GPRC6A regulates prostate cancer progression

Abstract

Background: GPRC6A is a nutrient sensing GPCR that is activated in vitro by a variety of ligands, including amino acids, calcium, zinc, osteocalcin (OC), and testosterone. The association between nutritional factors and risk of prostate cancer, the finding of increased expression of OC in prostate cancer cells, and the association between GPRC6A and risk of prostate cancer in Japanese men implicates a role of GPRC6A in prostate cancer.

Methods: We examined if GPRC6A is expressed in human prostate cancer cell lines and used siRNA-mediated knockdown GPRC6A expression in prostate cancer cells to explore the function of GPRC6A in vitro. To assess the role of GPRC6A in prostate cancer progression in vivo, we intercrossed Gprc6a(-/-) mice onto the TRAMP mouse prostate cancer model.

Results: GPRC6A transcripts were markedly increased in prostate cancer cell lines 22Rv1, PC-3, and LNCaP, compared to the normal prostate RWPE-1 cell line. In addition, a panel of GPRC6A ligands, including calcium, OC, and arginine, exhibited in prostate cancer cell lines a dose-dependent stimulation of ERK activity, cell proliferation, chemotaxis, and prostate specific antigen and Runx2 gene expression. These responses were inhibited by siRNA-mediated knockdown of GPRC6A. Finally, transfer of Gprc6a deficiency onto a TRAMP mouse model of prostate cancer significantly retarded prostate cancer progression and improved survival of compound Gprc6a(-/-) /TRAMP mice.

Conclusions: GPRC6A is a novel molecular target for regulating prostate growth and cancer progression. Increments in GPRC6A may augment the ability of prostate cancer cells to proliferate in response to dietary and bone derived ligands.

Figure 1. GPRC6A over-expression of human prostate cancer cell lines and human prostate cancer tissue

A). GPRC6A expression in normal human prostate gland. PCR products were amplified from normal human multiple tissue cDNAs: prostate, intestine and colon using human specific intron-spanning primers. B). GPRC6A over-expression of human prostate cancer cell lines by RT-PCR. RWPE-1 is human prostate epithelial cell. 22Rv1, PC-3 and LNCaP are human prostate cancer cell lines. The primers for GPRC6A application are described in Methods. We used the house-keeping control gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH) for a positive control of RNA integrity.

Figure 2. The ligands of GPRC6A stimulated ERK activation in human prostate cancer cell lines

A). Dose-dependent effects of extracellular calcium, zinc, OC, arginine, and testosterone-BSA on GPRC6A-mediated EKR activation in HEK cells transfected with GPRC6A. B). Dose-dependent effects of extracellular calcium, zinc, OC, arginine and testosterone on EKR activation in human prostate cancer cell lines 22Rv1 and PC-3. The HEK cells transfected with GRPC6A or without the plasmid cDNA of GPRC6A or 22Rv1 or PC-3 were incubated in Dulbecco’s modified Eagle’s medium /F-12 containing 0.1% bovine serum albumin quiescence media and exposed to the extracellular calcium, OC, arginine, or testosterone-BSA at indicated concentrations for 5 min, and ERK activation was determined as described under Materials and methods. Representative blots are shown, and the results were verified in at least three independent experiments.

Figure 3. The ligands of GPRC6A stimulated human prostate cancer cell proliferation and gene expression

Human prostate cancer cells, 22Rv1 or PC-3 (10³ cells/well) grown under subconfluent conditions were cultured in triplicate in a 96-well flat-bottomed microculture dish using RPMI 1640 containing 10% CFBS with various concentrations of GPRC6A ligands: calcium (A) and OC (B) for 72 h. Cell proliferation was determined by counting cells in a hemocytometer method as described in Materials and methods. In all of the above studies, values for relative cell proliferation (expressed as percent of control) represent the mean ± SEM of a minimum of three separate experiments. * indicates a significant difference from control and stimulation at p<0.05, respectively.

Figure 4. The ligands of GPRC6A stimulated human prostate cancer cells gene expression

OC, arginine and R1881 stimulated PSA and Runx 2II gene expression in human prostate cancer 22Rv1 (A and B) and PC-3 cells (C and D). * indicates a significant difference from control and stimulation at p<0.05, respectively.

Figure 5. GPRC6A siRNAs inhibited GPRC6A-mediated activation of phosph-ERK in human prostate cancer cell lines

A). GPRC6A siRNAs, hGPRC6A siRNA-202 and siRNA-514 inhibited GPRC6A mRNA expression in 22Rv1 and PC-3 cells. B) GPRC6A-mediated OC and testosterone stimulated phospho-ERK activation blocked by transfecting hGPRC6A siRNA-202 and siRNA-514 in 22Rv1 and PC-3 cells. C). GPRC6A-mediated calcium, testosterone, arginine and OC stimulated phospho-ERK activation blocked by hGPRC6A siRNA-202 in 22Rv1 cells. Representative blots are shown, and the results were verified in at least three independent experiments.

Figure 6. GPRC6A siRNAs inhibited GPRC6A-mediated stimulation gene expression of PSA and Runx2 and activation of cell chemotaxis in human prostate cancer cell lines

The ligands of GPRC6A, OC, arginine and R1881 stimulated gene expression of PSA (A) and Runx2 (B) were attenuated by GPRC6A siRNA in 22Rv1, human prostate cancer cells. C). Extracellular calcium and OC stimulated cell chemotaxis were inhibited by GPRC6A siRNA in 22Rv1, human prostate cancer cells. The experiments were described in Materials and methods. In all of the above studies, values for relative cell proliferation (expressed as percent of control) represent the mean ± SEM of a minimum of three separate experiments.

Figure 7. Effects of superimposed Gprc6a deficiency in the TRAMP mouse

(A) The gross appearance of whole prostatic glands (Upper panel) and hematoxylin/eosin stained histological sections of ventral prostate (Middle panel, X5 magnification; Lower panel, X20 magnification) from Gprc6a^−/−, TRAMP and Gprc6a^−/−/TRAMP mice at 30 weeks-of-age. Values (inset, upper panel) represent Mean±SEM of prostate gland weights/body weights. Arrow (Lower panel) shows intraepithelial hyperplasia. (B) Comparison of the survival rates in TRAMP and compound Gprc6a^−/−/TRAMP mice.