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WO2006039671A9 - Marqueurs de genes de metastase tumorale - Google Patents

Marqueurs de genes de metastase tumorale

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Publication number
WO2006039671A9
WO2006039671A9 PCT/US2005/035539 US2005035539W WO2006039671A9 WO 2006039671 A9 WO2006039671 A9 WO 2006039671A9 US 2005035539 W US2005035539 W US 2005035539W WO 2006039671 A9 WO2006039671 A9 WO 2006039671A9
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WIPO (PCT)
Prior art keywords
protein
factor
receptor
antigen
mucin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2005/035539
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English (en)
Other versions
WO2006039671A2 (fr
WO2006039671A3 (fr
Inventor
Masato Nakamura
Yasuyuki Ohnishi
Hiroshi Suemizu
Makoto Monnai
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Central Institute for Experimental Animals
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Central Institute for Experimental Animals
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Priority claimed from US10/955,192 external-priority patent/US20050249666A1/en
Priority claimed from US10/954,495 external-priority patent/US20070092881A1/en
Application filed by Central Institute for Experimental Animals filed Critical Central Institute for Experimental Animals
Publication of WO2006039671A2 publication Critical patent/WO2006039671A2/fr
Publication of WO2006039671A9 publication Critical patent/WO2006039671A9/fr
Publication of WO2006039671A3 publication Critical patent/WO2006039671A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • 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

Definitions

  • the present invention relates to the identification of genes associated with tumor metastasis, using a transgenic mouse model, hi particular, the invention concerns establishment and testing of human cancer cell lines with high metastatic potential in NOD/SClD/ ⁇ cnull (NOG) mice, and the identification of genes selectively expressed in strongly metastatic cancer cells.
  • NOG NOD/SClD/ ⁇ cnull
  • mice such as athymic nude mice, C.B-17/severe combined immunodeficiency (scid) mice and NOD/SCID mice have been widely used as animal models in cancer metastasis research (Bruns et al, Int. J. Cancer 10:102(2):101-8 (2002); Ohta et al, Jpn. J. Cancer Chemother. 23:1669-72 (1996); Jimenez et al, Ann. Surg. 231 :644-54 (2000)).
  • NOD/SCID/y c n ull also referred to as NOD/ShiJic-scid with ⁇ c null , or NOG
  • NOG transgenic mice have been described as an excellent recipient mouse model for engraftment of human cells (Ito et al, Blood 100:3175-82 (2002)), and for the study of the in vivo development of human T cells from CD34(+) cells (Saito et al, Int. Immunol. 14:1113-24 (2002)).
  • CBSC human cord blood stem cells
  • Metastasis including hepatic metastasis, is often observed in human cancer, including pancreatic cancer even in early stage, cancers of the digestive tract, including colorectal cancer and gastrointestinal cancer, lung cancer, and the like, and is one of the most frequent causes of cancer deaths.
  • New strategies are necessary to manage cancer metastases, which, in turn, require the availability of appropriate gene markers of metastasis, including, but not limited to, metastasis in the liver.
  • the present invention concerns identification of genes that are selectively expressed in tumor cells with high tendency to metastasize relative to tumor cells with low metastatic potential.
  • the invention concerns tumor markers, in particular, gene markers of metastatic cancer, such as metastatic pancreatic cancer.
  • gene markers are selected from the group consisting of TISl IB protein; prostate differentiation factor (PDF); glycoproteins hormone ⁇ -subunit; thrombopoietin (THPO); manic fringe homology (MFNG); complement component 5 (C5); jagged homolog 1 (JAGl); interleukin enhancer-binding factor (ILF); PCAF-associated factor 65 alpha; interleukin-12 ⁇ -subunit (IL-12- ⁇ ); nuclear respiratory factor 1 (NRFl); stem cell factor (SCF); transcription factor repressor protein (PRDI-BFl); small inducible cytokine subfamily A member 1 (SCYAl).
  • transducin ⁇ 2 subunit X-ray repair complementing defective repair -in Chinese hamster cells 1; putative renal organic anion transporter 1; Gl/S-specif ⁇ c cyclin E (CCNE); retinoic acid receptor- ⁇ (RARG); S-100 calcium-binding protein Al; neutral amino acid transporter A (SATT); dopachrome tautomerase; ets transcription factor (NERF2); calcium- activated potassium channel ⁇ -subunit; CD27BP; keratin 10; 6-O-methylguanine-DNA- methyltransferase (MGMT); xeroderma pigmentosum group A complementing protein (XPA); CDC6-related protein; cell division protein kinase 4; nociceptin receptor; cytochrome P450 XXVIIBl; N-myc proto-oncogene; solute carrier family member 1 (SLC2A1); membrane- associated kinase mytl; casper, a FADD- and caspase-related
  • the gene markers are selected from the group consisting of TISl IB protein; prostate differentiation factor (PDF); glycoproteins hormone ⁇ -subunit; thrombopoietin (THPO); manic fringe homology (MFNG); complement component 5 (C5); jagged homolog 1 (JAGl); interleukin enhancer-binding factor (ILF); PCAF-associated factor 65 alpha; interleukin-12 ⁇ -subunit (IL-12- ⁇ ); nuclear respiratory factor 1 (NRFl); stem cell factor
  • SCF transcription factor repressor protein
  • PRDI-BFl transcription factor repressor protein
  • SCYAl small inducible cytokine subfamily A member 1
  • the gene markers are selected from the group consisting of transducin ⁇ 2 subunit; X-ray repair complementing defective repair in Chinese hamster cells 1; putative renal organic anion transporter 1; Gl/S-specific cyclin E (CCNE); retinoic acid receptor- ⁇ (RARG); S-100 calcium-binding protein Al; neutral amino acid transporter A (SATT); dopachrome tautomerase; ets transcription factor (NERF2); calcium-activated potassium channel ⁇ -subunit; CD27BP; keratin 10; 6-O-methylguanine-DNA-methyltransferase (MGMT); xeroderma pigmentosum group A complementing protein (XPA); CDC6-related protein; cell division protein kinase 4; nociceptin receptor; cytochrome P450 XXVIIBl; N-myc proto-oncogene; solute carrier family member 1 (SLC2A1); membrane-associated kinase mytl; casper,
  • the gene markers are selected from the group consisting of interleukin 1 receptor-like 1, parathyroid hormone-like hormone, parathyroid hormone- like peptide, regulator of G-protein signaling 4, gap junction protein beta 6, neuregulin 1 isoform SMDF, fungal sterol-C5-desaturase homolog, G protein-coupled receptor, METHl protein (ADAMTSl), METHl protein (near ADAMTS 15), BH-protocadherin (brain-heart), upregulated by 1,25-dihydroxyvitamin D-3, lipocalin 2 (oncogene 24p3) (LCN2), argininosuccinate synthetase (ASS), extrecellular matrix protein 1 (ECMl), S100 calcium-binding protein A4 (S100Z4), solute carrier family 6 (neurotransmitter transporter) meml, serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), tissue plasminogen
  • the array displays at least 2, or at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30, or at least 35, or at least 40, or at least 45, or at least 50, or at least 55, or at least 60, etc. of the listed genes, or their expression products.
  • all genes that are overexpressed in tumor metastasis, or their expression products are displayed.
  • all genes that are under-expressed in tumor metastasis, or their expression product are displayed.
  • the invention concerns a method for predicting the likelihood of tumor metastasis in a patient, comprising determining the expression level of the RNA transcript of one or more genes selected from the group consisting of TISl IB protein; prostate differentiation factor (PDF); glycoproteins hormone ⁇ -subunit; thrombopoietin (THPO); manic fringe homology (MFNG); complement component 5 (C5); jagged homolog 1 (JAGl); interleukin enhancer-binding factor (ILF); PCAF-associated factor 65 alpha; interleukin-12 oc- subunit (IL-12- ⁇ ); nuclear respiratory factor 1 (NRFl); stem cell factor (SCF); transcription factor repressor protein (PRDI-BFl); small inducible cytokine subfamily A member 1 (SCYAl), transducin ⁇ 2 subunit; X-ray repair complementing defective repair in Chinese hamster cells 1 ; putative renal organic anion transporter 1; Gl/S-specific cyclin E (CCNE);
  • SATT dopachrome tautomerase
  • NERF2 ets transcription factor
  • CD27BP calcium-activated potassium channel ⁇ -subunit
  • keratin 10 6-O-methylguanine-DNA-methyltransferase
  • MGMT xeroderma pigmentosum group A complementing protein
  • XPA xeroderma pigmentosum group A complementing protein
  • SLC2A1 solute carrier family member 1
  • membrane-associated kinase mytl casper, a FADD- and caspase-related inducer of apoptosis; and C-src proto-oncogene, or their expression products, in a test biological sample comprising cancer cells obtained from said patient, relative to a reference biological sample, wherein differential expression is indicative of an increased likelihood of tumor metastasis.
  • the invention concerns a method for predicting the likelihood of tumor metastasis in a patient, comprising determining the expression level of the RNA transcript of one or more genes selected from the group consisting of interleukin 1 receptor- like 1, parathyroid hormone-like hormone, parathyroid hormone-like peptide, regulator of G-protein signaling 4, gap junction protein beta 6, neuregulin 1 isoform SMDF, fungal sterol-C5- desaturase homolog, G protein-coupled receptor, METHl protein (ADAMTSl), METHl protein (near ADAMTS 15), BH-protocadherin (brain-heart), upregulated by 1,25-dihydroxyvitamin D-3, lipocalin 2 (oncogene 24p3) (LCN2), argininosuccinate synthetase (ASS), extrecellular matrix protein 1 (ECMl), S100 calcium-binding protein A4 (S100Z4), solute carrier family 6 (neurotransmitter transporter)
  • the invention concerns screening assay, comprising
  • the candidate molecule is identified as an anticancer agent, if (i) it is capable of suppressing the expression of one or more gene selected from the group consisting of TIS1 1B protein; prostate differentiation factor (PDF); glycoproteins hormone ⁇ -subunit; thrombopoietin (THPO); manic fringe homology (MFNG); complement component 5 (C5); jagged homolog 1 (JAGl); interleukin enhancer-binding factor (ILF); PCAF-associated factor 65 alpha; interleukin- 12 ⁇ -subunit (IL-12- ⁇ ); nuclear respiratory factor 1 (NRFl); stem cell factor (SCF); transcription factor repressor protein (PRDI-BFl); and small inducible cytokine subfamily A member 1 (SCYAl), or if (ii) it is capable of increasing the expression of one or more genes selected from the group consisting of transducin ⁇ 2 subunit; X-ray repair complement
  • the invention concerns a screening assay, comprising
  • a candidate molecule to a tumor cell expressing one or more gene selected from the group consisting of interleukin 1 receptor-like 1, parathyroid hormone- like hormone, parathyroid hormone-like peptide, regulator of G-protein signaling 4, gap junction protein beta 6, neuregulin 1 isoform SMDF, fungal sterol-C5-desaturase homolog, G protein-coupled receptor, METHl protein (ADAMTSl), METHl protein (near ADAMTS 15), BH-protocadherin (brain-heart), upregulated by 1,25-dihydroxyvitamin D-3, lipocalin 2 (oncogene 24p3) (LCN2), argininosuccinate synthetase (ASS), extrecellular matrix protein 1 (ECMl), S100 calcium-binding protein A4 (S100Z4), solute carrier family 6 (neurotransmitter transporter) meml, serine (or cysteine) proteinase inhibitor, clade B (
  • the candidate molecule is identified as an anticancer agent, if (i) it is capable of suppressing the expression of one or more gene selected from the group consisting of upregulated by 1,25-dihydroxyvitamin D-3, lipocalin 2 (oncogene 24p3) (LCN2), argininosuccinate synthetase (ASS), extrecellular matrix protein 1 (ECMl), SlOO calcium- binding protein A4 (S100Z4), solute carrier family 6 (neurotransmitter transporter) meml, serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), tissue plasminogen activator (PLAT), EST DKFZp666M1410, ClOO calcium-binding protein A8 (calgranulin A) (S100A8), EST (MGC: 10500), placenta-specific 8 (PLAC8), interferon-inducible
  • the invention concerns a screening assay, comprising determining the ability of a candidate molecule to modulate the expression of one or more gene selected from the group consisting of TISl IB protein; prostate differentiation factor (PDF); glycoproteins hormone ⁇ -subunit; thrombopoietin (THPO); manic fringe homology (MFNG); complement component 5 (C5); jagged homolog 1 (JAGl); interleukin enhancer-binding factor (ILF); PCAF-associated factor 65 alpha; interleukin- 12 ⁇ -subunit (IL- 12- ⁇ ); nuclear respiratory factor 1 (NRFl); stem cell factor (SCF); transcription factor repressor protein (PRDI-BFl); small inducible cytokine subfamily A member 1 (SCYAl), transducin ⁇ 2 subunit; X-ray repair complementing defective repair in Chinese hamster cells 1 ; putative renal organic anion transporter 1; Gl/S-specific cyclin E (CCNE); retinoic acid receptor- ⁇ (RA), IAA,
  • the invention further concerns a screening assay, comprising determining the ability of a candidate molecule to modulate the expression of one or more gene selected from the group consisting of interleukin 1 receptor-like 1, parathyroid hormone-like hormone, parathyroid hormone-like peptide, regulator of G-protein signaling 4, gap junction protein beta 6, neuregulin 1 isoform SMDF, fungal sterol-C5-desaturase homolog, G protein-coupled receptor, METHl protein (ADAMTSl), METHl protein (near ADAMTS 15), BH-protocadherin (brain-heart), upregulated by 1,25-dihydroxyvitamin D-3, lipocalin 2 (oncogene 24p3) (LCN2), argininosuccinate synthetase (ASS), extracellular matrix protein 1 (ECMl), S100 calcium- binding protein A4 (S100Z4), solute carrier family 6 (neurotransmitter transporter) meml, serine (or cysteine) protein
  • Figures IA and B illustrate the incidences of hepatic metastasis and the number of liver foci in NOG mice following the inoculation of 1x10 4 , 1x10 3 and 1x10 2 cells of the indicated pancreatic adenocarcinoma cells lines (MIAPaCa-2, AsPC-I, PANC-I, Capan-1, and BxPC-3.
  • Figure 4. Microscopic view of the cell lines BxPC-3 and BxPC-3LMl in vitro.
  • Figure 5. Overview of establishing and testing the cell line BxPC-3LMl .
  • Figure 6 is a scatter graph of microarray analysis of the BxPC-3LMl and BxPC-3 cell lines. Signal intensity of each spot of gene chip microarray (gene expression) was plotted. The genes in the blue bottom area was omitted from further analysis due to low intensity. The genes in the red are were subjected to further analysis.
  • Figure 7. To confirm the results of gene expression analysis, some genes which showed significant difference in their expression levels in BxPC-3 and BxPC-3LMl were subjected to amplification by RT-PCR. The Figure shows the results of the RT-PCR analysis after varying numbers of amplification cycles.
  • Table 1 Hepatic metastasis after intrasplenic injection of various human pancreatic adenocarcinoma cell lines.
  • Table 2 Genes differentially expressed in cell lines with high metastasis potential relative to cell lines with low metastatic potential.
  • BxPC-3 and BxPC-3LMl cell lines selected by microarray analysis.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • cancer examples include but are not limited to, carcinoma (epithelial), such as, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, gastrointestinal cancer, colon cancer, lung cancer, hepatocellular cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, melanoma, and brain cancer; and sarcoma (non-epithelial), such as, liposarcomas, leiomyosarcomas, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral nerve tumor, gastrointestinal stromal tumor, desmoid tumor, Ewing's sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma and myeloma.
  • carcinoma epidermatitisarcoma
  • prostate cancer such as, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, gastrointestinal
  • metastasis is used herein in the broadest sense and refers to the spread of tumor, e.g. cancer from one part of the body to another. Tumors formed from cells that have spread are called secondary tumors, and contain the same type of cells as the original (primary) tumor.
  • prostate cancer that has metastasized to liver or bone is not liver or bone cancer, rather metastasized prostate cancer, as it still contains prostate cancer cells, regardless of their location.
  • the "pathology" of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.
  • differential gene refers to a gene whose expression is at a higher or lower level in one cell or cell type relative to another, or one patient or test subject relative to another.
  • differential gene expression can occur in normal cell/tissue/patient relative to a corresponding diseased cell/tissue/patient, or can reflect differences is gene expression pattern between different cell types or cells in different stages of development.
  • the terms also include genes whose expression is activated to a higher or lower level at different stages of the same disease.
  • a differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alternative splicing to result in a different polypeptide product. Such differences may, for example, be evidenced by a change in mRNA levels, surface expression, or secretion or other partitioning of a polypeptide.
  • Differential gene expression may include a comparison of expression between two or more genes or their gene products, or a comparison of the ratios of the expression between two or more genes or their gene products, or a comparison of two differently processed products of the same gene.
  • “differential gene expression” is considered to be present when there is at least an about 2-fold, preferably at least about 2.5-fold, more preferably at least about 4-fold, even more preferably at least about 6-fold, most preferably at least about 10-fold difference between the expression of a given gene or gene product between the samples compared.
  • the "reference biological sample” can, for example, be a normal sample, or a sample of a tumor of the same type, having low metastatic potential.
  • microarray refers to an ordered arrangement of hybridizable array elements on a substrate. The term specifically includes polynucleotide microarrays, such as cDNA and oligonucleotide microarrays, and protein arrays.
  • a microarray is an array of thousands of individual gene (DNA) sequences immobilized in a known order on a solid support. RNAs from different tissues are hybridized to the DNA on the chips. An RNA molecule will only bind to the DNA from which it was expressed.
  • DNA gene
  • RNAs from different tissues are hybridized to the DNA on the chips.
  • An RNA molecule will only bind to the DNA from which it was expressed.
  • polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double- stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions, hi addition, the term "polynucleotide” as used herein includes triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritiated bases are included within the term "polynucleotides” as defined herein.
  • polynucleotide embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.
  • oligonucleotide refers to a relatively short polynucleotide, including, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs.
  • transgenic animal and “transgenic mouse” as well we their grammatical equivalents, are used to refer to animals/mice deliberately produced to carry a gene from another animal.
  • xenotransplantation is used in the broadest sense and refers to the transfer of living cells, tissues or organs from one animal species into another, including humans.
  • the present invention provides a sensitive and reliable transgenic animal model for the study of tumor metastasis.
  • the present invention provides a reproducible mouse model of hepatic metastasis, which involves the introduction of mammalian (e.g. human) cancer cells into NOG mice.
  • NOG mice were developed at the Central Institute for Experimental Animals (CIEA, Kawasaki, Japan), and are also described in co-pending U.S. application Serial No. 10/221,549 filed on October 25, 2001, and in PCT Publication No. WO 03/0182671, the entire disclosures of which are hereby expressly incorporated by reference.
  • NOD/SCID/ ⁇ c null mice double homozygous for the severe combined immunodeficiency (SCID) mutation and interleukin-2R ⁇ (IL-2R ⁇ ) allelic mutation ( ⁇ c null ) were generated by 8 backcross matings of C57BL/6J- ⁇ c null mice and NOD/Shi-scid mice.
  • SCID severe combined immunodeficiency
  • IL-2R ⁇ interleukin-2R ⁇ allelic mutation
  • NOD/SCID/ ⁇ c n ull mice are described in Schultz et al, J. ImmonoL, 174:6477-6489 (2005).
  • the NOD-scidIL2R ⁇ null mice described in the Schultz et al. paper are specifically included within the term "NOG" mice, as used herein.
  • NOG mice are a superior mouse model for the study of human cancer metastasis.
  • this model can be used, for example, to screen and evaluate anti-cancer drugs and anti-metastasis drug candidates, and for the detection/screening of genes related to cancer metastasis, which, in turn, find utility in the diagnosis and/or treatment of metastatic cancer, and related conditions, including gene therapy treatment of metastatic cancer.
  • the mouse model of the present invention is suitable for modeling and studying any kind of metastasis, including hepatic, bone, brain, and lung metastasis. Metastasis occurs in all types of cancers, including, without limitation, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, gastrointestinal cancer, colon cancer, lung cancer, hepatocellular cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, and brain cancer. Although the invention will be illustrated by analyzing hepatic metastasis of human pancreatic cancer, it is not so limited.
  • the NOG mouse model can also be used to study metastases originating from other types of cancer at any location, including liver, bone, brain and liver.
  • mammalian tumor specimens preferably human tumor specimens
  • the tumor specimens may be obtained by any method known in the art.
  • the tumor specimens are surgically resected, such as in a biopsy or in the process of surgery to remove the tumor from the mammal.
  • the tumor specimen is obtained by purifying circulating tumor cells from the mammals blood.
  • cancer cells are transplanted into mice via tail vein injection, with or without prior immune-suppression, such as a sublethal dose of whole body irradiation and/or the administration of an immunosuppressant.
  • the cancer cells may be introduced into the animals by intrasplenic (portal vein) injection using an appropriate indwelling catheter.
  • Pulmonary metastasis can be established, for example, by intravenous injection of tumor cells into the recipient animals, for example as described in Worth and
  • the tumor cells may originate from tumor (cancer) cell lines, and from primary tumors (e.g. cancer) obtained from human or non-human subjects.
  • tumor metastasis macroscopic fragments of human fetal bone or mouse bone, may be implanted into NOG mice.
  • human tumor (cancer) cell lines or cells of primary tumors (cancer) can be injected either intravenously (colonization assay), or directly into the implanted tissue fragments.
  • Tumor metastasis can be monitored by methods known in the art, including various imaging techniques and histologic examination.
  • Human xenografts are considered highly predictive of tumor behavior within the donor patient, as the xenograft grows as a solid tumor, differentiates, and develops a stroma, vasculature, and a central necrosis. In most cases, xenografts retain most of the molecular, histological, and pathophysiological characteristics of the fresh patient-derived tumor.
  • the NOG mice that have developed metastatic cancer can be treated with the test compound(s), and any change in the number, size or other properties of the metastatic nodules as a result of drug treatment, and the viability of the test animals are monitored relative to untreated and/or positive control, where the positive control typically is an animal treated with a know anti-metastatic compound.
  • the administration of the test compounds can be performed by any suitable route, including, for example, oral, transdermal, intravenous, infusion, intramuscular, etc. administration. Results obtained in this model can then be validated by follow-up pharmacokinetic, toxicologic, biochemical and immunologic studies, and ultimately human clinical studies.
  • the NOG mouse model can also be used to study targeted gene delivery to metastatic nodules in vivo, for example by portal vein infusion of a retroviral vector, hi particular, this NOG model can be used to study the feasibility of gene transfer to target tumor metastasis, to monitor the duration and level of gene expression and the degree of therapeutic effect, to optimize the dosing regimen and/or mode of administration, to study the dissemination of the gene transfer vector to non-targeted tissues (which provides information about potential toxicity), and the like.
  • Gene delivery most commonly is performed using retroviral vectors by techniques well known in the art. Retroviruses are enveloped viruses containing a single stranded RNA molecule as their genome.
  • the viral genome is reverse transcribed into double stranded DNA, which integrates into the host genome where it is expressed.
  • the viral genome contains at least three genes: gag (coding for core proteins), pol (coding for reverse transcriptase) and env (coding for the viral envelope protein).
  • LTRs long terminal repeats
  • Retroviral vectors used in mouse models are most frequently based upon the Moloney murine leukemia virus (Mo-MLV).
  • lentiviruses can, for example, be used for gene transfer into experimental animals, such as NOG mice.
  • Gene delivery can also be performed by adenoviral vectors.
  • Adenoviruses are non- enveloped, icosahedral viruses with linear double-stranded DNA genomes. Adenoviruses infect non-dividing cells by interacting with cell surface receptors, and enter cells by endocytosis. Since the genome of adenoviruses cannot integrate with the host cell genome, the expression from adenoviral vectors is transient.
  • NOG mice were used to study differential gene expression between cells exhibiting high tendency to metastasize and cells with low metastatic potential.
  • gene expression patterns in highly metastatic cell lines and cell lines with low metastatic potential were compared, and differentially expressed genes were identified.
  • Methods of gene expression profiling are well known in the art and include methods based on hybridization analysis of polynucleotides, methods based on sequencing of polynucleotides, and proteomics-based methods.
  • the most commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999));
  • RNAse protection assays Hod, Biotechniques 13:852-854 (1992)
  • PCR-based methods such as reverse transcription polymerase chain reaction (RT-PCR)
  • RT-PCR reverse transcription polymerase chain reaction
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
  • Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).
  • SAGE Serial Analysis of Gene Expression
  • MPSS massively parallel signature sequencing
  • One of the most sensitive and most flexible quantitative PCR-based gene expression profiling methods is RT-PCR, which can be used to compare mRNA levels in different sample populations, including various tumor cells or tissues, to characterize patterns of gene expression.
  • RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction.
  • the two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT).
  • AMV-RT avilo myeloblastosis virus reverse transcriptase
  • MMLV-RT Moloney murine leukemia virus reverse transcriptase
  • the reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling.
  • extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA), following the manufacturer's instructions.
  • the derived cDNA can then be used as a template in the subsequent PCR reaction.
  • the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5 '-3' nuclease activity but lacks a 3 '-5' proofreading endonuclease activity.
  • TaqMan® PCR typically utilizes the 5 '-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used.
  • Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction.
  • a third oligonucleotide, or probe is designed to detect nucleotide sequence located between the two PCR primers.
  • the probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe.
  • the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner.
  • the resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore.
  • One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
  • TaqMan® RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700TM Sequence Detection SystemTM (Perkin-Elmer-Applied Biosystems, Foster City, CA, USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany).
  • a more recent variation of the RT-PCR technique is the real time quantitative PCR, which measures PCR product accumulation through a dual-labeled fluorigenic probe (i.e., TaqMan® probe), and is described in Held et al, Genome Research 6:986-994 (1996).
  • Differential gene expression can also be identified, or confirmed using the microarray technique.
  • polynucleotide sequences of interest including cDNAs and oligonucleotides
  • the arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest.
  • the source of mRNA typically is total RNA isolated from human tumors or tumor cell lines, such as tumor cell lines with low and high metastatic potential.
  • microarray methods for large-scale analysis of gene expression makes it possible to search systematically for molecular markers of cancer classification (such as tumors with different metastatic potentials) and outcome prediction in a variety of tumor types.
  • differential gene expression between tumors with low and high metastatic potential can be determined or confirmed at the protein level, using proteomics techniques.
  • Proteomics techniques typically include the following steps: (1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, e.g. my mass spectrometry or N-terminal sequencing, and (3) analysis of the data using bioinformatics.
  • Proteomics methods are valuable supplements to other methods of gene expression profiling, and can be used, alone or in combination with other methods, to detect the products of the metastasis markers of the present invention.
  • tumor markers have been identified by RT-PCR, as described in the Examples, and are listed in Tables 2 and 6 below.
  • the genes over-expressed or under-expressed in tumor, such as metastatic tumor are useful diagnostic and prognostic markers of cancer, including metastatic cancer, and targets for therapeutic intervention to treat cancer, including metastatic cancer.
  • cancer can be diagnosed by detecting a differentially expressed gene or gene product by methods known in the art, such as, for example, by assays based on nucleic acid hybridization, or by using antibodies to the target gene products in various binding assays, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases (Zola, Monoclonal Antibodies:
  • the antibodies used in the diagnostic assays can be labeled with a detectable moiety.
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or 125 I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al.,
  • the differentially expressed genes or gene products can also be used for screening drug candidates for the treatment of cancer, including metastatic cancer.
  • the screened drug candidates can be peptides, polypeptides, non-peptide small organic or inorganic molecules, antibodies, including antibody fragments, antisense molecules, oligonucleotide decoy molecules, and the like, hi certain embodiments, the screening assays can be performed in a microarray format, which facilitates high throughput screening.
  • Example 1 Example 1
  • DMEM fetal bovine serum
  • MIAPaCa-2 and PANC-1 were maintained a culture of DMEM supplemented with 10% FBS.
  • BxPC-3, Capan-2 and PL45 were maintained a culture of RPMI1640 (SIGMA, Cat.No.D6046 or D5796) supplemented with 10% FBS. These were maintained at 37oC in humidified atmosphere with 5% CO2.
  • Experimental liver metastases were generated by intrasplenic/portal injection of cancer cells, as described previously (Khatib et al, Cancer Res. 62:242-50 (2002)). The animals were sacrificed 6-8 weeks later and liver metastases were enumerated immediately, without prior fixation.
  • metastases were apparent in 50-80% of NOG mice when IxIO 3 MIAPaCa-2,
  • NOG mic represent an effective cancer metastasis model, which properly reflects the clinical conditions and behavior of human pancreatic cancer. Accordingly, the well- organized and reproducible hepatic metastases seen in NOG mice are useful in the study of hepatic metastasis of human pancreatic cancer and are expected to become the preferred model for screening and developing new anti-metastasis drugs.
  • the data presented demonstrate that the NOD/SCID/ ⁇ c null mouse model has a high potential to engraft xenogenic cells.
  • this model for intrasplenic (portal vein) injection of cancer cells, reliable hepatic metastasis behavior of human pancreatic cells was observed.
  • Four out of seven cell lines showed high hepatic metastatic potential (>80% incidence), and three of the cell lines studied showed low metastatic potential ( ⁇ 20% incidence) in NOG mice 6 weeks after transplantation only with 1 x 10 4 cells.
  • hepatic metastases were apparent in NOG mice even when 1 x 10 2 cells of high metastatic cell lines were inoculated.
  • the Cy-3 labeled signals were detected and obtained and analyzed the corresponding images by a GM418 array scanner (Takara). The data processing was carried out using Imagene Version 5.5 software.
  • MIAPaCa-2 and Panel cell lines were classified into a highly metastatic group, while the other cell lines, Capan2 and PL45, were classified into a non-metastatic group.
  • the average of the signal values from the "highly metastatic group” array was divided by the average of the signal values from the "non-metastatic group” array. The resulting values are referred to as "gene expression levels", where a 10-fold difference and higher values were considered significant.
  • TISl IB protein prostate differentiation factor
  • PDF glycoproteins hormone ⁇ -subunit
  • thrombopoietin THPO
  • MFNG manic fringe homology
  • C5 complement component 5
  • JAGl interleukin enhancer- binding factor
  • INF PCAF-associated factor 65 alpha
  • NRFl nuclear respiratory factor 1
  • SCF stem cell factor
  • PRDI-BFl transcription factor repressor protein
  • SCYAl small inducible cytokine subfamily A member 1
  • transducin ⁇ 2 subunit X-ray repair complementing defective repair in Chinese hamster cells 1; putative renal organic anion transporter 1; Gl/S-specific cyclin E (CCNE); retinoic acid receptor- ⁇ (RARG); S-100 calcium- binding protein Al; neutral amino acid transporter A (SATT); dopachrome tautomerase; ets transcription factor (NERF2); calcium-activated potassium channel ⁇ -subunit; CD27BP; keratin 10; 6-O-methylguanine-DNA-methyltransferase (MGMT); xeroderma pigmentosum group A complementing protein (XPA); CDC6-related protein; cell division protein kinase 4; nociceptin receptor; cytochrome P450 XXVIIBl; N-myc proto-oncogene; solute carrier family member 1 (SLC2A1); membrane-associated kinase
  • Example 3 Establishment of a cell line with high metastatic potential
  • Example 1 describes the establishment of a hepatic metastatic panel using human pancreatic cancer cells xeno-transplanted into NOG mice. Using this panel, the metastatic potentials of several cell lines have been characterized as shown in Table 1. One of the cell lines characterized is BxPC-3. After intrasplenic injection of this cell line into NOG mice only one in eight mice developed hepatic metastasis, i.e. the metastatic potential of this cell line was only 12.5%.
  • the present Example describes the development of a cell line with high metastatic potential from BxPC-3.
  • BxPC-3 (1 x 10 5 cells) was injected into the spleens of NOG mice. This is barely the amount needed to induce metastasis. After 6-8 weeks, the mice were sacrificed and the livers with a few metastatic foci were harvested. A single cell suspension was prepared by mincing and enzymatic dissociation, and were then cultured in vitro for 4 weeks. The cells in this culture were designated BxPC-3LMl, and the procedure is illustrated in Figures 2 and 5. Results
  • Table 3 compares the metastatic potentials of the original cell line BxPC-3 and the sub-line BxPC-3LMl in the liver of the NOG mouse.
  • Table 4 compares the metastatic potentials of the original cell line BxPC-3 and the subline BxPC-3LMl in the liver of the NOD/SCID mouse.
  • 1 x 10 4 cells of BxPC-3 resulted in no metastasis in any of the six mice tested, while BxPC-3MLl resulted in weak metastasis in 3 of the 5 mice tested (60% incidence).
  • the injection of 1 x 10 cells of BxPC-3 still resulted in no metastasis in the six mice tested in this experiment, while in case of BxPC-3LMl all mice tested developed strong metastases.
  • the difference between the metastatic potential of BxPC-3 and BxPC-3LMl is remarkable, since the latter was obtained by a single transplantation of the BxPC-3 cells in NOG mice.
  • BxPC-3 and BxPC-3LMl were similar in their cell number doubling time, in the presence of micro-satellite markers (STR), and ras and p53 mutational status. Accordingly, in its key characteristics, BxPC-3LMl showed no difference relative to the parental cell line.
  • the gene expression profiles of BxPC-3 and BxPC-3LMl were then compared by microarray analysis (GeneChip Human Genome Ul 33 Plus 2.0 Array, Affymetrix), essentially following the procedure described in Example 2. Results

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Abstract

L'invention concerne l'étude d'une métastase tumorale dans un modèle de souris transgénique NOD/SCID/?cnull ainsi que l'identification de marqueurs de tumeurs, y compris de marqueurs de tumeurs présentant un potentiel métastatique élevé, au moyen d'informations obtenues dans ce modèle de souris.
PCT/US2005/035539 2004-09-29 2005-09-29 Marqueurs de genes de metastase tumorale Ceased WO2006039671A2 (fr)

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