Ye Ruisong, Pi Min, Cox John V, Nishimoto Satoru K, Quarles L Darryl
Department of Medicine, University of Tennessee Health Science Center, 19 S Manassas St., Memphis, TN, 38163, USA.
Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, 19 S Manassas St., Memphis, TN, 38163, USA.
J Exp Clin Cancer Res. 2017 Jun 28;36(1):90. doi: 10.1186/s13046-017-0561-x.
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.
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.
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.
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.
GPRC6A与前列腺癌的发病机制有关，但其作用仍不确定，因为在大多数人类中进化出的第3个细胞内环（IL）中由K..Y多态性取代所产生的所谓耐受基因变体，取代了40%非洲裔人类和所有其他物种中存在的祖先RKLP。
我们通过对第3个IL区域进行测序，确定人源前列腺癌细胞系中是否存在K..Y多态性，并通过免疫荧光评估含有K..Y序列的“人源化”小鼠GPRC6A的细胞定位。我们评估了GPRC6A在表达内源性GPRC6A的PC-3细胞和使用CRISPR/Cas9技术构建的GPRC6A缺陷型PC-3细胞中的功能。在野生型和PC-3缺陷型细胞系中体外评估GPRC6A对基础和配体刺激的细胞增殖及迁移的影响。在植入野生型和PC-3缺陷型细胞并用GPRC6A配体骨钙素处理的异种移植小鼠模型中评估编辑GPRC6A对前列腺癌生长和进展的体内影响。
我们发现所有测试的人前列腺癌细胞系在第3个IL中内源性表达“K..Y”多态性。将小鼠野生型GPRC6A与通过用“K..Y”序列取代“RKLP”构建的“人源化”小鼠GPRC6A构建体进行比较，发现两种受体均主要在细胞表面表达。然而，在HEK-293细胞中，转染的“人源化”GPRC6A受体与ERK信号相比，优先激活mTOR。相反，在表达具有“K..Y”多态性的内源性GPRC6A的PC-3细胞中，配体骨钙素刺激ERK、AKT和mTOR磷酸化，促进细胞增殖和迁移，并上调调节睾酮生物合成的基因。通过CRISPR/Cas9靶向PC-3细胞中的GPRC6A在体外显著阻断了这些反应。此外，与表达GPRC6A的对照PC-3细胞相比，GPRC6A缺陷型PC-3异种移植瘤的生长明显减少，并且对骨钙素诱导的前列腺癌进展具有抗性。
人GPRC6A是一种功能性骨钙素和睾酮感应受体，可促进前列腺癌进展。GPRC6A可能导致前列腺癌的种族差异，并且是开发拮抗剂治疗前列腺癌的潜在治疗靶点。
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