Regulation of KiSS-1 Metastasis Suppressor Gene Expression in Breast Cancer Cells by Direct Interaction of Transcription Factors Activator Protein-2α and Specificity Protein-1

Abstract

KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2α (AP-2α) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2α binding elements, AP-2α-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2α into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2α wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2α regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2α directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2α and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2α in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis. KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2α (AP-2α) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2α binding elements, AP-2α-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2α into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2α wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2α regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2α directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2α and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2α in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis. The vast majority of breast cancer deaths results from complications caused by tumor cell metastasis rather than as a consequence of the original tumor growth. Once tumorigenic cells enter into the vascular and lymphatic systems, they travel to peripheral regions where they invade tissues and form neoplasms. Metastasis is a process requiring detachment of cancer cells from the primary site, survival of sheer forces encountered in the circulation, migration to other organs, attachment to and invasion of tissues, proliferation of these cells at the secondary site, and finally the capacity to enlist neighboring capillaries to supply the tumor with nutrients as it develops (1Pantel K. Brakenhoff R.H. Nat. Rev. Cancer. 2004; 4: 448-456Crossref PubMed Scopus (1063) Google Scholar). Interference at any one of these steps can block this metastatic cascade thereby preventing the formation of metastatic tumor growths. Consequently, there is a growing interest in researching the metastatic process to identify possible ways to inhibit its progression. Metastasis suppressor genes, which inhibit the spread of cancers to secondary sites, have become the target of mounting clinical and basic cancer research. One such gene, KiSS-1 or KiSS1, was originally identified as a metastasis suppressor by microcell-mediated transfer in melanoma lines, by which it was found to reduce tumor cell invasive and migratory properties without affecting their tumorigenicity (2Lee J.H. Welch D.R. Int. J. Cancer. 1997; 71: 1035-1044Crossref PubMed Scopus (161) Google Scholar). Since then, KiSS-1 has been shown to act as a potent anti-metastatic agent either by treatment using synthesized KiSS-1 peptide or upon ectopic expression in highly metastatic cells (2Lee J.H. Welch D.R. Int. J. Cancer. 1997; 71: 1035-1044Crossref PubMed Scopus (161) Google Scholar, 3Masui T. Doi R. Mori T. Toyoda E. Koizumi M. Kami K. Ito D. Peiper S.C. Broach J.R. Oishi S. Niida A. Fujii N. Imamura M. Biochem. Biophys. Res. Commun. 2004; 315: 85-92Crossref PubMed Scopus (115) Google Scholar, 4Ohtaki T. Shintani Y. Honda S. Matsumoto H. Hori A. Kanehashi K. Terao Y. Kumano S. Takatsu Y. Masuda Y. Ishibashi Y. Watanabe T. Asada M. Yamada T. Suenaga M. Kitada C. Usuki S. Kurokawa T. Onda H. Nishimura O. Fujino M. Nature. 2001; 411: 613-617Crossref PubMed Scopus (1186) Google Scholar, 5Lee J.H. Welch D.R. Cancer Res. 1997; 57: 2384-2387PubMed Google Scholar). Loss or reduced expression of KiSS-1 has been found in a variety of tumor metastasis, including breast cancer, bladder cancer, pancreatic cancer, and esophageal squamous cell carcinoma (3Masui T. Doi R. Mori T. Toyoda E. Koizumi M. Kami K. Ito D. Peiper S.C. Broach J.R. Oishi S. Niida A. Fujii N. Imamura M. Biochem. Biophys. Res. Commun. 2004; 315: 85-92Crossref PubMed Scopus (115) Google Scholar, 5Lee J.H. Welch D.R. Cancer Res. 1997; 57: 2384-2387PubMed Google Scholar, 6Ikeguchi M. Yamaguchi K. Kaibara N. Clin. Cancer Res. 2004; 10: 1379-1383Crossref PubMed Scopus (115) Google Scholar, 7Sanchez-Carbayo M. Capodieci P. Cordon-Cardo C. Am. J. Pathol. 2003; 162: 609-617Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Together, these studies suggest that KiSS-1 is a human metastasis suppressor gene and that loss of KiSS-1 and its receptor may correlate with human tumor progression and metastasis. The KiSS-1 gene encodes a largely hydrophobic 145-amino acid protein highly expressed in the placenta (4Ohtaki T. Shintani Y. Honda S. Matsumoto H. Hori A. Kanehashi K. Terao Y. Kumano S. Takatsu Y. Masuda Y. Ishibashi Y. Watanabe T. Asada M. Yamada T. Suenaga M. Kitada C. Usuki S. Kurokawa T. Onda H. Nishimura O. Fujino M. Nature. 2001; 411: 613-617Crossref PubMed Scopus (1186) Google Scholar, 8Muir A.I. Chamberlain L. Elshourbagy N.A. Michalovich D. Moore D.J. Calamari A. Szekeres P.G. Sarau H.M. Chambers J.K. Murdock P. Steplewski K. Shabon U. Miller J.E. Middleton S.E. Darker J.G. Larminie C.G. Wilson S. Bergsma D.J. Emson P. Faull R. Philpott K.L. Harrison D.C. J. Biol. Chem. 2001; 276: 28969-28975Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar). The KiSS-1 gene product consists of a protein kinase phosphorylation domain, a secretory signal, and polyproline-rich region, and a number of potentially important motifs for post-translational modifications (8Muir A.I. Chamberlain L. Elshourbagy N.A. Michalovich D. Moore D.J. Calamari A. Szekeres P.G. Sarau H.M. Chambers J.K. Murdock P. Steplewski K. Shabon U. Miller J.E. Middleton S.E. Darker J.G. Larminie C.G. Wilson S. Bergsma D.J. Emson P. Faull R. Philpott K.L. Harrison D.C. J. Biol. Chem. 2001; 276: 28969-28975Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar, 9Lee J.H. Miele M.E. Hicks D.J. Phillips K.K. Trent J.M. Weissman B.E. Welch D.R. J. Natl. Cancer Inst. 1996; 88: 1731-1737Crossref PubMed Scopus (840) Google Scholar, 13Stafford L.J. Xia C. Ma W. Cai Y. Liu M. Cancer Res. 2002; 62: 5399-5404PubMed Google Scholar). Independently, three groups discovered and isolated the 54-amino acid C-terminally amidated fragment of KiSS1 protein (amino acids 68–121), termed metastin and kisspeptin, respectively (4Ohtaki T. Shintani Y. Honda S. Matsumoto H. Hori A. Kanehashi K. Terao Y. Kumano S. Takatsu Y. Masuda Y. Ishibashi Y. Watanabe T. Asada M. Yamada T. Suenaga M. Kitada C. Usuki S. Kurokawa T. Onda H. Nishimura O. Fujino M. Nature. 2001; 411: 613-617Crossref PubMed Scopus (1186) Google Scholar, 10Kotani M. Detheux M. Vandenbogaerde A. Communi D. Vanderwinden J.M. Le Poul E. Brezillon S. Tyldesley R. Suarez-Huerta N. Vandeput F. C. M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The function of KiSS-1 peptide is through with the a of the receptor (4Ohtaki T. Shintani Y. Honda S. Matsumoto H. Hori A. Kanehashi K. Terao Y. Kumano S. Takatsu Y. Masuda Y. Ishibashi Y. Watanabe T. Asada M. Yamada T. Suenaga M. 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C-terminal of the KiSS-1 peptide to strong binding with a of including increased and as as such as and formation (4Ohtaki T. Shintani Y. Honda S. Matsumoto H. Hori A. Kanehashi K. Terao Y. Kumano S. Takatsu Y. Masuda Y. Ishibashi Y. Watanabe T. Asada M. Yamada T. Suenaga M. Kitada C. Usuki S. Kurokawa T. Onda H. Nishimura O. Fujino M. Nature. 2001; 411: 613-617Crossref PubMed Scopus (1186) Google Scholar, 8Muir A.I. Chamberlain L. Elshourbagy N.A. Michalovich D. Moore D.J. Calamari A. Szekeres P.G. Sarau H.M. Chambers J.K. Murdock P. Steplewski K. Shabon U. Miller J.E. Middleton S.E. Darker J.G. Larminie C.G. Wilson S. Bergsma D.J. Emson P. Faull R. Philpott K.L. Harrison D.C. J. Biol. Chem. 2001; 276: 28969-28975Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar, 10Kotani M. Detheux M. Vandenbogaerde A. Communi D. Vanderwinden J.M. Le Poul E. Brezillon S. Tyldesley R. Suarez-Huerta N. Vandeput F. C. M. J. Biol. 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However, has been the possible of KiSS-1 expression in cancer the KiSS-1 transcriptional regulation is unknown as is the for its loss metastatic progression. breast cancer cells become expression levels of different to cell tumor cell and migratory are commonly found in breast cancer, including and and that the loss of or may an increased metastatic they are to block formation of C. P. W. M. R. D. Int. J. Cancer. 1996; PubMed Scopus Google Scholar, Y. P. Nature. 2001; 411: PubMed Scopus Google Scholar, Liu S. Cancer Res. PubMed Google Scholar, J. PubMed Scopus Google Scholar, S.E. J.M. J. 2003; PubMed Scopus Google Scholar, S. D. M. M. J. PubMed Scopus Google Scholar). that the expression of important and metastasis. protein a transcription that are important and metastatic J. S. J. D. T. Nature. 1996; PubMed Scopus Google Scholar, M. Cancer Metastasis Rev. PubMed Scopus Google Scholar). transcriptional regulation the and have a variety of such as human activator and vascular S. D. M. 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Cancer Res. 2003; Google Scholar). we that the expression of KiSS-1 metastasis suppressor gene in breast cancer cells is directly with the expression of transcription AP-2α and Sp1, and that AP-2α and Sp1 synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Furthermore, we that KiSS-1 expression is by AP-2α through direct with the transcription Sp1 at two tandem Sp1-binding sites rather than via with the sites of KiSS-1 promoter. results a for the loss of KiSS-1 gene expression commonly in metastatic breast cancers and by which AP-2α and Sp1 transcription and tumor progression. and and cells from the and with and from was from and kinase from for Sp1, and from and from The KiSS-1 promoter was from using of and sites for into the from and other KiSS-1 promoter and into and sites in the for AP-2B, and Sp1 as C. M. 2003; PubMed Scopus Google Scholar). The was into and was from the Sp1 with which the DNA-binding and of and of in or in or with cells gene using to the 1 of was into of a using a to of for a of was to the in in in which was from and cells in with and luciferase activity of protein was the for in cell cells with levels the from cells as M. R. R. S. Cancer Res. 2004; PubMed Scopus Google Scholar). of was determined using of protein and at cell lines in with of and The cells and in The cell was in of by at for at was into at 1 of protein and or as to the in primary was at by secondary at using the to the synthesized and and was using and of 1 of with or without and of was for was in for AP-2 was for complexes at at for complexes as or was the by the cells with AP-2α and Sp1 with into and in 1 1 and 1 A. was to of by The was with for 1 at with The and the was with to and Sp1 at The and of the KiSS-1 promoter was from the using the and the product was a and with of AP-2α and Sp1 in cells was by and by analysis. cells with 1 1 and 1 by with a and with the and by using protein with and by and to in and for the with the and with the was isolated from breast cancer cell lines with was using and to the for of KiSS-1 and in for and which in of for for was KiSS-1 using of by and using KiSS-1 in Cancer with AP-2α and other have that KiSS-1 expression is in highly metastatic breast cancer cells. loss of the transcription AP-2α and Sp1 was directly or with loss of the expression levels of AP-2α and Sp1 levels in highly metastatic and breast cells with the expression of KiSS-1 to the of a and KiSS-1 KiSS-1 expression in the breast cell lines was using and to the The breast cell expression as with the metastatic breast tumor cell lines and demonstrated that AP-2α expression was or in the metastatic cell lines, and The expression levels of Sp1 in these cells. shown in Sp1 expression was decreased in the metastatic cancer cell lines Together, these suggest that the expression of KiSS-1 was with the expression levels of the two transcription factors, in breast cancer cells. that KiSS-1 expression is by AP-2α and Sp1, we AP-2α and Sp1 into these cells of the KiSS-1 promoter by of AP-2α and Sp1 was from to the cell Although it is not of AP-2α and Sp1 activate KiSS-1 promoter in cells where these are expressed at we that of these two have an the of the KiSS-1 promoter with the we that the loss of AP-2α and Sp1 and directly with the loss of KiSS-1 expression in breast cancer cells. of the of the KiSS-1 by AP-2α and of found and or are commonly as an of a such as a promoter may the of to an important such as the binding of transcription for transcriptional regulation of that AP-2 and Sp1 the expression of KiSS-1 gene, we and the regions of KiSS-1 gene different to the we our promoter this region, which was into a luciferase of the highly to the KiSS-1 contains three AP-2α binding by multiple Sp1 sites that the KiSS-1 like other genes, contains multiple AP-2α binding and a in it a gene by complexes in tumor cells. AP-2α and Sp1 KiSS-1 AP-2 and Sp1 in transcriptional regulation of the KiSS-1 tumor metastasis suppressor gene, luciferase of the KiSS-1 promoter into breast cell lines with Sp1, or to for we the luciferase activity by the KiSS-1 promoter. that activity was increased upon of either AP-2 or Sp1 in KiSS-1 promoter activity was increased than in cells with AP-2 and Sp1 luciferase of the KiSS-1 promoter for upon of AP-2α and Sp1 to which regions of the KiSS-1 promoter transcriptional by AP-2α and shown in the KiSS-1 promoter luciferase was synergistically by co-transfection of AP-2α and Sp1 to results using the and luciferase the KiSS-1 promoter suggesting that the promoter contains the for transcriptional regulation by the AP-2α and Sp1 and that the promoter to AP-2α and Sp1 these sites and KiSS-1 of through with Sp1 at Sp1 the of KiSS-1 gene transcriptional regulation by AP-2α and Sp1, we direct binding of AP-2α and Sp1 is for the of KiSS-1 promoter. AP-2 and Sp1 lacking the DNA-binding of transcription and into and cells with the KiSS-1 promoter luciferase and for luciferase activity dominant negative to activate KiSS-1 luciferase activity with However, AP-2B, the AP-2α lacking the C-terminal DNA-binding domain, increased luciferase activity to the as the wild-type form of AP-2 with Sp1 transcription results suggest that the DNA-binding of AP-2α is not in KiSS-1 transcriptional activity in human breast cancer cells. the transcriptional binding sites in transcriptional regulation of we of and in the luciferase as shown in with AP-2α and Sp1 in breast cell lines for luciferase analysis. shown in the promoter and demonstrated the to transcription of AP-2α and Sp1 the activity of the to that by the did not any AP-2 The two Sp1 sites in identified as and these two Sp1 sites in the transcriptional of the KiSS-1 promoter by AP-2α and Sp1, of deletion or at these two sites as as at sites of the KiSS-1 promoter and in luciferase that mutation at either Sp1 the KiSS-1 promoter to the regulation of AP-2α and Sp1 transcription in cells suggesting that the two Sp1 sites are for the regulation of KiSS-1 promoter by AP-2α and Sp1 transcription Sp1 and AP-2α a at the Sp1 of KiSS-1 the formation of Sp1 and at the two Sp1 sites in regulation of the KiSS-1 gene was using the to of the KiSS-1 promoter of using AP-2α and cells that Sp1 to the this was in the of The negative in the of is shown in of a the migration of the was by the in a However, the to the was for the Sp1 the for to any to with this of the promoter fragment with in a of the by the that AP-2 may to this that AP-2α in the we using isolated from cells. of using to Sp1, and was by using that the the of the two Sp1 sites and shown in the and of the KiSS-1 promoter fragment the two Sp1 sites and using to a product AP-2α with Sp1 at an of the indicate that AP-2α and AP-2α C-terminal deletion lacking the DNA-binding synergistically of suggesting that the of AP-2α is through the with Sp1, of its to directly the AP-2 sites of the KiSS-1 promoter. the of AP-2α without the C-terminal DNA-binding directly with Sp1 in the breast cancer cells with Sp1, and or with to Sp1 as as to the of and for the of cell that AP-2α or by demonstrated the of Sp1 protein in the and as with the AP-2α and with the Sp1 and in cells that either cell did not show any of AP-2α and levels are shown and results the direct of the of AP-2α with the Sp1 transcription in breast cell of metastatic breast cancer cells with KiSS-1 peptide as as ectopic of KiSS-1 in these cells has been shown to alter and thus metastasis (2Lee J.H. Welch D.R. Int. J. Cancer. 1997; 71: 1035-1044Crossref PubMed Scopus (161) Google Scholar, 4Ohtaki T. Shintani Y. Honda S. Matsumoto H. Hori A. Kanehashi K. Terao Y. Kumano S. Takatsu Y. Masuda Y. Ishibashi Y. Watanabe T. Asada M. Yamada T. Suenaga M. Kitada C. Usuki S. Kurokawa T. Onda H. Nishimura O. Fujino M. Nature. 2001; 411: 613-617Crossref PubMed Scopus (1186) Google Scholar, 8Muir A.I. Chamberlain L. Elshourbagy N.A. Michalovich D. Moore D.J. Calamari A. Szekeres P.G. Sarau H.M. Chambers J.K. Murdock P. Steplewski K. Shabon U. Miller J.E. Middleton S.E. Darker J.G. Larminie C.G. Wilson S. Bergsma D.J. Emson P. Faull R. Philpott K.L. Harrison D.C. J. Biol. Chem. 2001; 276: 28969-28975Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar, 10Kotani M. Detheux M. Vandenbogaerde A. Communi D. Vanderwinden J.M. Le Poul E. Brezillon S. Tyldesley R. Suarez-Huerta N. Vandeput F. C. M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The expression of like other tumor is commonly reduced or completely ablated in a variety of different cancers (3Masui T. Doi R. Mori T. Toyoda E. Koizumi M. Kami K. Ito D. Peiper S.C. Broach J.R. Oishi S. Niida A. Fujii N. Imamura M. Biochem. Biophys. Res. Commun. 2004; 315: 85-92Crossref PubMed Scopus (115) Google Scholar, 5Lee J.H. Welch D.R. Cancer Res. 1997; 57: 2384-2387PubMed Google Scholar, 6Ikeguchi M. Yamaguchi K. Kaibara N. Clin. Cancer Res. 2004; 10: 1379-1383Crossref PubMed Scopus (115) Google Scholar, 7Sanchez-Carbayo M. Capodieci P. Cordon-Cardo C. Am. J. Pathol. 2003; 162: 609-617Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). the of KiSS-1 expression has been to survival and of metastatic KiSS-1 has become an for the metastasis of these cancers M. Capodieci P. Cordon-Cardo C. Am. J. Pathol. 2003; 162: 609-617Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). this we that the expression of KiSS-1 is directly with the expression of the AP-2α transcription metastatic breast cell lines to AP-2α the loss of the KiSS-1 gene in these cells. we found that AP-2α and Sp1 synergistically activate the transcriptional of KiSS-1 promoter in multiple breast cancer cell lines via direct of these two transcription at two tandem Sp1-binding sites of the promoter. Although levels of AP-2α and Sp1 expression to the other breast lines the in KiSS-1 promoter upon of transcription is to the caused by the of these in the cells with the KiSS-1 promoter The KiSS-1 promoter was found to multiple sites, of which by of Sp1 this was in KiSS-1 a for regulation by AP-2α and Sp1 of the KiSS-1 demonstrated that the regulation was through a of the promoter and with two Sp1 The of Sp1 to to this was in in which an this promoter as by its to the results for a of the by the thus that AP-2α may this using the of the promoter the two tandem Sp1 sites demonstrated that AP-2α and Sp1 of promoter at these two Sp1 sites demonstrated the binding of the at these two sites and the of these two sites in and transcriptional of Although AP-2α is to transcriptional regulation of the KiSS-1 our using a dominant negative form of AP-2α that the DNA-binding domain, and the demonstrated that the of AP-2α a protein to transcriptional of KiSS-1 and that the of AP-2α is of its our suggest a possible for transcriptional of KiSS-1 in breast cells in which AP-2α is the of AP-2α with Sp1, which to the Sp1-binding The AP-2α and Sp1 of the KiSS-1 promoter and to the expression of KiSS-1 gene AP-2α expression is as commonly found in cancer of transcription to a loss of KiSS-1 transcriptional to transcriptional regulation by in which AP-2α and to its DNA-binding T. R. PubMed Scopus Google our results a in which AP-2α regulation is through two tandem Sp1 to and direct of the of AP-2α with Sp1 KiSS-1 was originally identified to reduce melanoma metastasis by using microcell-mediated transfer of (2Lee J.H. Welch D.R. Int. J. Cancer. 1997; 71: 1035-1044Crossref PubMed Scopus (161) Google Scholar). Although KiSS-1 was to it was that this of the a and as KiSS-1 that KiSS-1 expression upon transfer into melanoma cells. AP-2α is in the of the to AP-2α may in the KiSS-1 a the loss of the AP-2α transcription and loss of the KiSS-1 metastasis suppressor in breast cancer cell Loss of AP-2α has been in the of other cancers invasive breast including and cancer, cell and KiSS-1 expression has been found to reduced in of these it is possible that KiSS-1 expression in these tissues is the of AP-2α with Sp1 and that the loss of AP-2α is by the loss of KiSS-1 such cells to to metastatic this our studies are these of the of AP-2α in KiSS-1 expression in other cancers to identify the loss of KiSS-1 expression in such tissues and cancers.