasm and nucleus, but higher expression in the cytoplasm.
  • Bovine Serum Albumin (BSA, 2mg/ml) is used as standard.
  • Filter paper, membrane, gel, filter paper Note: a. cut the membrane and filter paper the same size as gel; b. Avoid any bubbles between membrane and gel.
  • Tissues used for this analysis were obtained from Singapore National Cancer Center Tissue Repository after two examinations by independent histopathologist.
  • the normal breast tissues were from the cosmetic surgery patients.
  • Protein extract preparation was carried out essentially as described in Ou,K.,Kesuma,D.,Ganesan,K.,Yu,K. et al., J Proteome Res.2006, 5, 2194-2206. Briefly, sliced tissues were washed in PBS to remove blood and other contaminants.
  • FIG.15(A) shows result of Western Blotting on PA2G4 in Cancer sample and Normal sample using Anti-actin as a reference.
  • Each band in FIG. 15(A) is measured as an integrated value in absorbance unit ahd calculated by PDQuest software.
  • FIG.15(B) shows the resulting integrated value in absorbance unit of the band of PA2G4 respectively in the Cancer sample and Normal sample.
  • Both In FIG.15(A) and (B) 1 ER+ represents Cancer sample and TNT represents Normal sample.
  • the vertical axis in FIG.15(B) represents the integrated value in absorbance unit of the band. From the FIGs, breast cancer sample clearly shows over expression in comparison with normal sample. Similar up regulations in breast cancer tissue are also observed in individual sample and cell lines (Data not shown).

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Abstract

Cette invention concerne des biomolécules, et en particulier une méthode permettant de détecter chez un sujet la présence et/ou la gravité de troubles cellulaires prolifératifs ainsi qu'un prédisposition à de tels troubles. Cette invention concerne également une méthode de surveillance de l'efficacité d'un traitement contre des troubles cellulaires prolifératifs chez un sujet.
PCT/JP2007/064288 2006-07-13 2007-07-11 Biomolécules de diagnostic WO2008007807A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2548888A1 (fr) * 2010-03-17 2013-01-23 Kagoshima University Peptide spécifique d'une pathologie parodontale et traitement et diagnostic d'une pathologie parodontale l'incluant
EP2548888A4 (fr) * 2010-03-17 2013-07-31 Univ Kagoshima Peptide spécifique d'une pathologie parodontale et traitement et diagnostic d'une pathologie parodontale l'incluant
US10627412B2 (en) 2010-03-17 2020-04-21 Kagoshima University Periodontal-disease-specific peptide, and treatment and diagnosis of periodontal disease using same

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