Annals of Oncology Advance Access originally published online on December 12, 2006
Annals of Oncology 2007 18(3):605-607; doi:10.1093/annonc/mdl421
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2006 European Society for Medical Oncology
letters to the editor |
Metastin (KISS-1) and metastin-coupled receptor (GPR54) expression in transitional cell carcinoma of the bladder
KISS-1 (1q32) has been identified as a metastasis suppressor gene in melanomas and breast cancers [1]. The KISS-1 gene product was also recently identified as the endogenous agonist for the Gq/11-coupled receptor (19p13) known as GPR54 [2], OT7T175 [3] or AXOR12 [4]. The KISS-1 gene encodes a 145-amino acid peptide containing a secretory signal sequence at the N-terminus (amino acid 1–19), which indicates that KISS-1 functions as a secretory protein [5]. The full-length KISS-1 protein, however, has not been detected in a secreted form. Instead, three truncated fragments of KISS-1 occur naturally in human placenta and are termed metastatin (54 amino acids), kisspeptin-14 (14 amino acids) and kisspeptin-13 (13 amino acids) [3, 2]. The expression and functional effects of the metastin and the metastin receptor remain unclear in carcinogenesis. While KISS-1 has been found to be a possible metastasis suppressor gene in human melanoma and breast carcinoma cells as well as in human ovarian cancer [6], its function is actually controversial in breast cancers and in other types of tumors. Studies in thyroid [7], breast [8] and hepatocellular [9] cancers showed an association between increased KISS-1 levels and disease progression. In bladder cancer, the only previous report indicates an association between a loss of KISS-1 expression and bladder cancer progression or a worse clinical outcome [10].Given this incomplete information, this study investigates for the first time the coupled expression of GPR54 and KISS-1 genes in human bladder transitional cell carcinoma (TCC).
KISS-1 and GPR54 expressions were examined using real-time RT-PCR in six bladder cancer cell lines (1207, T24, 647V, J82, RT112 and SD48) and in 64 human bladder TCCs (38 superficial and low-grade bladder carcinomas and 26 invasive high-grade bladder carcinomas) in relation to normal human bladder tissue (using a pool of RNA made up from five donors). RNA was isolated and converted to complementary DNA (cDNA). Total RNA was extracted from frozen bladder cancer specimens (100–300 µg of frozen tissue) using the acid–phenol guanidium method. Tumoral bladder epithelia were dissected under histological control. Deoxyribonuclease (DNAse) treatment was carried out using DNAse I (Invitrogen Life Technologies). One microgram of total RNA was converted to cDNA using the Superscript Reverse Transcriptase Kit (Invitrogen Life Technologies; Carlsbad, CA, USA). First-strand cDNA was synthesized in a total volume of 20 µl by heating at 42°C for 1 h. Then, real-time quantitative RT-PCR (qRT-PCR) was carried out and the data were analyzed on an Applied Biosystems 7000 Sequence Detection System instrument (Applied Biosystems, Foster City, CA) as described previously [11]. Reference genes ppia [12] and rplo [12] were utilized for gene normalization for relative quantification of target genes. The qRT-PCRs were carried out using the reagents and protocol contained in the SYBR® GREEN PCR Master Mix (Applied Biosystems) and using the following primers: KISS-1 (F) ACT CAC TGG TTT CTT GGC AGC T, (R) CAG AGG CCA CCT TTT CTA ATG G; GPR54 (F) CGA CTT CAT GTG CAA GTT CGT C, (R) CAC ACT CAT GGC GGT CAG AG. Reactions were run in a volume of 20 µl that contained 5 µl of cDNA (1 : 20 dilution of reverse transcription product) and 1 µl of each primer mixture (from a stock solution at 10 pmol/µl). For comparison of gene expression levels, we used the nonparametric Mann–Whitney for quantitative values and KI2 test for qualitative values using the SPSS 14.0 Software. A P value <0.05 was considered to be significant.
A strong correlation between the target gene expression levels using reference genes rplpo and ppia was observed (for KISS-1: r2 = 0.97 and P < 0.0001; for GPR54: r2 = 0.64 and P < 0.0001).
In the cell lines, the 2–
Ct expressions of KISS-1 and of GPR54 (both referred to ppia) were, respectively, 0.3 and 0.1/0.3 and 0.0/0.6 and 0.1/0.8 and 0.8/1.1 and 0.0/25.9 and 2.1 for RT112/T24/J82/1207/SD48/647V. Sanchez-Carbayo et al. [10] have previously reported that low expression of KISS-1 was observed in cell lines derived from the most advanced bladder carcinomas such as T24 or J82. In their report and in ours, the ratio of T24/J82 KISS-1 expression is roughly 0.5. Only the cell line 1207, derived from a high-grade infiltrating tumor, and the cell line 647V, derived from a low-grade superficial tumor, expressed significant levels of both KISS-1 and GPR54 genes. Interestingly, we have recently characterized all these six cell lines for their response to the growth inhibitor Gefitinib® (Astra Zeneca, Macclesfield, UK), and shown that only 647V and 1207 were sensitive to this epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor without dependence of the EGFR expression level [13].
In the TCC from bladder specimens, the levels of KISS-1 expression showed significant increase (mean ± standard deviation, low grade 2.54 ± 3.12, high grade 4.07 ± 12.94) compared with normal bladder tissue (where 2–Ct = 1). Between low- and high-grade groups, the results indicated that the mean KISS-1/PPIA and KISS-1/RPLO ratios were not significantly different (P = 0.37). GPR54 also showed high levels of expression in bladder carcinomas compared with normal bladder tissue, and the difference between low- and high-grade groups was significant (low grade 4.32 ± 5.95, high grade 29.03 ± 114.94, P = 0.03) (Figure 1). When we categorized levels of expression (<0.5 = low; 0.5–1.5 = normal; >1.5 = high), we observed an association between an increase of the aggressiveness of the tumor ranges and the expression level of gpr54 (referenced to ppia and rplo, P = 0.005 and P = 0.001). No significant association was found for KISS-1 (P = 0.377 and P = 0.105) even though our results (low KISS-1 expression in 26% and high KISS-1 expression in 52% of high-grade tumors) were close to the observations of Sanchez-Carbayo et al. [10] who reported, using in situ hybridization: low KISS-1 expression in 34% and high KISS-1 expression in 45% of high-grade tumors. No correlation (r2 = 0.001, P = 0.104; r2 = 0.04', P = 0.20) was observed between GPR54 and KISS-1 expression, respectively.
|
Tyrosine kinase receptors and G protein-coupled receptors are signaling pathways involved in cell growth, cell migration and cell adhesion, and they are highly activated in cancer progression. The molecular mechanisms by which KISS-1 is involved in the invasive/metastatic phenotype have not been elucidated. Overexpression of the GPR54 gene in thyroid cancer was reported and has been shown to activate the intracellular growth signal pathway [7]. Stafford et al. [14] found that activation of the receptor by the KISS-1 peptide leads to activation of G protein-activated phospholipase C (PLC) and subsequently to inhibition of cell proliferation and cell migration. Jiang et al. [6] showed that KISS-1 expression exerts its effect (inhibition of cell migration without affecting cell proliferation) by inhibiting protein kinase C alpha (PKC-
). These results are conflicting because PLC is an enzyme-producing diacylglycerol (DAG) which activates PKC. In human esophageal squamous cell carcinoma, loss of KISS-1 and GPR54 expression was observed and was not correlated with tumor size or degree of tumor invasion but was found to be a significant predictor of lymph node metastasis [15]. In human ovarian cancer, as well as in melanoma, KISS-1 was also found to act as a metastasis suppressor [6]. Conversely, in hepatocellular carcinoma, overexpression of KISS-1 and GPR54 genes was observed and correlated with tumor progression but not with suppression of metastasis [9]. Martin et al. [8] showed that KISS-1 was overexpressed in human breast cancer tissues, particularly in patients with aggressive tumors and with mortality; these data were in opposition to the previous study of Lee and Welch [1] in breast cancer. Our results are in accordance with the study of Martin et al. [8] in breast cancer, and with the study of Ikeguchi et al. [9] in hepatocellular carcinoma. Ohtaki et al. [3] reported that significantly high GPR54 messenger RNA (mRNA) expression levels with low KISS-1 mRNA expression levels were detected in some ovarian cancers. They proposed metastin treatment of patients who have tumors with a high expression level of GPR54 mRNA.
Finally, 95% of TCC analyzed in our study expressed abnormal levels of KISS-1 or GPR54. Moreover, our results have demonstrated that GPR54 expression is highly deregulated in invasive and high-grade tumors more often than in superficial and low-grade tumors. Thus, deregulation in the couple KISS-1/GPR54 is a frequent event in bladder transitional cell carcinogenesis, indicating further functional investigations in order to clarify the signals mediated by KISS-1 and GPR54 interactions in cancer cell progression.
1 Institut National de la Santé et de la Recherche Médicale EMI 03-37, Université Paris XII, Faculté de Médecine, Créteil
2 Department of Pathology, La Pitié Salpêtrière Hospital, AP-HP, GHU-Est
3 CeRePP, Tenon Hospital
4 Department of Urology, Tenon Hospital, AP-HP, GHU-Est, Paris, France
* E-mail: olivier.cussenot{at}tnn.ap-hop-paris.fr
Acknowledgements
This research was funded by INSERM, University Paris 12 and Fondation pour la Recherche Médicale.
References
1. Lee JH and Welch DR. (1997) Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KISS-1. Cancer Res 57:122384–2387.
2. Kotani M, Detheux M, Vandenbogaerde A, et al. (2001) The metastasis suppressor gene KISS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 276:3734631–34636.
3. Ohtaki T, Shintani Y, Honda S, et al. (2001) Metastasis suppressor gene KISS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 411:6837613–617.[CrossRef][Medline]
4. Muir AI, Chamberlain L, Elshourbagy NA, et al. (2001) AXOR12, a novel human G protein-coupled receptor, activated by the peptide KISS-1. J Biol Chem 276:3128969–28975.
5. Harms JF, Welch DR, Miele ME. (2003) KISS1 metastasis suppression and emergent pathways. Clin Exp Metastasis 20:111–18.[CrossRef][Web of Science][Medline]
6. Jiang Y, Berk M, Singh LS, et al. (2005) KiSS1 suppresses metastasis in human ovarian cancer via inhibition of protein kinase C alpha. Clin Exp Metastasis 22:5369–376.[CrossRef][Web of Science][Medline]
7. Ringel MD, Hardy E, Bernet VJ, et al. (2002) Metastin receptor is overexpressed in papillary thyroid cancer and activates MAP kinase in thyroid cancer cells. J Clin Endocrinol Metab 87:52399.
8. Martin TA, Watkins G, Jiang WG. (2005) KISS-1 expression in human breast cancer. Clin Exp Metastasis 22:6503–511.[CrossRef][Web of Science][Medline]
9. Ikeguchi M, Hirooka Y, Kaibara N. (2003) Quantitative reverse transcriptase polymerase chain reaction analysis for KISS-1 and orphan G-protein-coupled receptor (hOT7T175) gene expression in hepatocellular carcinoma. J Cancer Res Clin Oncol 129:9531–535.[CrossRef][Web of Science][Medline]
10. Sanchez-Carbayo M, Capodieci P, Cordon-Cardo C. (2003) Tumor suppressor role of KISS-1 in bladder cancer: loss of KISS-1 expression is associated with bladder cancer progression and clinical outcome. Am J Pathol 162:2609–617.
11. Fromont G, Chene L, Vidaud M, et al. (2005) Differential expression of 37 selected genes in hormone-refractory prostate cancer using quantitative taqman real-time RT-PCR. Int J Cancer 114:2174–181.[CrossRef][Web of Science][Medline]
12. Chene L, Giroud C, Desgrandchamps F, et al. (2004) Extensive analysis of the 7q31 region in human prostate tumors supports TES as the best candidate tumor suppressor gene. Int J Cancer 111:5798–804.[CrossRef][Web of Science][Medline]
13. Nicolle G, Daher A, Maille P, et al. (2006) Gefitinib inhibits the growth and invasion of urothelial carcinoma cell lines in which Akt and MAPK activation is dependent on constitutive epidermal growth factor receptor activation. Clin Cancer Res 12:92937–2943.
14. Stafford LJ, Xia C, Ma W, et al. (2002) Identification and characterization of mouse metastasis-suppressor KiSS1 and its G-protein-coupled receptor. Cancer Res 62:195399–5404.
15. Ikeguchi M, Yamaguchi K, Kaibara N. (2004) Clinical significance of the loss of KISS-1 and orphan G-protein-coupled receptor (hOT7T175) gene expression in esophageal squamous cell carcinoma. Clin Cancer Res 10:41379–1383.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||