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WO1997013778A1 - Gene suppresseur de metastases - Google Patents

Gene suppresseur de metastases Download PDF

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Publication number
WO1997013778A1
WO1997013778A1 PCT/US1996/016414 US9616414W WO9713778A1 WO 1997013778 A1 WO1997013778 A1 WO 1997013778A1 US 9616414 W US9616414 W US 9616414W WO 9713778 A1 WO9713778 A1 WO 9713778A1
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Danny Ray Welch
Jeong-Hyung Lee
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The Penn State Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • a cDNA molecule from the KiSS-l gene has a novel nucleotide sequence and encodes a protein that suppresses metastasis in melanoma cancers.
  • the gene is useful as a marker to distinguish metastatic from non-metastatic cells, a characteristic forming the basis of diagnostic tests, and to provide criteria for disease staging, and planning therapeutic strategies.
  • the KiSS-l nucleotide sequence is a candidate for use in gene therapy, and pharmaceutical compositions include the gene product of KiSS-l .
  • Metastasis the spread of tumor cells from a primary tumor to form discontinuous tumors at nearby or distant sites is the major life-threatening complication to cancer cure. Metastasis is responsible for the majority of cancer-related deaths. In order to improve treatments and in order to more accurately diagnose and stage cancer, a greater understanding of the metastatic process at the molecular level is required. New modalities of cancer therapy are needed particularly to treat metastatic cancer. However, dissecting the specific genetic events controlling metastasis has been complicated by three factors. First, metastasis involves multiple steps, suggesting involvement of multiple genes (Liotta, 1992; Nicolson, 1993; Radinsky, 1991) .
  • metastatic cells are highly aneuploid; therefore, identifying structurally altered genes or abnormally functioning genes over background genomic instability in tumors has proven extremely difficult.
  • the ability of assays to identify metastasis-associated genes has been hampered by tumor heterogeneity and technical limitations. Nonetheless, the current paradigm of human tumor progression proposes that a finite number of nonrandom chromosomal and genetic alterations are responsible for each stage of neoplastic evolution
  • metastasis-regulatory genes remain elusive.
  • Metastasis is known to be controlled at two levels.
  • the latter types of genes are termed "metastasis-suppressor genes.”
  • Neuroectodermal cancers such as neuroblastoma, small cell lung carcinoma, and cutaneous and ocular malignant melanomas are examples of cancers for which metastasis is a serious problem.
  • Melanoma currently accounts for only 1-5% of all cancers, but its incidence has increased more than 4% annually since 1970 (Armstrong, 1994; Koh, 1991) .
  • the major complication preventing cure of melanoma is metastasis (Frost, 1992) .
  • Neoplastic progression toward malignancy of cutaneous melanoma can be described by relatively discrete stages - melanocyte tumorigenesis precedes formation of benign nevi (atypical moles) which presumably become fully tumorigenic before expanding radially (radial growth phase) then invading vertically (vertical growth phase) into the skin. Some cells then acquire the ability to metastasize. Despite some defined criteria, it is still not always possible to correctly stage and predict a primary tumor's propensity to metastasize. Less subjective criteria are needed.
  • chromosome 9 is the multiple tumor suppressor gene, MTSl, which encodes for the cyclin-dependent kinase CDKN2 on chromosome 9 (Kamb, 1994; Parker, 1994; Skolnick, 1994; Hussussian, 1994) .
  • MMCT Microcell-mediated chromosome transfer
  • SOD2 (which maps to 6q25 (Church, 1992) and encodes manganese superoxide dismutase) is thought to be a melanoma tumor-suppressor since transfection into the UACC-903 blocked tumor formation (Church, 1993) .
  • C8161 and all neo6/C8161 hybrids retain S0D2 and express MnSOD; therefore, MnSOD is not apparently responsible for tumor suppression in this model (Welch, 1994) .
  • transfection of SOD2 into C8161 clones does not suppress tumorigenicity or metastasis (Miele and Welch, 1995) .
  • Metastatic suppressor genes Genes expressed in nonmetastatic cells, but not in their metastatic counterparts, are termed "metastasis suppressor genes.” Further support for this classification is obtained from the ability of a particular gene or gene product to suppress metastasis in a model system when the gene is introduced into metastatic cells. The availability of paired cell lines -metastatic and non-metastatic with otherwise the same genetic background, allowed the analysis of differentially expressed genes. Using two techniques to identify the differentially expressed genes, differential display(a PCR-based technique) and subtraction libraries, novel human metastatic suppressor genes have been found.
  • KiSS-l was identified using subtractive hybridization comparing parental, metastatic melanomas with chromosome 6-melanoma hybrid cells.
  • the gene maps to chromosome lq32 - q41. It is likely to be regulated by gene(s) on chromosome 6.
  • Candidate genes were identified by searching for a minimum 10-fold increase in mRNA expression in nonmetastatic chromosome 6-C8161 hybrids cells (neo6/C8161.1) compared to highly metastatic parental C8161 cells grown under similar conditions.
  • a resulting oligonucleotide probe was used to screen Northern blots of Meljuso, C8161 and neo/Meljuso cell lines.
  • KiSS-l The gene designated KiSS-l was found to be more highly expressed in neo ⁇ / hybrids than metastatic parental cells. Further support for the role of the gene product was that transfection of a KiSS-l cDNA into human melanoma cells suppressed metastatis in athymic mice, a model for metastasis.
  • a full-length KiSS-l cDNA (about 0.784 kb) has a novel nucleotide sequence. (SEQ ID No: 1)
  • Other sequences within the scope of the invention include truncated sequences and fragments of the full length sequence.
  • Characteristics of a cDNA with SEQ ID No:l include about 0.784 kb and an open reading frame (ORF) of 0.495 kb.
  • the cDNA encodes a protein of a predicted size of about 18 kD molecular weight and about 164 amino acids in length.
  • the cDNA predicts a predominantly hydrophilic, 164 amino acid protein with a polyproline rich domain indicative of an SH3 ligand and a putative protein kinase - 6 -
  • a nucleotide sequence of the KiSS-l gene is useful for diagnosis and staging of breast cancer and neuroectodermal cancers including melanomas, small cell carcinoma of the lung and neuroblastomas.
  • the gene is a candidate for gene therapy protocol, e.g. gene replacement.
  • the protein encoded by the KiSS-l cDNA is also suitable for diagnostic tests and planning treatment strategies, as well as for use in pharmaceutical compositions.
  • Monoclonal and polyclonal antibodies directed to proteins or polypeptides having amino acid sequences as shown in SEQ ID NOS: 2, 3, 4, 5 and 6 respectively are useful to detect the sequences.
  • a probe for the KiSS-l gene or at least the active fragment thereof is applied to a biological test sample such as a skin or tumor biopsy under conditions suitable for hybridization.
  • the probe may be labeled for ease of detection if hybridization occurs.
  • Diagnostic methods also include detecting expression of a tumor suppressor gene or fragment thereof by detecting KiSS-l in mRNA or its gene product or fragments or truncations thereof.
  • Nucleotide sequences that are useful as probes to detect by hybridization related sequences in other cancers are components of recombinant expression systems.
  • Recombinant expression systems are useful to produce the KiSS-l gene product or a fragment or truncation thereof, for example, for use in preparation of pharmaceutical compositions or for gene therapy.
  • Suitable recombinant express systems comprise regulatory elements that allow expression of the KiSS-l gene or a fragment or truncation thereof, such as a nucleotide sequence in accordance with SEQ ID NOS: 1, 7, 8, 9 or 10.
  • a nucleotide sequence in accordance with SEQ ID NOS: 1, 7, 9, 10 or a functional equivalent is combined with suitable promoters, enhancers and inducible elements.
  • liposomes are used as gene carriers.
  • suitable means of gene transfer are particle bombardment methods via a "gene gun," and electrofection.
  • molecules with amino acid sequences in accordance with SEQ ID NOS: 2, 3, 4, 5, or 6 are combined with suitable diluents in pharmaceutical compositions and administered to a mammal intradermally or systemically.
  • FIG. 1 shows the nucleotide sequence (SEQ ID NO: 1) of a cDNA from a gene designated KiSS-l .
  • FIG. 2 shows the predicted protein sequence of KiSS-l .
  • Proteins and “polypeptides” are used interchangeably herein. Abbreviations for amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  • the KiSS-l gene product has a polyproline-rich domain and a putative protein kinase C- ⁇ phosphorylation site.
  • amino acids 81-100 show the PXXP consensus SH3 binding motif.
  • KiSS-l Several candidate metastasis-suppressor cDNAs were identified by subtraction hybridization and differential display comparing C8161 and neo6/C8161 cells (Lee, 1996) .
  • One clone, designated KiSS-l was expressed only in nonmetastatic neo6/C8161 cells.
  • Full-length KiSS-l was obtained by cDNA library screening using a neo6/ C8161.1 ⁇ ZAP-cDNA library (7.5 X IO 6 plaques) . After third round screening, seven positive clones were isolated and identified as a same gene by restriction mapping and sequence analysis.
  • the longest cDNA insert was sequenced on both strands by the dideoxy chain termination method with Sequenase version 2.0 (Amersham) .
  • the cDNA designation combines interim laboratory nomenclature for putative Suppressor Sequences with acknowledgment of the gene' s discovery in Hershey. KiSS-l cDNA sequence was submitted to GenBank as a novel gene with an accession number of U43527.
  • KiSS-l mRNA was sought in cell clones derived from C8161, neo6/C8161, and neo6(del) (q21- q23)/C8161 cell clones.
  • neo6/C8161 hybrids were prepared using MCH262A1.D6 as a microcell chromosome donor (Trent, 1990) and neo6 (del) (q21-q23) hybrids were prepared using a related variant A9/6q " C1.1.2 (Gualandi, 1994) as a microcell chromosome donor.
  • Poly (A) + -enriched mRNA was isolated from 80-90% confluent cells using FastTrack mRNA isolation kit (Invitrogen) .
  • Poly(A) + mRNA (2.5 ⁇ g) was loaded and electrophoresed on 1% agarose gel containing 2.2M formaldehyde at 78V for 3.5 hr.
  • the RNA was transferred onto a nylon membrane using the TurboblotterTM system (Schleicher & Schuell) and fixed using a UV crossiinker (Stratagene) .
  • KiSS-l cDNA probe was radiolabeled using random priming kit (Amersham) .
  • Prehybridization was done in 48% formamide, 4.8X SSC (IX SSC; 0.15M NaCl, 0.015M sodium citrate), 20 mM Tris [pH 7.6], IX Denhart ⁇ s solution (0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 10% dextran sulfate, and 0.1% SDS.
  • Hybridization was carried out in the same buffer plus herring sperm DNA (100: ⁇ g) for 24 hrs at 42°C.
  • the membrane was rinsed twice with 2X SSC/0.1% SDS for 15 min at 42°C, followed by twice washing with 0.1X SSC/0.1% SDS for 15 min at 42°C.
  • the rinsed membrane was exposed to X-ray film (Dupont-NEN) . KiSS-l was undetectable in any cell line possessing metastatic potential, whether low or high.
  • KiSS-l mRNA was sought in human metastatic melanoma cell lines: C8161; C8161cl.8; C8161cl.9; nonmetastatic hybrid cell clone neo6/C8161.1; Meljuso; A375P; A375M; MeWo; MeWo-3S5; MeWo-70W; M24met; and OM431.
  • Poly (A) + RNA was isolated from each cell culture using a Micro-FastTrack mRNA isolation kit (Invitrogen) . All cell lines having metastatic potential did not express KiSS-l .
  • KiSS-l mRNA was sought in normal human tissues.
  • a multiple-tissue RNA blot (2 ⁇ g of poly (A) + RNA per lane) was purchased from Clontech. The blot was hybridized using full-length of KiSS-l probe. Detection of KiSS-l message (1.0 kb) in placenta was possible following overnight exposure. KiSS-l mRNA was detectable in pancreas (0.8 kb) and kidney (0.9 kb) only after 3-7 day exposure at -70°C with two intensifying screens. Besides differential organ expression, these results suggest that alternative splice variants of KiSS- 1 may exist in different cells.
  • KiSS-l cDNA A full-length of KiSS-l cDNA was cloned into pcDNA3 (Invitrogen) in which transcription is driven by a human cytomegalovirus promoter.
  • the resultant plasmid pcDNA3- KiSS-l was transfected into C8161 cells using Lipofectin (GIBCO-BRL) .
  • Individual transfectants were isolated and cloned following growth in the neomycin analog G-418 (600 ⁇ g/ml) .
  • Poly(A)mRNA was isolated from each transfectant using Micro-FastTrack mRNA isolation kit (Invitrogen) and northern blot analysis was done.
  • C8161neomix is an uncloned population of C8161 cells transfected with pcDNA3 vector only. Two different sized transcripts are observed. KiSS-l (1.0 kb) is smaller than "exogenous" KiSS-l (1.3 kb) due to vector sequences in the latter. Lanes labeled Pl and P2 are uncloned populations of C8161 cells transfected with pcDNA3-fCiSS-l construct.
  • K ⁇ SS-1 is not expressed in metastatic malanomA cells
  • KiSS-l The expression pattern of KiSS-l was confirmed by northern blot analysis using an extensive panel of cell lines derived from the C8161 melanoma with widely ranging metastatic potentials. KiSS-l mRNA expression could not be detected in any metastatic melanoma cell line. Expression was not detectable, even in overexposed blots or by RT-PCR, in parental C8161 or two subclones,
  • KiSS-l mRNA expression was also undetectable in C8161cl.9 microcell hybrids containing human chromosome 6 bearing a complex deletion within the region 6q21-q23 which remained metastatic in nude mice.
  • KiSS-l transcript (1.0 kb) was found in placenta, with very weak expression in kidney (detectable only after exposure for three or more days using two intensifying screens) . Upon longer exposure, different transcript sizes of KiSS-l were detected in pancreas (0.8 Kb) and kidney (0.9 kb) , suggesting that alternative splicing may take place in different tissues. Expression of KiSS-l could also be detected in normal human melanocytes by RT-PCR.
  • KiSS-l cDNA (FIG. 1) has a single open reading frame that encodes a protein of 164 amino acids with a predicted molecular mass of 18 kDa.
  • the initiation codon and surrounding nucleotides fit the Kozak consensus and there is a consensus polyadenylation site downstream of the termination codon.
  • vi tro transcription and translation of a full-length KiSS-l cDNA resulted in a single band at 18 kDa.
  • EBL European Molecular Biology Laboratory
  • KiSS-l suppresses metastasis when transfected into C8161 human melanoma cells
  • KiSS-l cDNA expression vector was transfected into the C8161 human malignant melanoma cell line.
  • Full-length cDNA of KiSS-l was subcloned into the pcDNA3 constitutive expression vector and transfected into C8161.
  • the vector alone was transfected as a negative control.
  • Several clones were randomly selected and analyzed for expression of KiSS-l by RNA blotting.
  • the KiSS-l transcript in transfectants is larger (1.3 kb) due to additional sequences in the expression vector.
  • the in vi tro growth rates of the selected clones were not significantly different compared to parental metastatic C8161 cell clones.
  • KiSS-l transfectant clones 2, 3 and 9 were less able to colonize lung or regional lymph nodes than concomitantly injected C8161 cells (Table 1) . Differences in metastatic potential could not be explained by slower in vivo primary tumor growth rate since several more metastatic subclones of C8161 grow more slowly than KiSS-l transfectants (Welch, 1994) .
  • KiSS-lcl . 2 which highly expressed KiSS-l RNA, produced an average of only 1 metastasis per mouse. None of the mice had more than 3 metastases and only two had lymph node metastases. KiSS-lcl . 3 , produced a mean of 3 metastases per mouse (all mice yielded less than 6 metastasis) and slightly higher incidences of lymph node metastases. Similar results were obtained following direct inoculation into the lateral tail veins of athymic nude mice (experimental metastasis assay, Table 2) . he predicted KiSS-l protein is an SH3 ligand or is phosphorylated by PKC- ⁇
  • KiSS-l The predicted protein sequence of KiSS-l cDNA was analyzed and the following homologies were found: a putative protein kinase C- ⁇ phosphorylation site (single- letter amino acid code, SMR corresponding to amino acid positions 154 to 156, ) and a proline-rich region with five overlapping minimal SH3 binding domains (Cohen, 1995; Alexandropoulos, 1995) . (PXXP motif, FIG. 2, corresponding to amino acids 85-97 where X can be any amino acid) . This suggests that KiSS-l is a ligand for a protein possessing an SH3 domain and/or is a substrate for PKC- ⁇ phosphorylation.
  • the N-terminal 70 amino acids contain seven serines and a tyrosine (Y60) as possible phosphorylation sites.
  • Y60 tyrosine
  • KiSS-l is an SH3 ligand (Cohen, 1995; Alexandropoulos, 1995; Hennessey, 1991, Musacchio, 1994) . Rules governing specificity of SH3 binding are currently being elucidated (Cohen, 1995; Alexandropoulos, 1995; Joseph, 1995; Feng, 1994; Mayer, 1995; Rickles, 1995; Ishino, 1995); but, for the most part, they remain unknown. However, the putative KiSS-l SH3 binding domain shares many features of the RLP-type (class I) ligand orientation.
  • SH3 domains are 50-70 amino acid modules present in a variety of intracellular proteins that mediate protein- protein interactions important for intracellular signaling and cytoskeletal organization (Koch, 1991; Musacchio, 1992; Pawson, 1992; Pawson, 1993; Feller,
  • KiSS-l may suppress metastasis by regulating key signalling molecules important to one or more of these steps. KiSS-l transfectants did not display significantly influenced adhesion to the extracellular matrix components type-I collagen or fibronectin, nor was adhesion to a complex basement membrane-like mixture, Matrigel affected.
  • KiSS-l maps to chromosome Iq32-q41 by fluorescence in si tu hybridization. Its mapping to another location within the genome leads to the conclusion that KiSS-l is regulated by the gene(s) on chromosome 6. KiSS-l is an important downstream effector of a gene(s) encoded on chromosome 6 which is consistent with metastasis suppression following introduction of chromosome 1 into MelJuSo melanoma cells. KiSS-l mRNA is not detected in metastatic neo6 (del) (q21-q23) /C8161 hybrids.
  • KiSS-l mutations (2) KiSS-l deletions in subclones of C8161, or (3) mutation or deletion of regulatory gene(s) mapping to 6q21-q23.
  • the latter explanation is consistent with high frequency loss of heterozygosity observed in late-stage melanomas (Trent, 1989; Guan, 1992; Milliken, 1991) .
  • C8161 is an amelanotic human melanoma cell line that metastasizes widely following intradermal, subcutaneous or intravenous injection into athymic nude or SCID mice Welch 1994; 1991) .
  • Subclones C8161cl.9 and C8161cl.8 were isolated from C8161 by limiting dilution and were chosen for these studies because they have the highest and lowest metastatic potentials, respectively (Welch, 1994) .
  • neo6/C8161.1, neo6/C8161.2 and neo6/C8161.3 were derived from microcell-mediated chromosome transfer of a single copy of a neomycin-tagged human chromosome 6 using the MCH262A1.D6 donor cell line (Welch, 1994; Trent, 1990) .
  • Four additional, independently-derived neo6/C8161 hybrid cell clones (neo6/C8161.4, neo6/C8161.5b, neo6/C861.6 and neo6/C8161.8) were subsequently developed.
  • neo6/C8161 hybrid cell clones None of the neo6/C8161 hybrid cell clones were metastatic in athymic nude mice. neo6(del) (q21-q23) hybrid cell clones were prepared using a related chromosome 6 microcell donor variant A9/6q ⁇ cl.l.2 (Gualandi, 1994) and are metastatic in nude mice.
  • Cell line nomenclature has been chosen to identify the origin and nature of each cell line as unambiguously as possible.
  • Single cell clones are preceded by a ".” (e.g., C8161cl.9 is a single cell clone) and pooled, uncloned populations are identified by a "-" (e.g., C8161-K1SS-1-P1 is an uncloned population #1 of C8161 cells transfected with KiSS-l ) .
  • Microcell hybrids are identified by the tagged chromosome followed by a "/"
  • neo6/C8161.3 is single cell clone 3 derived from a fusion with a neo-tagged human chromosome 6; neo6 (del) (q21-q23) /C8161cl.8 is single cell clone 8 derived from a microcell fusion with a neo-tagged chromosome 6 containing deletion of the q21-q23 bands) . All cells were grown in DME-F12 (Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal bovine serum (CDME-F12) .
  • DME-F12 Irvine Scientific, Santa Ana, CA
  • CDME-F12 10% fetal bovine serum
  • neo-resistant cells, neo6/C8161 and neo6 (del) (q21-q23) /C8161 hybrids were maintained in cDME- F12 containing 500 ⁇ g/ml geneticin (G-418, GIBCO-BRL, Gaithersburg, MD) .
  • Nearly confluent cultures (70-90%) were passaged in Corning (Oneata, NY) tissue culture dishes following detachment with a solution of calcium- and magnesium-free Dulbecco' s phosphate buffered saline containing 2 mM EDTA at split ratios of 1:10 to 1:20. All cultures were routinely tested and found to be negative for Mycoplasma spp . infection using a PCR-based test kit (PanVera, Madison, WI) .
  • Subtractive hybridization was done as described (Lee, 1991; Duguid, 1988; Schweinfest, 1990) with minor modifications. Briefly, mRNAs were isolated from 80-90% confluent cells using FastTrack mRNA isolation kit (Invitrogen Corp., San Diego, CA) . A cDNA library was constructed from neo6/C8161.1 cells using ⁇ ZAP-cDNA Gigapack II GoldTM cloning kit (Stratagene, La Jolla, CA) . Subtractive hybridization was carried out between the first strand cDNA from nonmetastatic neo6/C8161.1 cells and mRNA from metastatic parental C8161 cells.
  • cDNA was synthesized from 1 ⁇ g poly(A) + enriched RNA from neo6/C8161.1 using oligo(dT) primers. Biotinylation of mRNA from parental C8161 cells was performed using PhotobiotinTM labeling system (GIBCO-BRL) according to manufacturers instructions. To subtract, biotinylated mRNA (20 ⁇ g) was mixed with cDNA (1 ⁇ g) from neo6/C8161.1 and ethanol precipitated.
  • the pellet was resuspended in DEPC-treated water (20.5 ⁇ l) then added to 2X hybridization buffer [80% formamide, 100 mM hydroxyethylpiperazine ethane sulfonic acid (pH 7.5), 2% sodium dodecyl sulfate, 22.5 ⁇ l] .
  • 2X hybridization buffer [80% formamide, 100 mM hydroxyethylpiperazine ethane sulfonic acid (pH 7.5), 2% sodium dodecyl sulfate, 22.5 ⁇ l] .
  • This mixture was boiled for 2 min, chilled on ice for 5 min followed by addition of NaCl (5 M, 2 ⁇ l) . Hybridization was performed at 42°C for 48 hr.
  • the hybridization mixture was added to streptavidin (25 ⁇ g, Sigma) and incubated at room temperature for 5 min before extraction with phenol:chloroform: isoamylalcohol (25:24:1)
  • the organic phase was twice back-extracted with 50 ⁇ l streptavidin- binding buffer [100 mM Tris-HCl pH 8.0; 1 M EDTA, 500 mM NaCl] . Then the aqueous phases were pooled. Unhybridized cDNA target was ethanol precipitated. This subtracted cDNA was used as a probe for the neo6/C8161.1 cDNA library screening following random primer labeling (Amersham) . Positive clones were isolated and used for evaluation of expression in northern blot analysis.
  • Candidate cDNAs were initially evaluated for differential expression by Northern blotting using poly(A) + enriched mRNA from C8161 and neo6/C8161.1 cells. If expression was greater in neo6/C8161.1 cells by at least 10-fold, the same cDNA was used to probe a more extensive blot containing RNAs from a panel of cell lines with differing metastatic potentials. If the pattern of expression correlated with metastatic potential, full- length cDNAs were isolated and further characterized. Full-length KiSS-l was obtained from a ⁇ ZAP-cDNA library (7.5 X IO 6 plaques) prepared from neo6/C8161.1 cells.
  • poly (A) + enriched mRNA was isolated from 80-90% confluent cells using FastTrack mRNA isolation kit (Invitrogen) .
  • Poly(A) + mRNA (2.5 ⁇ g) was loaded and electrophoresed on 1% agarose gel containing 2.2M formaldehyde at 78V for 3.5 hr.
  • RNA was transferred onto a nylon membrane using the TurboblotterTM system (Schleicher & Schuell) and fixed by UV crosslinking (Stratagene) .
  • Full-length KiSS-l cDNA probe was radiolabeled using random priming (Amersham) .
  • Prehybridization was done in 48% formamide, 4.8X SSC (IX SSC; 0.15M NaCl, 0.015M sodium citrate), 20 mM Tris [pH 7.6], IX Denhart's solution (0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 10% dextran sulfate, and 0.1% SDS.
  • Hybridization was carried out in the same buffer plus herring sperm DNA (100: ⁇ g) for 24 hrs at 42°C. The membrane was rinsed twice with 2X SSC/0.1% SDS for 15 min at 42°C, followed by twice washing with 0.1X SSC/0.1% SDS for 15 min at 42°C. The rinsed membrane was exposed to X-ray film (Dupont-NEN) for desired intensity.
  • KiSS-l Expression of KiSS-l in normal human tissues was evaluated using a mutiple-tissue RNA blot (2 ⁇ g of poly (A) RNA per lane) purchased from Clontech. The blot was hybridized using full-length KiSS-l probe as above. Expression of KiSS-l in normal human melanocytes (Clonetics) was determined by RT-PCR.
  • Transfectants were evaluated for growth in vi tro .
  • Cells (2 x 10 4 ) were inoculated into 24-well tissue culture plates (Corning) in CDME-F12 medium. After 24, 48, 72, and 96 hr, cells were trypsinized and counted using a hemacytometer.
  • mice For spontaneous metastasis assays, cells (1 x IO 6 ) were injected intradermally into the dorsolateral flank of athymic nude mice (4-12 mice per group) . Three- to four-week old female mice (Harlan Sprague Dawley, Madison, WI) were used for these studies. Animals were maintained under the guidelines of the National Institutes of Health and The Pennsylvania State University College of Medicine. All protocols were approved by the Institutional Animal Care and Use Committee. Food and water were provided ad libi t um . Tumor size was measured weekly by taking orthogonal measurements and expressed as mean tumor diameter. Mean tumor diameter was calculated as described (Welch, 1994; Claas, 1996) . After the mean tumor diameter reached 1.5- 2.0 cm, mice were necropsied, and visible metastases were counted (Welch 1991; 1983) .
  • lungs from each mouse were removed and fixed in a mixture of formalin and Bouin's fixative (5:1 v/v) and examined under a dissecting stereomicroscope (Welch, 1983) . Unless otherwise noted, all other tissues were examined and found to be free of metastases.
  • the number of lung metastases was compared in KiSS-l transfectants and parental C8161 cells.
  • One-way ANOVA followed by Tukey' s Honestly Significant Difference post-test was used.
  • For spontaneous metastasis assays a Kruskal-Wallis ANOVA of ranks procedure was used. Calculations were performed using SigmaStat statistical analysis software (Jandel Scientific) . Statistical significance was defined as P ⁇ 0.05.
  • microcell hybrids are made by treating proliferating (80% confluent) donor cells with colcemid (0.15 ⁇ g/ml) for 48 h. Chromosomes recondense and form micronuclei because spindle formation is blocked. Each micronucleus contains one or more chromosomes which are then enucleated by treatment with cytochalasin B (2 ⁇ g/ml) and centrifugation. The resulting microcells are sequentially filtered through 8, 5, and 3 ⁇ m polycarbonate filters to obtain a population containing mostly single chromosomes.
  • microcells are fused using polyethylene glycol to a semi-confluent recipient C8161 cell culture. After fusion and recovery (24 h) , cells are grown under selective conditions, i.e., CDME-F12 containing G-418 (600 ⁇ g/ml) . Only recipient cells that contain the neo-tagged chromosome will survive. Individual colonies were isolated.
  • PCR-RFLP Verification of Chromosome 6 in C8161 and neo6 hybrid cell lines and tumors Genomic DNA from cell lines, tumors, lung metastases and donor cell line was isolated (Sambrook, 1989) and digested with EcoRI.
  • DNA 50 ng was added to a 30 ⁇ l PCR reaction mix and amplified for 28 cycles (94°C, 1 min; 55°C, 1 min; 72°C, 1 min) .
  • An apparently intact added chromosome was found in all cell lines, tumors and metastases evaluated.
  • RNA used for Northern blot analysis and cDNA library construction was isolated from 80-90% confluent cells using FastrackTM mRNA isolation kit (Invitrogen, San Diego, CA) .
  • a cDNA library from neo6/C8161.1 cells was constructed using ⁇ ZAP-cDNA Gigapack II GoldTM cloning kit (Stratagene, La Jolla, CA) .
  • the first strand cDNA was synthesized using a hybrid oligo (dT) linker-primer, containing an Xhol restriction site, from 5 ⁇ g poly(A) + RNA isolated from neo6/C8161.1 cells.
  • an EcoRI adapter was ligated. and cDNA fragments were digested with Xhol, and size- fractionated by Sephacryl S-400 spin column.
  • Fractions containing cDNA fragments larger than 500 bp were pooled and used for ligation into the EcoRI-XhoI site of UniZap II arms.
  • Ligation mixtures were packaged in vi tro with Gigapack Gold extracts, and the resulting phage library was titered and amplified on the E. col i XLl blue cells. After a single round of amplification, the phage library titer was 5 x 10 10 plaque-forming unit (pfu) /ml.
  • Recombinant plaques were plated a density of 10,000 pfu/150 mm plate. Lifting of plaque onto a membrane (Colony/Plaque ScreenTM Hybridization Membrane, Dupont- NEN, Boston, MA) was performed according to the manufacturer's protocol. Prehybridization of the immobilized plaques was carried out for 1 hr at 42°C in 48% formamide/4.8X SSCV20 mM Tris (pH 7.6) /IX Denhart's solution/10% dextran sulfate/0.1% SDS. Hybridization was carried out for 16-24 hrs at 42°C in prehybridization mix plus 100 ⁇ g/ml herring sperm DNA, plus radiolabeled cDNA probe. After full-length KiSS-l had been obtained, screening was performed using recombinant plaques plated at a density of 50,000 pfu/150 mm plate.
  • the plasmid DNA was isolated using WizardTM miniprep kit (Promega, Madison, WI) . DNA sequences were determined by double stranded DNA sequencing using the dideoxy chain termination method with Sequenase version 2.0 (Amersham) . Computer-assisted DNA sequence analysis
  • '1XSSC 0.15M sodium citrate
  • Denhardt's solution 0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone. was performed with FASTA program to search sequence identity.
  • a biological sample is obtained from the subject.
  • Suitable samples include skin and tumor biopsies.
  • the sample is processed by methods known to those of skill in the art, e.g. tissues are sectioned, fixed and mounted, so that histochemical staining or in situ hybridization is facilitated.
  • Antibodies to polypeptides encoded by the KiSS-l gene are prepared and labeled for detection in the biological sample.
  • Suitable polypeptides include those having amino acid sequences in accordance with SEQ ID NOS: 2, 3,4,5 of the present invention.
  • Suitable antibodies are preferably monoclonal antibodies.
  • Riboprobes (antisense molecules) directed to nucleotide sequences as shown in SEQ ID NOS: 1,7, 8, 9, and possibly 10, are used for in situ hybridization to detect the expression of the KiSS-l gene (Ausubel, 1990) .
  • Suitable labels include S-35, biotin -substituted nucleotides, digoxygenin.
  • the three possible types of outcomes for either the assays for the polypeptide products of KiSS-l expression, or for mRNA complementary to the KiSS-l gene, and the interpretation of those outcomes are: 1. If there is evidence of KiSS-l expression , the biological sample is classified as non-metastatic; 2. If there is no evidence of KiSS-l expression, the biological sample is classified as metastatic; and 3. If either there is evidence of KiSS-l expression in some cells or portions of a biological sample, but not in other cells or portions of the tissue, a decision on whether to classify the sample as metastatic or non-metastatic requires a comparison to percentages ( or levels of expression in a fluid sample) of expression developed from normal control samples and from samples of the cancer in different stages. Staging of cancers is discussed in Hollev et el. (1991) and is cancer-type specific.
  • EXAMPLE 1 Detection of KiSS-l mRNA by Riboprobes to Stage Malignant Melanoma
  • KiSS-l gene expression was detected in 6 cell lines.
  • the following cell lines were tested for KiSS-l gene expression using RT-PCR (reverse transcriptase polymerase chain reaction) .
  • a cell line (WM35) derived from a melanoma in the radial growth phase in which gene expression was detected; 3.
  • a cell line (WM793) derived from a melanoma in the early vertical growth phase gave inconclusive results of gene expression; 4.
  • a metastatic melanoma cell line (WM115) in the vertical growth phase showed no evidence of gene expression; and 5.
  • Two malignant melanoma cell lines (1205-LU and WM239A) showed no gene expression.
  • KiSS-l suppresses spontaneous metastasis of C8161 human melanoma cells
  • mice (1 x 10 s ) were injected intradermally into the dorsolateral flank of 3-4 week old, female athymic nude mice (7-8 mice per group, Harlan Sprague Dawley). When mean tumor diameter (square root ofthe product of orthogonal measurements) reached 1.5-2.0 cm, mice were killed. All organs were examined for presence of metastases and many were confirmed by microscopic examination of hematoxylin- and eosin-stained paraffin-embedded sections (4-6 ⁇ m). Macroscopic lung metastases were quantified after staining with in a mixture of formalinrBouin's fixative (5:1) and counting with the aid of a dissecting microscope as previously described (Welch, 1991).
  • KiSS-l suppresses experimental metastasis of C8161 human melanoma cells
  • Genomics 14:680-684, 1992. Hennessey C, Henry JA, May FEB, Westley BR, Angus B, Lennard TWJ. Expression of the antimetastatic gene nm23 in human breast cancer: an association with good prognosis. J Natl Cancer Inst 1991; 83:281-285.
  • Pawson T Gish GD. SH2 and SH3 domains: from structure to function. Cell 1992; 71:359-362. Pawson T, Schlessinger J. SH2 and SH3 domains. Curr Biol 1993; 3:434-442.

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Abstract

Un nouveau gène humain suppresseur de métastases a été cloné par hybridation soustractive par comparaison des cellules métastatiques et non métastatiques. La perte d'expression correspond à un potentiel métastatique dans les cellules humaines, l'expression du gène entraînant la suppression notable de métastases chez des souris nues athymiques. Un ADNc a été isolé, le gène a été appelé KiSS-1 et localisé dans le chromosome 1. La protéine KiSS-1 prédite comporte une région riche en proline par homologie au domaine de liaison SH3 qui prédit un mécanisme de suppression des métastases. KiSS-1 et ses produits sont utilisés comme marqueurs pour classifier par stades cliniques les mélanomes, en vue d'un diagnostic ou d'une thérapie.
PCT/US1996/016414 1995-10-11 1996-10-10 Gene suppresseur de metastases WO1997013778A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002072816A1 (fr) * 2001-02-26 2002-09-19 Takeda Chemical Industries, Ltd. Proteine de recepteur murin de type kiss-1 et adn correspondant
US6699965B1 (en) 1998-10-27 2004-03-02 Takeda Chemical Industries, Ltd. Peptides that activate the G-protein coupled receptor protein, 0T7T175
EP1239037A4 (fr) * 1999-12-17 2004-03-31 Takeda Chemical Industries Ltd PROCEDE DE PRODUCTION DE PEPTIDE KiSS-1
WO2004096855A3 (fr) * 2003-04-28 2005-06-23 Wyeth Corp Procedes et compositions permettant de moduler le recepteur 54 couple a une proteine g

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023814A1 (fr) * 1994-03-04 1995-09-08 National Jewish Center For Immunology And Respiratory Medicine Produit et procede de modulation de l'activite des lymphocytes t

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023814A1 (fr) * 1994-03-04 1995-09-08 National Jewish Center For Immunology And Respiratory Medicine Produit et procede de modulation de l'activite des lymphocytes t

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PROCEEDINDS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, March 1996, Vol. 37, LEE et al., "Cloning of a Novel Gene, KiSS-1, which is Responsible for Metastasis Suppression in Chromosome 6/Human Melanoma Hybrid Cells", page 76, Abstract No. 531. *
PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, March 1996, Vol. 37, LEE et al., "Use of Differential Display and Subtractive Hybridization to Identify Highly Expressed Genes in Metastasis-Suppressed Chromosome 6/Human Melanoma Hybrids", page 76, Abstract No. 530. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6699965B1 (en) 1998-10-27 2004-03-02 Takeda Chemical Industries, Ltd. Peptides that activate the G-protein coupled receptor protein, 0T7T175
US7314754B2 (en) 1998-10-27 2008-01-01 Takeda Pharmaceutical Company Limited Polynucleotide encoding the hOT7T175 G protein coupled receptor
US7608409B2 (en) 1998-10-27 2009-10-27 Takeda Pharmaceutical Company Limited Screening assay using G-protein coupled receptor protein OT7T175
EP1239037A4 (fr) * 1999-12-17 2004-03-31 Takeda Chemical Industries Ltd PROCEDE DE PRODUCTION DE PEPTIDE KiSS-1
US6838259B2 (en) * 1999-12-17 2005-01-04 Takeda Chemical Industries, Ltd. Process for producing kiss-1 peptide
WO2002072816A1 (fr) * 2001-02-26 2002-09-19 Takeda Chemical Industries, Ltd. Proteine de recepteur murin de type kiss-1 et adn correspondant
WO2004096855A3 (fr) * 2003-04-28 2005-06-23 Wyeth Corp Procedes et compositions permettant de moduler le recepteur 54 couple a une proteine g

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