expression of GPR54 and KiSS1 in human breast cancer
Authors: Leah M. Prentice, Melinda A. Miller, Dmitry A. Turbin, Samuel A. J. R. Aparicio, David G. Huntsman
Journal: Cancer Research (2006)

4317   Metastatic breast cancer is the second most common cause of death due to cancer in women. Metastin is a 54 amino acid kisspeptide that was characterized as anti-metastatic using  in vitro  and  in vivo  studies in a human melanoma cell line. The kisspeptins (Kp10, Kp13, Kp14, and Kp54) are encoded by the  KiSS1  gene which is located on chromosome 1q32. The kisspeptin receptor is  GPR54 , a G coupled receptor, whose gene is located at chromosome 19p13. Mutated  GPR54  is found in humans with hypogonadotropic hypogonadism and  GPR54  null mice do not undergo puberty. The kisspeptins stimulate release of LH and FSH through the release of GnRH via the hypothalamo-pituitary axis in mice and humans. As well as having an endocrine function, these genes have also been demonstrated to have an autocrine/paracrine function. Both are expressed in syncytiotrophoblast cells and are thought to control trophoblast invasion during pregnancy. Therefore, metastasis development in breast cancer may in part be determined by  GPR54  and  KiSS1  expression. Using tissue microarray (TMA) technology, 438 cases of clinical breast cancers with linked outcome data have been assessed for gene copy number of  KiSS1  and  GPR54  using florescent in situ hybridization (FISH). Using the  KiSS1  fluorescent probe, it was discovered that 29/233 breast cancer cases were amplified (12.4%), while only 3 cases showed loss of gene copy number. There was no correlation with survival or ER status. For the  GPR54  fluorescent probe, 27/191 cases showed gene loss (14%) while 4 cases were amplified.  GPR54  loss correlates with poor survival in node positive patients (p=0.0071). To further assess gene expression, the breast cancer cell lines MDA-MB-231 and MCF-7 were assessed for  GPR54  and  KiSS1  mRNA levels using real time PCR. Non-metastatic MCF-7 breast cancer cells showed high levels of both  GPR54  and  KiSS1  expression as compared to the immortalized H-tert breast cell line. Metastatic MDA-MB-231 breast cancer cell line demonstrated increased  KiSS1  expression as compared to H-terts but relatively little  GPR54  expression as compared to MCF-7 cells or H-tert cells. Western blot analysis confirmed these findings, demonstrating visible  GPR54  expression in the MCF-7 protein lysates only. FISH was used on metaphases of MDA-MB-231, MCF-7 and H-tert cells to determine if gene copy number reflected the expression levels. The MDA-MB-231 cells showed three copies of  KiSS1  and two to three copies of  GPR54 . The MCF-7 cells showed five copies of  KiSS1  and two copies of  GPR54 , whereas the H-tert cells demonstrated the expected normal two copies of each gene. Loss of  GPR54  gene copy number in clinical breast cancer cases of node positive patients and the loss of  GPR54  expression in the MDA-MB-231 cell line supports the hypothesis that breast cancer metastasis is in part regulated by  GPR54  expression. For the MDA-MB-231 cell line, loss of  GPR54  expression may be determined by epigenetic factors.