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Journal of Experimental & Clinical Cancer Research
Published in: Journal of Experimental & Clinical Cancer Research 1/2017

Open Access 01-12-2017 | Research

CRISPR/Cas9 targeting of GPRC6A suppresses prostate cancer tumorigenesis in a human xenograft model

Authors: Ruisong Ye, Min Pi, John V. Cox, Satoru K. Nishimoto, L. Darryl Quarles

Published in: Journal of Experimental & Clinical Cancer Research | Issue 1/2017

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Abstract

Background

GPRC6A is implicated in the pathogenesis of prostate cancer, but its role remains uncertain because of a purported tolerant gene variant created by substitution of a K..Y polymorphism in the 3rd intracellular loop (IL) that evolved in the majority of humans and replaces the ancestral RKLP present in 40% of humans of African descent and all other species.

Methods

We determined whether the K..Y polymorphism is present in human-derived prostate cancer cell lines by sequencing the region of the 3rd IL and assessed the cellular localization of a “humanized” mouse GPRC6A containing the K..Y sequence by immunofluorescence. We assessed functions of GPRC6A in PC-3 cells expressing endogenous GPRC6A and in GPRC6A-deficient PC-3 cells created using CRISPR/Cas9 technology. The effect of GPRC6A on basal and ligand stimulated cell proliferation and migration was evaluated in vitro in wild-type and PC-3-deficient cell lines. The effect of editing GPRC6A on prostate cancer growth and progression in vivo was assessed in a Xenograft mouse model implanted with wild-type and PC-3 deficient cells and treated with the GPRC6A ligand osteocalcin.

Results

We found that all of the human prostate cancer cell lines tested endogenously express the “K..Y” polymorphism in the 3rd IL. Comparison of mouse wild-type GPRC6A with a “humanized” mouse GPRC6A construct created by replacing the “RKLP” with the “K..Y” sequence, found that both receptors were predominantly expressed on the cell surface. The transfected “humanized” GPRC6A receptor, however, preferentially activated mTOR compared to ERK signaling in HEK-293 cells. In contrast, in PC-3 cells expressing the endogenous GPRC6A with the “K..Y” polymorphism, the ligand osteocalcin stimulated ERK, AKT and mTOR phosphorylation, promoted cell proliferation and migration, and upregulated genes regulating testosterone biosynthesis. Targeting GPRC6A in PC-3 cells by CRISPR/Cas9 significantly blocked these responses in vitro. In addition, GPRC6A deficient PC-3 xenografts exhibited significantly less growth and were resistant to osteocalcin-induced prostate cancer progression compared to control PC-3 cells expressing GPRC6A.

Conclusions

Human GPRC6A is a functional osteocalcin and testosterone sensing receptor that promotes prostate cancer progression. GPRC6A may contribute to racial disparities in prostate cancer, and is a potential therapeutic target to develop antagonists to treat prostate cancer.
Appendix
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Literature
1.
2.
Huang W-C, Xie Z, Konaka H, Sodek J, Zhau HE, Chung LW. Human osteocalcin and bone sialoprotein mediating osteomimicry of prostate cancer cells: role of cAMP-dependent protein kinase A signaling pathway. Cancer Res. 2005;65:2303–13.CrossRefPubMed
3.
4.
Pi M, Chen L, Huang M-Z, Zhu W, Ringhofer B, Luo J, Christenson L, Li B, Zhang J, Jackson PD. GPRC6A null mice exhibit osteopenia, feminization and metabolic syndrome. PLoS One. 2008;3:e3858.CrossRefPubMedPubMedCentral
5.
Pi M, Kapoor K, Ye R, Nishimoto SK, Smith JC, Baudry J, Quarles LD. Evidence for osteocalcin binding and activation of GPRC6A in β-cells. Endocrinology. 2016;157:1866–80.CrossRefPubMedPubMedCentral
6.
Oury F, Sumara G, Sumara O, Ferron M, Chang H, Smith CE, Hermo L, Suarez S, Roth BL, Ducy P. Endocrine regulation of male fertility by the skeleton. Cell. 2011;144:796–809.CrossRefPubMedPubMedCentral
7.
Mizokami A, Yasutake Y, Higashi S, Kawakubo-Yasukochi T, Chishaki S, Takahashi I, Takeuchi H, Hirata M. Oral administration of osteocalcin improves glucose utilization by stimulating glucagon-like peptide-1 secretion. Bone. 2014;69:68–79.CrossRefPubMed
8.
Oya M, Kitaguchi T, Pais R, Reimann F, Gribble F, Tsuboi T. The G protein-coupled receptor family C group 6 subtype A (GPRC6A) receptor is involved in amino acid-induced glucagon-like peptide-1 secretion from GLUTag cells. J Biol Chem. 2013;288:4513–21.CrossRefPubMed
9.
Otani T, Mizokami A, Hayashi Y, Gao J, Mori Y, Nakamura S, Takeuchi H, Hirata M. Signaling pathway for adiponectin expression in adipocytes by osteocalcin. Cell Signal. 2015;27:532–44.CrossRefPubMed
10.
Mera P, Laue K, Ferron M, Confavreux C, Wei J, Galán-Díez M, Lacampagne A, Mitchell SJ, Mattison JA, Chen Y. Osteocalcin signaling in myofibers is necessary and sufficient for optimum adaptation to exercise. Cell Metab. 2016;23:1078–92.CrossRefPubMedPubMedCentral
11.
Mera P, Laue K, Wei J, Berger JM, Karsenty G. Osteocalcin is necessary and sufficient to maintain muscle mass in older mice. Mol Metabol. 2016;5:1042–7.CrossRef
12.
13.
Lee NK, Sowa H, Hinoi E, Ferron M, Ahn JD, Confavreux C, Dacquin R, Mee PJ, McKee MD, Jung DY. Endocrine regulation of energy metabolism by the skeleton. Cell. 2007;130:456–69.CrossRefPubMedPubMedCentral
14.
Bhindi B, Xie WY, Kulkarni GS, Hamilton RJ, Nesbitt M, Finelli A, Zlotta AR, Evans A, van der Kwast TH, Alibhai SM. Influence of metabolic syndrome on prostate cancer stage, grade, and overall recurrence risk in men undergoing radical prostatectomy. Urology. 2016;93:77–85.CrossRefPubMed
15.
Oury F, Ferron M, Huizhen W, Confavreux C, Xu L, Lacombe J, Srinivas P, Chamouni A, Lugani F, Lejeune H. Osteocalcin regulates murine and human fertility through a pancreas-bone-testis axis. J Clin Invest. 2013;123:2421–33.CrossRefPubMedPubMedCentral
16.
Pi M, Faber P, Ekema G, Jackson PD, Ting A, Wang N, Fontilla-Poole M, Mays RW, Brunden KR, Harrington JJ. Identification of a novel extracellular cation-sensing G-protein-coupled receptor. J Biol Chem. 2005;280:40201–9.CrossRefPubMedPubMedCentral
17.
18.
Wei J, Hanna T, Suda N, Karsenty G, Ducy P. Osteocalcin promotes β-cell proliferation during development and adulthood through Gprc6a. Diabetes. 2014;63:1021–31.CrossRefPubMedPubMedCentral
19.
Hill H, Grams J, Walton R, Liu J, Moellering D, Garvey W. Carboxylated and uncarboxylated forms of osteocalcin directly modulate the glucose transport system and inflammation in adipocytes. Horm Metab Res. 2014;46:341–7.CrossRefPubMedPubMedCentral
20.
Liu M, Zhao Y, Yang F, Wang J, Shi X, Zhu X, Xu Y, Wei D, Sun L, Zhang Y. Evidence for a role of GPRC6A in prostate cancer metastasis based on case-control and in vitro analyses. Eur Rev Med Pharmacol Sci. 2016;20:2235–48.PubMed
21.
Takata R, Akamatsu S, Kubo M, Takahashi A, Hosono N, Kawaguchi T, Tsunoda T, Inazawa J, Kamatani N, Ogawa O. Genome-wide association study identifies five new susceptibility loci for prostate cancer in the Japanese population. Nat Genet. 2010;42:751–4.CrossRefPubMed
22.
Long Q-Z, Du Y-F, Ding X-Y, Li X, Song W-B, Yang Y, Zhang P, Zhou J-P, Liu X-G. Replication and fine mapping for association of the C2orf43, FOXP4, GPRC6A and RFX6 genes with prostate cancer in the Chinese population. PLoS One. 2012;7:e37866.CrossRefPubMedPubMedCentral
23.
Haiman CA, Han Y, Feng Y, Xia L, Hsu C, Sheng X, Pooler LC, Patel Y, Kolonel LN, Carter E. Genome-wide testing of putative functional exonic variants in relationship with breast and prostate cancer risk in a multiethnic population. PLoS Genet. 2013;9:e1003419.CrossRefPubMedPubMedCentral
24.
Nimptsch K, Rohrmann S, Nieters A, Linseisen J. Serum undercarboxylated osteocalcin as biomarker of vitamin K intake and risk of prostate cancer: a nested case-control study in the Heidelberg cohort of the European prospective investigation into cancer and nutrition. Cancer Epidemiol Prev Biomarkers. 2009;18:49–56.CrossRef
25.
Hayashi Y, Kawakubo-Yasukochi T, Mizokami A, Takeuchi H, Nakamura S, Hirata M. Differential roles of carboxylated and uncarboxylated osteocalcin in prostate cancer growth. J Cancer. 2016;7:1605.CrossRefPubMedPubMedCentral
26.
Pi M, Kapoor K, Wu Y, Ye R, Senogles SE, Nishimoto SK, Hwang D-J, Miller DD, Narayanan R, Smith JC. Structural and functional evidence for testosterone activation of GPRC6A in peripheral tissues. Mol Endocrinol. 2015;29:1759–73.CrossRefPubMedPubMedCentral
27.
Zarif JC, Miranti CK. The importance of non-nuclear AR signaling in prostate cancer progression and therapeutic resistance. Cell Signal. 2016;28:348–56.CrossRefPubMedPubMedCentral
28.
Jacobsen SE, Nørskov-Lauritsen L, Thomsen ARB, Smajilovic S, Wellendorph P, Larsson NH, Lehmann A, Bhatia VK, Bräuner-Osborne H. Delineation of the GPRC6A receptor signaling pathways using a mammalian cell line stably expressing the receptor. J Pharmacol ExpTher. 2013;347:298–309.CrossRef
29.
Rueda P, Harley E, Lu Y, Stewart GD, Fabb S, Diepenhorst N, Cremers B, Rouillon M-H, Wehrle I, Geant A. Murine GPRC6A mediates cellular responses to L-amino acids, but not osteocalcin variants. PLoS One. 2016;11:e0146846.CrossRefPubMedPubMedCentral
30.
MacArthur DG, Balasubramanian S, Frankish A, Huang N, Morris J, Walter K, Jostins L, Habegger L, Pickrell JK, Montgomery SB. A systematic survey of loss-of-function variants in human protein-coding genes. Science. 2012;335:823–8.CrossRefPubMedPubMedCentral
31.
Shahabi A, Lewinger JP, Ren J, April C, Sherrod AE, Hacia JG, Daneshmand S, Gill I, Pinski JK, Fan JB. Novel gene expression signature predictive of clinical recurrence after radical prostatectomy in early stage prostate cancer patients. Prostate. 2016;76:1239–56.CrossRefPubMed
32.
Kinsey‐Jones JS, Alamshah A, McGavigan AK, Spreckley E, Banks K, Cereceda Monteoliva N, Norton M, Bewick GA, Murphy KG. GPRC6a is not required for the effects of a high‐protein diet on body weight in mice. Obesity. 2015;23:1194–200.CrossRefPubMedPubMedCentral
33.
Jørgensen S, Have CT, Underwood CR, Johansen LD, Wellendorph P, Gjesing AP, Jørgensen CV, Quan S, Rui G, Inoue A. Genetic Variations in the Human GPRC6A Receptor Control Cell Surface Expression and Function. J Biol Chem. 2017;292:1524–34.
34.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.CrossRefPubMedPubMedCentral
35.
Kuang D, Yao Y, Lam J, Tsushima RG, Hampson DR. Cloning and characterization of a Family C orphan G‐protein coupled receptor. J Neurochem. 2005;93:383–91.CrossRefPubMed
36.
Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343:84–7.CrossRefPubMed
37.
Pi M, Wu Y, Lenchik NI, Gerling I, Quarles LD. GPRC6A mediates the effects of L-arginine on insulin secretion in mouse pancreatic islets. Endocrinology. 2012;153:4608–15.CrossRefPubMedPubMedCentral
38.
Consortium GP. A global reference for human genetic variation. Nature. 2015;526:68–74.CrossRef
39.
Thomsen AR, Plouffe B, Cahill TJ, Shukla AK, Tarrasch JT, Dosey AM, Kahsai AW, Strachan RT, Pani B, Mahoney JP. GPCR-G protein-β-arrestin super-complex mediates sustained G protein signaling. Cell. 2016;166:907–19.CrossRefPubMed
40.
Pi M, Nishimoto SK, Quarles LD. GPRC6A: Jack of all metabolism (or master of none). Molecular Metabolism. 2017;6:185–93.
41.
Fujiwara T, Kanazawa S, Ichibori R, Tanigawa T, Magome T, Shingaki K, Miyata S, Tohyama M, Hosokawa K. L-arginine stimulates fibroblast proliferation through the GPRC6A-ERK1/2 and PI3K/Akt pathway. PLoS One. 2014;9:e92168.CrossRefPubMedPubMedCentral
42.
Dehghan A, Dupuis J, Barbalic M, Bis JC, Eiriksdottir G, Lu C, Pellikka N, Wallaschofski H, Kettunen J, Henneman P. Meta-analysis of genome-wide association studies in > 80 000 subjects identifies multiple loci for C-reactive protein levels clinical perspective. Circulation. 2011;123:731–8.CrossRefPubMedPubMedCentral
43.
Lv X, Liu J, Shi Q, Tan Q, Wu D, Skinner JJ, Walker AL, Zhao L, Gu X, Chen N. In vitro expression and analysis of the 826 human G protein-coupled receptors. Protein Cell. 2016;7:325–37.CrossRefPubMedPubMedCentral
44.
De Toni L, Guidolin D, De Filippis V, Tescari S, Strapazzon G, Santa Rocca M, Ferlin A, Plebani M, Foresta C. Osteocalcin and Sex hormone binding globulin compete on a specific binding site of GPRC6A. Endocrinology. 2016;157:4473–86.CrossRefPubMed
45.
46.
Kinkade CW, Castillo-Martin M, Puzio-Kuter A, Yan J, Foster TH, Gao H, Sun Y, Ouyang X, Gerald WL, Cordon-Cardo C. Targeting AKT/mTOR and ERK MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model. J Clin Invest. 2008;118:3051–64.PubMedPubMedCentral
47.
Ge C, Zhao G, Li Y, Li H, Zhao X, Pannone G, Bufo P, Santoro A, Sanguedolce F, Tortorella S. Role of Runx2 phosphorylation in prostate cancer and association with metastatic disease. Oncogene. 2016;35:366.CrossRefPubMed
48.
Gupta A, Zhou CQ, Chellaiah MA. Osteopontin and MMP9: Associations with VEGF expression/secretion and angiogenesis in PC3 prostate cancer cells. Cancers. 2013;5:617–38.CrossRefPubMedPubMedCentral
49.
Alimirah F, Chen J, Basrawala Z, Xin H, Choubey D. DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation. FEBS Lett. 2006;580:2294–300.CrossRefPubMed
50.
Mostaghel EA. Steroid hormone synthetic pathways in prostate cancer. Translational Androl Urol. 2013;2:212–27.
51.
Chun JY, Nadiminty N, Dutt S, Lou W, Yang JC, Kung H-J, Evans CP, Gao AC. Interleukin-6 regulates androgen synthesis in prostate cancer cells. Clin Cancer Res. 2009;15:4815–22.CrossRefPubMedPubMedCentral
52.
Thulin MH, Nilsson ME, Thulin P, Céraline J, Ohlsson C, Damber J-E, Welén K. Osteoblasts promote castration-resistant prostate cancer by altering intratumoral steroidogenesis. Mol Cell Endocrinol. 2016;422:182–91.CrossRefPubMed
53.
Logothetis CJ, Lin S-H. Osteoblasts in prostate cancer metastasis to bone. Nat Rev Cancer. 2005;5:21–8.CrossRefPubMed
54.
Yeung F, Law WK, Yeh C-H, Westendorf JJ, Zhang Y, Wang R, Kao C, Chung LW. Regulation of human osteocalcin promoter in hormone-independent human prostate cancer cells. J Biol Chem. 2002;277:2468–76.CrossRefPubMed
55.
56.
Dagogo-Jack S. Ethnic disparities in type 2 diabetes: pathophysiology and implications for prevention and management. J Natl Med Assoc. 2003;95:774.PubMedPubMedCentral
57.
Powell IJ. Epidemiology and pathophysiology of prostate cancer in African-American men. J Urol. 2007;177:444–9.CrossRefPubMed
Metadata
Title
CRISPR/Cas9 targeting of GPRC6A suppresses prostate cancer tumorigenesis in a human xenograft model
Authors
Ruisong Ye
Min Pi
John V. Cox
Satoru K. Nishimoto
L. Darryl Quarles
Publication date
01-12-2017
Publisher
BioMed Central
Published in
Journal of Experimental & Clinical Cancer Research / Issue 1/2017
Electronic ISSN: 1756-9966
DOI
https://doi.org/10.1186/s13046-017-0561-x

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