Expression And Function Of GPR116in Breast Cancer Tumorgenesis And Progression

Breast cancer is one of the most common malignant tumors that threats the health of women. The occurrence of breast cancer metastasis often leads to eventual death in many cancer patients, and patients with advanced breast cancer always have other complications because of the cancer metastasis, and thus the quality of their life is seriously affected. Therefore the identification of genes related to breast cancer metastasis is of great importance to cancer diagnosis and the therapy.To this day, G protein coupled receptors (GPCRs) remain important status of pharmacological target in cancer therapy. These receptors control a large range of cell signalings, and their misregulation often results in various diseases. The adhesion GPCR family is a new GPCR sub-family. A number of studies have reported that many adhesion GPCRs express aberrantly in cancer cell.This study aims to find an adhesion GPCR that is related to breast cancer tumorigenesis and progression. Ultimately, this information will facilitate the development of new and targeted drugs and be useful as diagnostic or prognostic targets.So far there is still no relevant research reports about human GPR116. We found GPR116only express in the highly aggressive breast cancer cell line, and has low or no expression in the low/non-aggressive breast cancer cell lines. Further more, we used RT-PCR, Real-time PCR and Western Blot to confirm this result in six breast cancer cell lines with gradiently descent metastasis ability, we found both GPR116messenger RNA and protein expression correlates with breast cancer poor prognosis. The comparison of the expression of GPR116in primary tumor and adjacent non-malignant tissues from24cases of lymphnode-positive breast cancer patients as well as the comparsion of its expression in75cases of breast cancer specimens with different aggressive levels and normal breast tissues, strongly support our findings that GPR116is closely related to the breast cancer tumorigenesis and progression: The expression of GPR116can only be found in aggressive breast cancer samples, and hard to be detected in the control samples taken from adjacent non-malignant tissues or the normal breast tissues.We constructed the vectors containing GPR116full length gene. On the other hand, we found the effective small interfering RNA (siRNA) targeting GPR116, and constructed four short hairpin RNA (shRNA) for lentivirus interference system.Using the vectors mentioned above, we overexpressed GPR116both in the ER positive (MCF-7) and ER negative (SK-BR-3) breast cancer cell lines to obtain two cell lines overexpressing GPR116. Meanwhile, using the effective siRNA and shRNA, we obtained MDA-MB-231breast cancer cell lines transiently/stably knocking down GPR116.Using the stable cell lines mentioned above, we found:knockdown of GPR116in MDA-MB-231decreased the migration and invasion ability in vitro (P<0.01) and the ability of metastasis to mouse lung in vivo (P<0.001). On the other hand, overexpress GPR116in MCF-7and SK-BR-3increased their migration and invasion ability in vitro (P<0.01). We also found GPR116can alter the cytoskeleton of breast cancer cell: GPR116increases the filamentous actin (F-actin) to form stress fibers, and increases the formation of focal adhesion in the leading end of the cell. It seems GPR116also negatively mediates the expression of β-catenin.In order to explore the mechanism behind the phenotype, RT-PCR was performed to assess the expression levels of28genes known to be involved in breast cancer metastasis suppression or promotion. We found the expression of CTGF was correlated with GPR116. As CTGF had already been reported to be both sufficient and essential for the stimulation of migration of breast cancer cell, we considered the possibility that GPR116signaling might promote migration through the induction of CTGF. The results of luciferase reporter gene assays demonstrated that GPR116can induce CTGF expression and maybe in a synergistic manner with TGF-β.In a word, this study found the expression of human GPR116correlated with breast cancer poor prognosis. GPR116regulates breast cancer cell cytoskeleton, adhesion protein and β-catenin to promote breast cancer metastasis. GPR116signaling might promote breast cancer metastasis through the induction of CTGF, and/or the synergization of TGF-β induced CTGF expression.