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

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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.

Original languageEnglish (US)
Article number90
JournalJournal of Experimental and Clinical Cancer Research
Volume36
Issue number1
DOIs
StatePublished - Jun 28 2017

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Clustered Regularly Interspaced Short Palindromic Repeats
Heterografts
Prostatic Neoplasms
Carcinogenesis
Osteocalcin
Ligands
Cell Line
Cell Movement
Cell Proliferation
HEK293 Cells
Androgen Receptors
Growth
Genes
Fluorescent Antibody Technique
Testosterone
Phosphorylation
Technology

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

Cite this

@article{be32260934734b00aaf953a87dfd62d7,
title = "CRISPR/Cas9 targeting of GPRC6A suppresses prostate cancer tumorigenesis in a human xenograft model",
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.",
author = "Ruisong Ye and Min Pi and John Cox and Satoru Nishimoto and Leigh Quarles",
year = "2017",
month = "6",
day = "28",
doi = "10.1186/s13046-017-0561-x",
language = "English (US)",
volume = "36",
journal = "Journal of Experimental and Clinical Cancer Research",
issn = "0392-9078",
publisher = "BioMed Central",
number = "1",

}

TY - JOUR

T1 - CRISPR/Cas9 targeting of GPRC6A suppresses prostate cancer tumorigenesis in a human xenograft model

AU - Ye, Ruisong

AU - Pi, Min

AU - Cox, John

AU - Nishimoto, Satoru

AU - Quarles, Leigh

PY - 2017/6/28

Y1 - 2017/6/28

N2 - 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.

AB - 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.

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U2 - 10.1186/s13046-017-0561-x

DO - 10.1186/s13046-017-0561-x

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AN - SCOPUS:85021425874

VL - 36

JO - Journal of Experimental and Clinical Cancer Research

JF - Journal of Experimental and Clinical Cancer Research

SN - 0392-9078

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M1 - 90

ER -