Abstract
Many studies have suggested a role for the members of the G12 family of heterotrimeric G proteins (Galpha12 and Galpha13) in oncogenesis and tumor cell growth. However, few studies have examined G12 signaling in actual human cancers. In this study, we examined the role of G12 signaling in prostate cancer. We found that expression of the G12 proteins is significantly elevated in prostate cancer. Interestingly, expression of the activated forms of Galpha12 or Galpha13 in the PC3 and DU145 prostate cancer cell lines did not promote cancer cell growth. Instead, expression of the activated forms of Galpha12 or Galpha13 in these cell lines induced cell invasion through the activation of the RhoA family of G proteins. Furthermore, inhibition of G12 signaling by expression of the RGS domain of the p115-Rho-specific guanine nucleotide exchange factor (p115-RGS) in the PC3 and DU145 cell lines did not reduce cancer cell growth. However, inhibition of G12 signaling with p115-RGS in these cell lines blocked thrombin- and thromboxane A2-stimulated cell invasion. These observations identify the G12 family proteins as important regulators of prostate cancer invasion and suggest that these proteins may be targeted to limit invasion- and metastasis-induced prostate cancer patient mortality.
Highlights
Studies over the past three decades have clearly established that signaling through cell surface G protein-coupled receptors (GPCRs)3 controls many physiologic and pathophysiologic processes [2, 3]
G12 Proteins Are Up-regulated in Invasive, Tumorigenic Prostate Cancer Cell Lines—To assess the biologic significance of the G12 family of heterotrimeric G proteins in prostate cancer, we compared expression of G␣12 and G␣13 in an immortalized prostate epithelial cell line (PrEC LHS) [31] and in the three commonly used prostate cancer cell lines: LNCaP, DU145, and PC3
G␣12 Is Up-regulated in Pathologic Specimens of Adenocarcinoma of the Prostate—To determine whether the in vitro findings that G␣12 expression is elevated in aggressive prostate cancer cells extended to actual human tissues, we performed immunohistochemical analysis of G␣12 expression in histopathologic specimens taken from patients with adenocarcinoma of the prostate
Summary
Antibodies and Reagents—Antibodies to RhoA, G␣q, G␣12, and G␣13 and the blocking peptide for the G␣12 antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-Myc antibody was obtained from Zymed Laboratories (San Francisco, CA). Cell Lines—The PC3, DU145, and LNCaP cell lines were obtained from the Duke University Medical Center Cell Culture Facility. The immortalized prostate epithelial cells (PrECLHS) [31] were obtained from Dr Phillip Febbo (Duke University, Durham, NC). The PC3 cell line was maintained in F-12K nutrient mixture (Invitrogen) supplemented with 10% fetal bovine serum. The LNCaP and DU145 cell lines were maintained in RPMI (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum. Sections were incubated with G␣12 antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100 in phosphatebuffered saline for 1 h, followed by biotinylated goat anti-rabbit antisera (Vector Laboratories, Burlingame, CA) diluted 1:200 in phosphate-buffered saline for 30 min, followed by horseradish peroxidase-labeled streptavidin (Jackson ImmunoResearch, West Grove Park, PA) for 30 min, all at room temperature. Western blotting was performed using the Odyssey System (LICOR, Lincoln, Nebraska) according to the manufacturer’s instructions
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