Tumor Suppressor Role of KiSS-1 in Bladder Cancer
2003; Elsevier BV; Volume: 162; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)63854-0
ISSN1525-2191
AutoresMarta Sänchez‐Carbayo, Paola Capodieci, Carlos Cordon‐Cardo,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe expression profiles of nine bladder cancer cell lines were compared against a pool containing equal total RNA quantities of each of them. Lower expression of KiSS-1 was revealed in cells derived from the most advanced bladder tumors. When comparing 15 primary bladder tumors versus a pool of four bladder cancer cell lines, lower transcript levels of KiSS-1 were observed in the invasive bladder carcinomas as compared to superficial tumors. KiSS-1 expression ratios provided prognostic information. The expression pattern of KiSS-1 transcripts was analyzed using in situ hybridization in nine bladder cancer cells, paired normal urothelium and bladder tumor samples (n = 25), and tissue microarrays of bladder tumors (n = 173). We observed complete loss of KiSS-1 in all invasive tumors under study as compared to their respective normal urothelium. The expression of KiSS-1 was found to be significantly associated with histopathological stage. Patients with lower KiSS-1 expression showed a direct correlation with overall survival in a subset of bladder tumors whose follow-up was available (n = 69). We did not observe any significant differential KiSS-1 expression along cell cycle by sorting analysis. A potential tumor suppressor role in bladder cancer was revealed for KiSS-1. Moreover, it showed predictive value by identifying patients with poor outcome. The expression profiles of nine bladder cancer cell lines were compared against a pool containing equal total RNA quantities of each of them. Lower expression of KiSS-1 was revealed in cells derived from the most advanced bladder tumors. When comparing 15 primary bladder tumors versus a pool of four bladder cancer cell lines, lower transcript levels of KiSS-1 were observed in the invasive bladder carcinomas as compared to superficial tumors. KiSS-1 expression ratios provided prognostic information. The expression pattern of KiSS-1 transcripts was analyzed using in situ hybridization in nine bladder cancer cells, paired normal urothelium and bladder tumor samples (n = 25), and tissue microarrays of bladder tumors (n = 173). We observed complete loss of KiSS-1 in all invasive tumors under study as compared to their respective normal urothelium. The expression of KiSS-1 was found to be significantly associated with histopathological stage. Patients with lower KiSS-1 expression showed a direct correlation with overall survival in a subset of bladder tumors whose follow-up was available (n = 69). We did not observe any significant differential KiSS-1 expression along cell cycle by sorting analysis. A potential tumor suppressor role in bladder cancer was revealed for KiSS-1. Moreover, it showed predictive value by identifying patients with poor outcome. Transitional cell carcinomas (TCCs) have been classified into two groups with distinct clinical behavior and different molecular profiles. Low-grade tumors are always papillary and usually superficial, whereas high-grade tumors can be either papillary or nonpapillary and are often invasive. Patients diagnosed with localized TCC have a 5-year relative survival rate of 93%. However, patients presenting with regional and distant disease spread have 5-year relative survival rates of 49% and 6%, respectively.1: Cancer Facts and Figures 2000.: American Cancer Society 2000Google Scholar Bladder cancer progression and development of secondary metastases follow complex sequential steps characterized by alterations of many genes involved in critical cell functions, definitively not being completely understood.2Fidler IJ Critical factors in the biology of human cancer metastasis.Cancer Res. 1990; 50: 6130-6138PubMed Google Scholar It has been reported that genes on chromosome 6 could regulate expression of metastasis suppressor genes encoded elsewhere within the genome.3Welch DR Chen P Miele ME McGary CT Bower JM Weissman BE Standbridge EJ Microcell-mediated transfer of chromosome 6 into metastatic human C8161 melanoma cells suppresses metastasis but does not inhibit tumorigenicity.Oncogene. 1994; 9: 255-262PubMed Google Scholar, 4Miele ME Robertson G Lee JH Coleman A McGarry CT Fisher PB Lugo TG Welch DR Metastasis suppressed, but tumorigenicity and local invasiveness unaffected in the human melanoma cell line MelJuSo after introduction of human chromosomes 1 or 6.Mol Carcinog. 1996; 15: 284-299Crossref PubMed Scopus (66) Google Scholar, 5Lee JH Miele ME Hicks DJ Phillips KK Trent JM Weissman BE Welch DR KiSS-1, a novel human malignant melanoma metastasis-suppressor gene.J Natl Cancer Inst. 1996; 88: 1731-1737Crossref PubMed Scopus (853) Google Scholar, 6Lee JH Welch DR Identification of highly expressed genes in metastasis-suppressed chromosome 6/human malignant melanoma hybrid cells using substractive hybridization and differential display.Int J Cancer. 1997; 71: 1035-1044Crossref PubMed Scopus (162) Google Scholar, 7Lee JH Welch DR Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1.Cancer Res. 1997; 57: 2384-2387PubMed Google Scholar, 8West A Votja PJ Welch DR Weissman BE Chromosome localization and genomic structure of the KiSS-1 metastasis suppressor gene.Genomics. 1998; 54: 145-148Crossref PubMed Scopus (160) Google Scholar KiSS-1, mapping to chromosome 1q32,8West A Votja PJ Welch DR Weissman BE Chromosome localization and genomic structure of the KiSS-1 metastasis suppressor gene.Genomics. 1998; 54: 145-148Crossref PubMed Scopus (160) Google Scholar is one of these genes regulated at chromosome 6. KiSS-1 expression was discovered to be reduced in metastatic melanoma.3Welch DR Chen P Miele ME McGary CT Bower JM Weissman BE Standbridge EJ Microcell-mediated transfer of chromosome 6 into metastatic human C8161 melanoma cells suppresses metastasis but does not inhibit tumorigenicity.Oncogene. 1994; 9: 255-262PubMed Google Scholar, 4Miele ME Robertson G Lee JH Coleman A McGarry CT Fisher PB Lugo TG Welch DR Metastasis suppressed, but tumorigenicity and local invasiveness unaffected in the human melanoma cell line MelJuSo after introduction of human chromosomes 1 or 6.Mol Carcinog. 1996; 15: 284-299Crossref PubMed Scopus (66) Google Scholar, 5Lee JH Miele ME Hicks DJ Phillips KK Trent JM Weissman BE Welch DR KiSS-1, a novel human malignant melanoma metastasis-suppressor gene.J Natl Cancer Inst. 1996; 88: 1731-1737Crossref PubMed Scopus (853) Google Scholar, 6Lee JH Welch DR Identification of highly expressed genes in metastasis-suppressed chromosome 6/human malignant melanoma hybrid cells using substractive hybridization and differential display.Int J Cancer. 1997; 71: 1035-1044Crossref PubMed Scopus (162) Google Scholar Experimental and clinical studies have revealed its role as a functionally active metastasis suppressor gene in certain tumors.5Lee JH Miele ME Hicks DJ Phillips KK Trent JM Weissman BE Welch DR KiSS-1, a novel human malignant melanoma metastasis-suppressor gene.J Natl Cancer Inst. 1996; 88: 1731-1737Crossref PubMed Scopus (853) Google Scholar, 6Lee JH Welch DR Identification of highly expressed genes in metastasis-suppressed chromosome 6/human malignant melanoma hybrid cells using substractive hybridization and differential display.Int J Cancer. 1997; 71: 1035-1044Crossref PubMed Scopus (162) Google Scholar, 7Lee JH Welch DR Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1.Cancer Res. 1997; 57: 2384-2387PubMed Google Scholar, 8West A Votja PJ Welch DR Weissman BE Chromosome localization and genomic structure of the KiSS-1 metastasis suppressor gene.Genomics. 1998; 54: 145-148Crossref PubMed Scopus (160) Google Scholar, 9Ringel MD Hardy E Bernet VJ Burch HB Schuppert F Burman KD Saji M Metastin receptor is overexpressed in papillary thyroid cancer and activated MAP kinase in thyroid cancer cells.J Clin Endocrinol Metab. 2002; 87: 2399-2402Crossref PubMed Scopus (128) Google Scholar Regulation of events downstream of cell-matrix adhesion involving cytoskeleton reorganization has been attributed to KiSS-1.7Lee JH Welch DR Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1.Cancer Res. 1997; 57: 2384-2387PubMed Google Scholar, 10Yan C Wang H Boyd DD KiSS-1 represses 92-kDa type IV collagenase expression by down-regulating NF-kappa B binding to the promoter as a consequence of Ikappa Balpha-induced block of p65/p50 nuclear translocation.J Biol Chem. 2001; 276: 1164-1172Crossref PubMed Scopus (188) Google Scholar, 11Ohtaki 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 Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor.Nature. 2001; 411: 613-617Crossref PubMed Scopus (1205) Google Scholar, 12Kotani M Detheux M Vandenbogaerde A Communi D Vanderwinden JM Le Poul E Brezillon S Tyldesley R Suarez-Huerta N Vandeput F Blanpain C Schiffmann SN Vassart G Parmentier M The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54.J Biol Chem. 2001; 276: 34631-34636Crossref PubMed Scopus (1251) Google Scholar, 13Hori A Honda S Asada M Ohtaki T Oda K Watanabe T Shintani Y Yamada T Suenaga M Kitada C Onda H Kurokawa T Nishimura O Fujino M Metastin suppresses the motility and growth of CHO cells transfected with its receptor.Biochem Biophys Res Commun. 2001; 286: 958-963Crossref PubMed Scopus (95) Google Scholar, 14Muir AI Chamberlain L Elshourbagy NA Michalovich D Moore DJ Calamari A Szekeres PG Sarau HM Chambers JK Murdock P Steplewski K Shabon U Miller JE Middleton SE Darker JG Larminie CG Wilson S Bergsma DJ Emson P Faull R Philpott KL Harrison DC AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1.J Biol Chem. 2001; 276: 28969-28975Crossref PubMed Scopus (765) Google Scholar However, the mechanism by which KiSS-1 is involved in the invasive/metastatic phenotype has not been elucidated. A differential expression of KiSS-1 and a clone representing the regulatory counterpart of KiSS-1 on chromosome 63Welch DR Chen P Miele ME McGary CT Bower JM Weissman BE Standbridge EJ Microcell-mediated transfer of chromosome 6 into metastatic human C8161 melanoma cells suppresses metastasis but does not inhibit tumorigenicity.Oncogene. 1994; 9: 255-262PubMed Google Scholar, 4Miele ME Robertson G Lee JH Coleman A McGarry CT Fisher PB Lugo TG Welch DR Metastasis suppressed, but tumorigenicity and local invasiveness unaffected in the human melanoma cell line MelJuSo after introduction of human chromosomes 1 or 6.Mol Carcinog. 1996; 15: 284-299Crossref PubMed Scopus (66) Google Scholar were identified through molecular profiling analysis of bladder cancer cell lines and primary bladder tumors. We further evaluated the role of KiSS-1 in bladder cancer progression by characterizing its mRNA expression levels in a comprehensive collection of bladder cancer cell lines, as well as in normal urothelium, and in a large cohort of bladder tumors. Nine bladder cancer cell lines including T24, J82, 5637, HT-1376, RT4, SCaBER, TCCSUP, UMUC-3, and HT1197, were obtained from the American Type Culture Collection (Rockville, MD) and maintained following standard procedures. All cells were grown and harvested at 75 to 90% confluence no longer than four to six passages for the extraction of total RNA using RNeasy protocol (Qiagen, Valencia, CA). Cytospins were also prepared and later used for analysis of the expression of the targets under study. Total RNA from bladder tumors was isolated in two steps using TRIzol (Life Technologies, Inc., Grand Island, NY), followed by RNeasy (Qiagen) purification. Two separate studies of expression profiling in bladder cancer on bladder cancer cell lines and clinical material using different sets of cDNA microarrays were performed. First, a set of 8976 sequence-verified human IMAGE cDNA clones, representing both known genes and expressed sequence tags, was used to compare the expression profiles of nine bladder cancer cell lines (T24, J82, 5637, HT-1376, RT4, SCaBER, TCCSUP, UMUC-3, and HT1197) against a pool containing equal RNA quantities of each of them. The second set contained 17,842 known genes and expressed sequence tags and was used to study the transcriptome of 15 bladder cancer tumors against a pool containing equal RNA quantities of four selected bladder cancer cell lines: T24, J82, RT4, and HT1197. Clones were polymerase chain reaction amplified and spotted onto poly-l-lysine-coated microscope slides using a custom robot designed and built at Albert Einstein College of Medicine, New York, NY (sequence.aecom.yu.edu/bioinf/funcgenomic.html).15Cheung VG Morley M Aguilar F Massimi A Kucherlapati R Childs G Making and reading microarrays.Nat Genet. 1999; 21: S15-S19Crossref PubMed Scopus (567) Google Scholar In the first gene expression analysis of the bladder cancer cell lines, 10 μg of total RNA of each cell line was labeled with Cy5 and hybridized against 10 μg of total RNA of a pool containing equal RNA quantities of each of these cell lines labeled with Cy3.16Stears RL Getts RC Gullans SR A novel, sensitive detection system for high-density microarrays using dendrimer technology.Physiol Genomics. 2000; 3: 93-99Crossref PubMed Scopus (189) Google Scholar In the expression-profiling study of the bladder tissues, 5 μg of total RNA of each bladder tissue and pool of cell lines was linearly amplified using a single amplification round.17Wang E Miller LD Ohnmacht GA Liu ET Marincola FM High-fidelity mRNA amplification for gene profiling.Nature Biotechnol. 2000; 18: 457-459Crossref Scopus (582) Google Scholar Amplified cRNA obtained from bladder tissues (normal or tumor) were labeled with Cy5 and hybridized against amplified cRNA obtained from a pool containing equal RNA quantities of the four cell lines labeled with Cy3. After hybridization, slides were washed, dried, and scanned by an Axon automated laser scanner. GenePix software was used for gridding and calculation of red and green signal intensities.18Eisen MB Spellman PT Brown PO Botstein D Cluster analysis and display of genome-wide expression patterns.Proc Natl Acad Sci USA. 1998; 95: 14863-14868Crossref PubMed Scopus (13311) Google Scholar Before any analysis, plots of the fold change versus the average intensity were examined to look for abnormalities in single-array data. Samples were normalized using this intensity-dependent normalization using the Splus function lowess.19Felsenstein J Confidence limits on phylogenies: an approach using the bootstrap.Evolution. 1985; 39: 783-791Crossref PubMed Google Scholar We focused on the expression results in these two independent studies of two clones representing KiSS-1 and 6q16.2-21, whose accession numbers were AA464595 and N76881, respectively. We collected the red to green ratio (R/G) expression data of these two clones for the analysis of this study. Twenty-five paired normal urothelium and respective bladder tumors were first evaluated. All these tumors presented high tumor grade. Three different bladder cancer microarrays also were constructed for this study. All these specimens were obtained under Institutional Review Board-approved protocols. Normal and tumor tissues were embedded in paraffin and 5-μm sections were stained with hematoxylin and eosin to identify viable, morphologically representative areas of the specimen from which needle core samples were taken, using a precision instrument (Beecher Instruments, Silver Spring, MD). From each specimen triplicate cores with diameters of 0.6 mm were punched and arrayed on the recipient paraffin block.20Hoos A Urist MJ Stojadinovic A Mastorides S Dudas ME Leung DHY Kuo D Brennan MF Lewis JJ Cordon-Cardo C Validation of tissue microarrays for immunohistochemical profiling of cancer specimens using the example of human fibroblastic tumors.Am J Pathol. 2001; 158: 1245-1251Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar Five-μm sections of these tissue array blocks were cut and placed on charged poly-l-lysine-coated slides and used for in situ hybridization analysis. These tissue microarrays included a total of 173 bladder primary TCC tumors. Tumor stage and grade were defined according to consensus criteria.21American Joint Committee on Cancer Staging of cancer at genitourinary sites.in: American Joint Committee on Cancer: Manual for Staging of Cancer. ed 3. J. B. Lippincott Co., Philadelphia1988: 194-195Google Scholar, 22Mostofi FK Histological Typing of Urinary Bladder Tumors. World Health Organization, Geneva1973Google Scholar A total of 40 superficial (TIS, pTa, T1) and 64 invasive (pT2, pT3, pT4) TCC tumors were analyzed in two microarrays. These tumors corresponded to 14 grade 1, 8 grade 2, and 82 grade 3 lesions. The third tissue microarray comprised a cohort of 69 well-characterized bladder primary TCC cases with known p53 and pRB status, and consisted of two superficial (TIS, pTa, T1) and 67 invasive lesions (pT2, pT3, pT4). Human KiSS-1 cDNA sequence (GenBank accession number, AA464595) was obtained from the set of clones used in the cDNA microarrays. Recombinant plasmid (1 μg) was linearized by EcoRI and NotI. Riboprobes were generated with T7 and T3 polymerases for 2 hours at 37°C in 1× transcription buffer (Boehringer Mannheim, Indianapolis, IN) containing 20 U of RNase inhibitor; 1 mmol/L each of ATP, GTP, and CTP; 6.5 mmol/L of UTP; and 3.5 mmol/L of digoxigenin-UTP. Deparaffinized tissue sections were rinsed in water and phosphate-buffered saline for 10 minutes. Slides were digested in prewarmed in citrate buffer (pH = 6) for 5 minutes in microwave at full power. Prehybridization was performed for 30 minutes at 45°C in 50% deionized formamide and 2× standard saline citrate. For hybridization, 10 pmol/L of digoxigenin-labeled riboprobe was added to 50 μl of hybridization buffer [50% deionized formamide (v/v), 10% dextran sulfate, 2× standard saline citrate, 1% sodium dodecyl sulfate, and 0.25 mg/ml of herring sperm DNA]. After overnight incubation at 45°C, slides were washed twice for 20 minutes in prewarmed 2× standard saline citrate at 42°C, followed by two washes in prewarmed 1× standard saline citrate at 42°C for 20 minutes. Slides were then incubated in normal sheep serum diluted in buffer 1 (2 mol/L Tris-HCl, 5 mol/L NaCl, pH 7.5) for 30 minutes followed by incubation in the same buffer with antibody digoxigenin-AP (Boehringer Mannheim) at a dilution of 1:500 for 2 hour at room temperature. Visualization was accomplished by nitro blue tetrazolium 5-bromo-4-chloro-3-indolylphosphate. The slides were counterstained with methyl green and mounted.23Capodieci P Magi-Galluzi C Moreira Jr, G Zeheb R Loda M Automated in situ hybridization: diagnostic and research applications.Diagn Mol Pathol. 1998; 7: 69-75Crossref PubMed Scopus (14) Google Scholar Protein patterns of expression of p53, pRB, and certain adhesion molecules were assessed at the microanatomical level, using both cytospin from all cell lines and tissue samples outlined above. Standard immunoperoxidase procedures were used for immunohistochemistry. The following antibodies were used: E-cadherin, mouse monoclonal clone 36 at 1:1000 (2.5 μg/ml) (BD Transductions Labs, Lexington, KY); moesin, mouse monoclonal clone 38/87 at 1:50 (4 μg/ml) with microwave pretreatment of the slides (Neomarkers, Fremont, CA); zyxin, mouse monoclonal clone 21 at 1:25 (10 μg/ml) with microwave pretreatment of the slides (Transduction Labs); underphosphorylated pRB, mouse monoclonal clone G99-549 at a final concentration of 10 μg/ml (PharMingen, San Diego, CA); total RB, mouse monoclonal clone 3C8 at a final concentration of 1.2 μg/ml (QED Bioscience, San Diego, CA); p53 mouse monoclonal clone 1801 at 0.2 μg/ml (Calbiochem, Cambridge, MA); p21/WAF1, mouse monoclonal Ab-1 at 5 μg/ml (Calbiochem); cyclin D1, mouse monoclonal 1 μg/ml (Calbiochem); cyclin E, purified rabbit antiserum (1:500, supplied as a tissue culture supernatant by Dr. A. Koff, Memorial Sloan-Kettering Cancer Center). Staining conditions were optimized on sections from formalin-fixed, paraffin-embedded tissue controls for each antibody as specified by manufacturers. Antibody reactivity was detected by using diaminobenzidine as chromogen, and sections were counterstained with hematoxylin. The primary antibody was omitted for negative controls. All TCCs (n = 173) were used for the analysis of association between KiSS-1 p53, pRB, and other cell-cycle and adhesion molecules evaluated by immunohistochemistry. These cases were also used for evaluating KiSS-1 mRNA expression versus histopathological stage and tumor grade, using the nonparametric Wilcoxon-Mann-Whitney and Kruskall-Wallis tests.24Dawson-Saunders B Trapp RG Basic and Clinical Biostatistics. ed 2. Appleton & Lange, Norwalk1994Google Scholar The consensus value of the three representative cores from each tumor sample arrayed was used for statistical analyses. KiSS-1 expression values were displayed as negative to low (0 to 20%), intermediate (20 to 40%), and high (>40%). These cutoffs were selected to represent low different degrees of expression of KiSS-1. The prognostic value of the ratios of KiSS-1 and 6q16.2-21 was evaluated in the subsets of 15 patients to whom cDNA microarray analysis were performed. The relationship of the in situ hybridizations of KiSS-1 to outcome was also evaluated using a subset of 69 TCC cases for which follow-up was available. Specific of disease overall survival time was defined as the months elapsed between transurethral resection (two superficial lesions) or cystectomy (rest of cases) and death from disease (or the last follow-up date). Patients who were alive at the last follow-up or lost to follow-up were censored. For survival analysis of the patients to whose tumors expression profiles were analyzed using cDNA microarrays, ratios of KiSS-1 and 6q16.2-21 and mRNA levels of KiSS-1 were analyzed as categorical variables taking the cutoffs of 1.5 and 4.0, respectively. When we evaluated the prognostic value of the mRNA levels of KiSS-1 obtained by in situ hybridization on the cohort of 69 patients, we used the expression cutoff of 40% to define the expression of KiSS-1 as high or positive. This cutoff was selected to represent low and high degrees of expression of KiSS-1. The association of the KiSS-1 expression levels with overall survival was analyzed using the log-rank test. Survival curves were plotted using standard Kaplan-Meier methodology.24Dawson-Saunders B Trapp RG Basic and Clinical Biostatistics. ed 2. Appleton & Lange, Norwalk1994Google Scholar Associations between biomarkers were analyzed using Kendall's tau test using the SPSS statistical package (version 8.0). Cells were grown and collected with trypsin at 80% confluency. They were washed with phosphate-buffered saline and stained with Hoechst 33342 for 2 hours at 37°C. Cell cycle subpopulations G1, S, and G2M were sorted using a MoFlow sorter. Cytospins of each cell cycle phase were fixed with methanol:acetone (1:1 v/v) during 10 minutes at −20°C and kept at −80°C until the analysis of KiSS-1 by in situ hybridization. The present analysis was conducted under four major sets of experiments. Initially, bladder cancer cell lines and primary bladder tumor samples were analyzed using cDNA microarrays and KiSS-1 was identified as a differentially expressed gene between distinct histopathological tumor types and stages of the disease. Paired normal urothelium and tumor were evaluated to assess the potential involvement of KiSS-1 in the progression of the disease. Third, tissue microarrays were used to validate the potential clinical significance of KiSS-1 at the microanatomical detail using in situ hybridization on tissue samples of well-characterized cases. A cohort of superficial and invasive bladder neoplasms was used to evaluate the association between KiSS-1 with histopathological stage and grade. An additional tissue microarray, containing bladder tumors from cases with annotated follow-up, was used to delineate associations of KiSS-1 with clinical outcome. Finally, KiSS-1 expression along cell cycle was explored using cytospins containing enriched cell-cycle populations of the bladder cancer cell lines under study obtained by sorting analysis. We identified differential expression of KiSS-1 in bladder cancer in two independent profiling approaches. First, the expression profiling of nine bladder cancer cell lines: T24, J82, 5637, HT-1376, RT4, SCaBER, TCCSUP, UMUC-3, and HT1197, was compared against a pool containing equal RNA quantities of each of them using cDNA arrays containing 8976 clones. This analysis revealed low expression of KiSS-1 in most of the cell lines derived from the most advanced bladder carcinomas as compared to the ones obtained from a papillary tumor (RT4) (Table 1).Table 1Differential Red-to-Green Ratios of KiSS-1 and 6q16.2-21 among Bladder Cancer Cell Lines Out of the cDNA Microarrays Comparing Each of the Cell Lines (Cy5) Versus a Pool Containing Equal RNA Quantities of Each of Them (Cy3)Bladder cancer cell lineR/G ratio of KiSS-1 (AA464595)R/G ratio of 6q16.2-21 (N76881)UMUC-30.90.9856371.221.15HT11971.030.88HT13760.740.97J822.421.1T241.041.05SCABER0.761.32TCCSUP0.81.04RT42.821.88 Open table in a new tab The expression profiles of 15 bladder cancer tumors was analyzed versus a pool containing equal RNA quantities of four selected bladder cancer cell lines: T24, J82, RT4, and HT1197 using cDNA microarrays containing 17,842 known genes and expressed sequence tags (Table 1). We observed that transcripts of KiSS-1 were highly expressed in superficial noninvasive tumors when compared to organ-confined invasive and primary invasive TCCs of patients that developed metastatic lesions (Table 2). We also observed that messenger expression levels of the 6q16.2-21 clone displayed similar variations as the KiSS-1 gene in both tumor cell lines and clinical samples analyzed (Table 1, Table 2) .Table 2Differential Red-to-Green Ratios (R/G) of KiSS-1 and 6q16.2-21 Obtained from the cDNA Microarray Analysis Comparing Bladder Tumors (R) and a Pool of Bladder Cancer Cell Lines (G)Patient IDKiSS-1 R/G (AA464595)6q16.2-21 R/G (N76881)AgeSexTNMCarcinoma in situSmoking habitFamilial cancer historyFollow-up (months)Clinical outcome1744.5812.7767MTISG3N0+−−12NED1602.607.1083MTAG1N0+−−20NED1573.0115.3661MTISG3N0+−−44NED1342.6015.6480MT3BG3NO+++13NED1700.921.5055MT4G3N0M1++NK4DOD1681.512.7161MT4BG3N1M2++NK1DOD1691.984.1175FTAG3NO−−NK41NED1651.414.2675MTAG1NO−−−11NED1631.693.5465MTAG1NO−++17NED1623.3419.2660MTISG3NO+++15NED1411.255.1849FTAG3M1−++3DOD1351.481.9064FT4BG3N1+++11DOD1331.275.4383MT3BG3NO+++1DOD1301.112.3072MT3BG3N0−−−13NED1241.822.0059FT3AG3N1M0−+−9NEDAccession numbers of KiSS-1 and 6q16.2-21 are included in brackets. NK, not known; NED, no evidence of disease; DOD, death of disease. Open table in a new tab Accession numbers of KiSS-1 and 6q16.2-21 are included in brackets. NK, not known; NED, no evidence of disease; DOD, death of disease. We evaluated whether the expression ratios of KiSS-1 and 6q16.2-21 obtained from the cDNA microarray analysis of the patients with bladder tumors could provide additional predictive information. The overall survival analysis revealed that KiSS-1 ratios were significantly associated with overall survival in this subset of 15 bladder lesions (Figure 1). Patients whose tumor samples were subjected to gene profiling had a median follow-up of 12 months (mean, 14.3 months; range, 1 to 44 months) (Table 2). We further investigated the prognostic utility of KiSS-1 for overall survival in a separate set of patients with bladder cancer (see below). The differential expression of KiSS-1 in the progression of bladder cancer was evaluated in 25 paired normal urothelium and respective bladder tumors. High positive expression of KiSS-1 was observed in all normal urothelium samples (Figure 2). All superficial tumors showed intermediate or high expression of KiSS-1. Three invasive patients displayed intermediate expression of KiSS-1, whereas undetectable to low expression was observed in 80% (14 of 17) of the invasive tumors. All primary bladder tumors that developed distant metastases showed complete loss of KiSS-1. KiSS-1 expression levels between superficial and invasive tumors were found to reach a significant association (Mann-Whitney, P = 0.001). All these tumors presented high tumor grade. We evaluated the potential correlation between KiSS-1 expression and the suspicion of vascular invasion, positive nodes, multifocality, squamous differentiation, and carcinoma in situ (Table 3). Interestingly, we only observed a significant association of KiSS-1 expression with those patients in which vascular invasion was reported (Mann-Whitney, P = 0.012).Table 3Messenger Expression of KiSS-1 by in Situ Hybridization in the Paired Normal Urothelium (NU) and Bladder Tumors (TM) Regarding Their Histopathological Stage, Grade, the Presence of Positive Lymph Nodes (LN) and Vascular Invasion (VI), Multifocality (MF), Carcinoma in Situ (IS), and Squamous Differentiation (SQ)IDKiSS-1 NUKiSS-1 TMAgeSexStageLNVIMFISSQ1>40% 40%20–40%42MPTA−−−−−3>40%20–40%58FPT3B++−++4>40%20–40%64MPT3B+−−+−5>40% 40%>40%43MIS−−++−7>40% 40%20–40%78FPT3B−−−++9>40%>40%75MPT1−−−−−10>40%>40%73MIS−−−+−11>40% 40% 40%>40%(NT)61MPT1−−−−−14>40%>20%66MPT1−−−−−15>40% 40% 40% 40% 40%<20%6
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