Abstract
BackgroundGonadotropin-releasing hormone receptor (GnRHR) transmits its signal via two major Gα-proteins, primarily Gαq and Gαi. However, the precise mechanism underlying the functions of Gαs signal in prostate cancer cells is still unclear. We have previously identified that GV1001, a fragment of the human telomerase reverse transcriptase, functions as a biased GnRHR ligand to selectively stimulate the Gαs/cAMP pathway. Here, we tried to reveal the potential mechanisms of which GV1001-stimulated Gαs-cAMP signaling pathway reduces the migration and metastasis of prostate cancer (PCa) cells.MethodsThe expression of epithelial-mesenchymal transition (EMT)-related genes was measured by western-blotting and spheroid formation on ultra-low attachment plate was detected after GV1001 treatment. In vivo Spleen-liver metastasis mouse model was used to explore the inhibitory effect of GV1001 on metastatic ability of PCa and the transwell migration assay was performed to identify whether GV1001 had a suppressive effect on cell migration in vitro. In order to demonstrate the interaction between androgen receptor (AR) and YAP1, co-immunoprecipitation (co-IP), immunofluorescence (IF) staining, chromatin immunoprecipitation (ChIP) were performed in LNCaP cells with and without GV1001 treatment.ResultsGV1001 inhibited expression of EMT-related genes and spheroid formation. GV1001 also suppressed in vivo spleen-liver metastasis of LNCaP cells as well as cell migration in vitro. GV1001 enhanced the phosphorylation of AR and transcription activity of androgen response element reporter gene through cAMP/protein kinase A pathway. Moreover, GV1001 increased Ser-127 phosphorylation of YAP1 and its ubiquitination, and subsequently decreased the levels of AR-YAP1 binding in the promoter region of the CTGF gene. In contrast, both protein and mRNA levels of NKX3.1 known for tumor suppressor gene and AR-coregulator were upregulated by GV1001 in LNCaP cells. YAP1 knockout using CRISPR/Cas9 significantly suppressed the migration ability of LNCaP cells, and GV1001 did not affect the cell migration of YAP1-deficient LNCaP cells. On the contrary, cell migration was more potentiated in LNCaP cells overexpressing YAP5SA, a constitutively active form of YAP1, which was not changed by GV1001 treatment.ConclusionsOverall, this study reveals an essential role of AR-YAP1 in the regulation of PCa cell migration, and provides evidence that GV1001 could be a novel GnRHR ligand to inhibit metastasis of PCa via the Gαs/cAMP pathway.
Highlights
Gonadotropin-releasing hormone receptor (GnRHR) transmits its signal via two major Gα-proteins, primarily Gαq and Gαi
Anti‐migratory and anti‐metastatic effects of GV1001 on human malignant prostate cancer (PCa) We recently reported that Gαs-cAMP signaling is selectively activated by GV1001 binding to GnRHR, but not by Leuprolide acetate (LA), a representative GnRHR agonist [22]
We revealed that the proliferation of PCa cells was slightly inhibited by both GV1001 and LA, but cell migration was only suppressed by GV1001 [22]
Summary
Gonadotropin-releasing hormone receptor (GnRHR) transmits its signal via two major Gα-proteins, primarily Gαq and Gαi. We tried to reveal the potential mechanisms of which GV1001-stimulated Gαs-cAMP signaling pathway reduces the migration and metastasis of prostate cancer (PCa) cells. GnRHR primarily interacts with Gαq and exerts reproductive effects such as synthesis and secretion of pituitary gonadotropin hormones, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) [7,8,9]. Protein or mRNA expression of GnRH and GnRHR was detected in pituitary cells and in extra-pituitary cells; ovarian, breast, prostate tissues, and various cancer cell lines including breast (MCF-7 and MDA-MB-468) and prostate cancer (PCa) cells (PC-3 and LNCaP) [11,12,13,14]. GnRH analogs counteracted the tumor growth of androgen receptor (AR)-negative DU145 xenografts, further supporting their direct and reproductive system-independent anti-cancer effects [20]. Cancer cell-expressing GnRHR is considered as a novel receptor-targeting anti-cancer strategy
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