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A Role for the G12 Family of Heterotrimeric G Proteins in Prostate Cancer Invasion

2006; Elsevier BV; Volume: 281; Issue: 36 Linguagem: Inglês

10.1074/jbc.m604376200

1083-351X

Patrick Kelly, Laura N. Stemmle, John F. Madden, Timothy A. Fields, Yehia Daaka, Patrick J. Casey,

Cancer, Hypoxia, and Metabolism

Many studies have suggested a role for the members of the G12 family of heterotrimeric G proteins (Gα12 and Gα13) 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 Gα12 or Gα13 in the PC3 and DU145 prostate cancer cell lines did not promote cancer cell growth. Instead, expression of the activated forms of Gα12 or Gα13 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. Many studies have suggested a role for the members of the G12 family of heterotrimeric G proteins (Gα12 and Gα13) 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 Gα12 or Gα13 in the PC3 and DU145 prostate cancer cell lines did not promote cancer cell growth. Instead, expression of the activated forms of Gα12 or Gα13 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. It is estimated that over 230,000 new cases of prostate cancer will be diagnosed in the United States this year (1Jemal A. Siegel R. Ward E. Murray T. Xu J. Smigal C. Thun M.J. CA-Cancer J. Clin. 2006; 56: 106-130Crossref PubMed Scopus (5521) Google Scholar). Although the prognosis for patients with early stage prostate cancer has improved, the treatment options for patients with locally advanced disease or metastasis remain few. For this reason, prostate cancer remains the second leading cause of cancer deaths in males and will claim the lives of greater than 25,000 American men this year alone (1Jemal A. Siegel R. Ward E. Murray T. Xu J. Smigal C. Thun M.J. CA-Cancer J. Clin. 2006; 56: 106-130Crossref PubMed Scopus (5521) Google Scholar). Therefore, it is imperative that new strategies be developed to treat patients with advanced prostate cancer, and this requires a better understanding of the molecular mechanisms that regulate prostate cancer invasion and metastasis. Studies over the past three decades have clearly established that signaling through cell surface G protein-coupled receptors (GPCRs) 3The abbreviations used are: GPCR, G protein-coupled receptor; PAR-1, protease-activated receptor-1; GFP, green fluorescent protein; GFR, growth factor-reduced; GST, glutathione S-transferase; RhoGEF, Rho-specific guanine nucleotide exchange factor. controls many physiologic and pathophysiologic processes (2Rohrer D.K. Kobilka B.K. Physiol. Rev. 1998; 78: 35-52Crossref PubMed Scopus (95) Google Scholar, 3Offermanns S. Oncogene. 2001; 20: 1635-1642Crossref PubMed Scopus (75) Google Scholar). However, it is only recently that the functional significance of GPCRs in prostate cancer invasion and metastasis has begun to be appreciated (4Daaka Y. Sci. STKE 2004. 2004; : RE2Google Scholar). GPCRs for thrombin (5Tantivejkul K. Loberg R.D. Mawocha S.C. Day L.L. John L.S. Pienta B.A. Rubin M.A. Pienta K.J. J. Cell Biochem. 2005; 96: 641-652Crossref PubMed Scopus (78) Google Scholar), thromboxane A2 (6Nie D. Che M. Zacharek A. Qiao Y. Li L. Li X. Lamberti M. Tang K. Cai Y. Guo Y. Grignon D. Honn K.V. Am. J. Pathol. 2004; 164: 429-439Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 7Dassesse T. de Leval X. de Leval L. Pirotte B. Castronovo V. Waltregny D. Eur. Urol. 2006; (in press)PubMed Google Scholar), bradykinin (8Taub J.S. Guo R. Leeb-Lundberg L.M. Madden J.F. Daaka Y. Cancer Res. 2003; 63: 2037-2041PubMed Google Scholar), lysophosphatidic acid (9Hwang Y.S. Hodge J.C. Sivapurapu N. Lindholm P.F. Mol. Carcinog. 2006; 45: 518-529Crossref PubMed Scopus (43) Google Scholar), and SDF-1 (10Taichman R.S. Cooper C. Keller E.T. Pienta K.J. Taichman N.S. McCauley L.K. Cancer Res. 2002; 62: 1832-1837PubMed Google Scholar) have all been implicated in prostate cancer invasion and metastasis. The best characterized example is the thrombin receptor, protease-activated receptor-1 (PAR-1). PAR-1 is preferentially expressed in aggressive prostate cancer lines and in metastatic prostate cancer specimens (5Tantivejkul K. Loberg R.D. Mawocha S.C. Day L.L. John L.S. Pienta B.A. Rubin M.A. Pienta K.J. J. Cell Biochem. 2005; 96: 641-652Crossref PubMed Scopus (78) Google Scholar, 11Liu J. Bastian M. Kohlschein P. Schuff-Werner P. Steiner M. Urol. Res. 2003; 31: 163-168Crossref PubMed Scopus (25) Google Scholar, 12Kaushal V. Kohli M. Dennis R.A. Siegel E.R. Chiles W.W. Mukunyadzi P. Prostate. 2005; 66: 273-282Crossref Scopus (50) Google Scholar). Moreover, studies suggest that the activation of PAR-1 increases prostate cancer cell resistance to apoptotic stimuli (5Tantivejkul K. Loberg R.D. Mawocha S.C. Day L.L. John L.S. Pienta B.A. Rubin M.A. Pienta K.J. J. Cell Biochem. 2005; 96: 641-652Crossref PubMed Scopus (78) Google Scholar), stimulates the expression of angiogenic factors (13Liu J. Schuff-Werner P. Steiner M. Biochem. Biophys. Res. Commun. 2006; 343: 183-189Crossref PubMed Scopus (47) Google Scholar), and promotes prostate cancer cell invasion (14Yoshida E. Verrusio E.N. Mihara H. Oh D. Kwaan H.C. Cancer Res. 1994; 54: 3300-3304PubMed Google Scholar, 15Shi X. Gangadharan B. Brass L.F. Ruf W. Mueller B.M. Mol. Cancer Res. 2004; 2: 395-402PubMed Google Scholar). Nevertheless, the pathways through which PAR-1 and the GPCRs mentioned above affect prostate cancer cell function are not fully understood. GPCRs alter cellular function primarily through the activation of heterotrimeric G proteins. Heterotrimeric G proteins consist of two functional signaling units, a guanine nucleotide binding α-subunit and a βγ-subunit dimer. The α-subunits of heterotrimeric G proteins can be divided into four families based on sequence homology: Gs,Gi,Gq, and G12 (4Daaka Y. Sci. STKE 2004. 2004; : RE2Google Scholar, 16Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (248) Google Scholar, 17Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell Biol. 2002; 3: 639-650Crossref PubMed Scopus (2104) Google Scholar). The last of the four families to be identified, the G12 family has been of particular interest to cancer researchers, since its members were found to promote the growth and oncogenic transformation of murine fibroblasts (18Chan A.M. Fleming T.P. McGovern E.S. Chedid M. Miki T. Aaronson S.A. Mol. Cell Biol. 1993; 13: 762-768Crossref PubMed Scopus (145) Google Scholar, 19Xu N. Bradley L. Ambdukar I. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6741-6745Crossref PubMed Scopus (174) Google Scholar). These findings led to the hypothesis that GPCRs may signal through the G12 proteins to promote tumorigenesis and tumor cell growth (20Radhika V. Dhanasekaran N. Oncogene. 2001; 20: 1607-1614Crossref PubMed Scopus (123) Google Scholar). Interestingly, however, studies that examined the role of the G12 proteins in development found that G12 proteins were not required for cell growth but were critical for cell movement in the developing embryo (21Parks S. Wieschaus E. Cell. 1991; 64: 447-458Abstract Full Text PDF PubMed Scopus (247) Google Scholar, 22Lin F. Sepich D.S. Chen S. Topczewski J. Yin C. Solnica-Krezel L. Hamm H. J. Cell Biol. 2005; 169: 777-787Crossref PubMed Scopus (89) Google Scholar, 23Offermanns S. Mancino V. Revel J.P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (294) Google Scholar, 24Ruppel K.M. Willison D. Kataoka H. Wang A. Zheng Y.W. Cornelissen I. Yin L. Xu S.M. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8281-8286Crossref PubMed Scopus (79) Google Scholar, 25Xu J. Wang F. Van Keymeulen A. Herzmark P. Straight A. Kelly K. Takuwa Y. Sugimoto N. Mitchison T. Bourne H.R. Cell. 2003; 114: 201-214Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). Since similar cellular movements underlie cancer cell invasion (26Yang J. Mani S.A. Donaher J.L. Ramaswamy S. Itzykson R.A. Come C. Savagner P. Gitelman I. Richardson A. Weinberg R.A. Cell. 2004; 117: 927-939Abstract Full Text Full Text PDF PubMed Scopus (3082) Google Scholar, 27Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5489) Google Scholar, 28Huber M.A. Kraut N. Beug H. Curr. Opin. Cell Biol. 2005; 17: 548-558Crossref PubMed Scopus (1558) Google Scholar, 29Brumby A.M. Richardson H.E. Nat. Rev. Cancer. 2005; 5: 626-639Crossref PubMed Scopus (183) Google Scholar), these findings suggest that G12 signaling may also play a role in cancer metastasis. Recently, we examined the role of the G12 proteins in human breast cancer. We found that the G12 proteins promote breast cancer metastasis by stimulating cancer cell invasion, not cancer cell growth (30Kelly P. Moeller B.J. Juneja J. Booden M.A. Der C.J. Daaka Y. Dewhirst M.W. Fields T.A. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8173-8178Crossref PubMed Scopus (132) Google Scholar). In this study, we investigated the role of G12 signaling in prostate cancer. We found that the G12 proteins are up-regulated in prostate cancer and that signaling through the G12 pathway does not increase prostate cancer cell growth. Rather, activation of G12 induces a striking increase in cancer cell invasiveness. These observations identify G12 family proteins as regulators of prostate cancer invasion and provide support for targeting these proteins in therapeutic strategies to limit invasion- and metastasis-induced patient morbidity and mortality. Antibodies and Reagents—Antibodies to RhoA, Gαq,Gα12, and Gα13 and the blocking peptide for the Gα12 antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-Myc antibody was obtained from Zymed Laboratories (San Francisco, CA). Polyclonal antisera to Gα12 and Gα13 were also obtained from Dr. Stefan Offermanns (University of Heidelberg). Polyclonal antiserum to RGS2 was from Dr. David Siderovski (University of North Carolina, Chapel Hill, NC). Polyclonal antiserum to Gαq was from Dr. Tom Gettys (Pennington Biomedical Research Center, Baton Rogue, LA). Recombinant human thrombin was from Enzyme Research Laboratories (South Bend, IN), and U46619 and tetanolysin were from Biomol (Plymouth Meeting, PA). Growth factorreduced Matrigel was from BD Biosciences, and the fibronectin, from human plasma, was from Sigma. 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) (31Berger R. Febbo P.G. Majumder P.K. Zhao J.J. Mukherjee S. Signoretti S. Campbell K.T. Sellers W.R. Roberts T.M. Loda M. Golub T.R. Hahn W.C. Cancer Res. 2004; 64: 8867-8875Crossref PubMed Scopus (154) Google Scholar) 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. The PrEC-LHS cells were maintained in a defined medium (PrEGM) from BioWhittaker (Rockland, ME). Adenoviral Infections—The dominant negative Rho kinase adenovirus was obtained from Dr. P. Vasantha Rao (Duke University, Durham, NC). The other recombinant adenoviruses were constructed by subcloning human Gαq(Q209L), Gα12(Q231L), Gα13(Q226L), and HA-RGS2, all from the UMR cDNA Resource (Rolla, MO), Myc-p115-RGS (gift of Dr. Tohru Kosaza, University of Illinois, Chicago, IL), and Mycp115RGS(E29K) (generated by site-directed mutagenesis of the Myc-p115) into the Adtrack-CMV vector (gift of Dr. Bert Vogelstein, Johns Hopkins University Medical Center, Baltimore, MD) and then recombining these with pAdEasy-1 in the BJ5183 strain of Escherichia coli (Stratagene, La Jolla, CA). The resulting DNA was transfected into HEK 293 cells with Lipofectamine (Invitrogen), and the viruses were serially amplified and purified using Adeno-X™ virus purification kits (BD Biosciences). Cell lines were infected at a multiplicity of infection of 5-50, for 6-24 h at 37 °C, and infection efficiencies ranged from 80 to 100% based on GFP expression. Retrovirus Production—Recombinant retroviruses were constructed by subcloning human Gα12(Q231L), Gα13(Q226L), and HA-RGS2, all obtained from the UMR cDNA Resource, and Myc-p115 into the pLXRN vector (Clontech). The DNA constructs were co-transfected into the GP2-293 packaging line (Clontech) using Fugene (Roche Applied Science). Viral supernatants were collected 48 h later, clarified by filtration, and concentrated by ultracentrifugation. The concentrated virus was used to infect 1 × 106 cells in a 60-mm dish with 8 μg/ml polybrene (Sigma). PC3 cells were selected with 400 μg/ml Geneticin (Invitrogen). Cell Invasion Assay—For invasion assays, transwell chamber filters (8-μm pore size, polycarbonate filter, 6.5-mm diameter; Costar) were coated with 50 μg of growth factor-reduced (GFR) Matrigel™. After infection with adenovirus, cells were starved for 12 h in Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin, detached with Cellstripper™ (Mediatech, Herndon, VA), and 5 × 105 cells in 100 μl were placed into the upper chamber of the transwell with or without agonists. For experiments using C3 toxin, 1 mg of purified C3, and 20 hemolytic units of tetanolysin were added to the cells for 1 h prior to harvesting the cells. For experiments with thrombin or U46619, the cells were treated at the indicated ligand concentration for 2 h prior to harvest and for the duration of the experiment. The upper well of the transwell was then transferred to a well containing 600 μl of 5 μg/ml of fibronectin diluted in Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin. Cells were incubated for 36 h at 37 °C in a humidified incubator. Cells in the top well were removed with cotton swabs. The membranes were then stained (Hema3 staining kit; Fisher), and the cells were counted using a phase-contrast microscope. Five randomly selected high powered fields were counted for each membrane. Preparation of GST Fusion Proteins—GST-C3 expression construct was obtained from Judith Meinkoth (University of Pennsylvania, Philadelphia, PA), and the GST-rhotekin-RBD construct was obtained from Robert Lefkowitz (Duke University Medical Center, Durham, NC). The GST-C3 and GST-rhotekin-RBD proteins were made in the BL21DE3 strain of E. coli (Invitrogen). Briefly, starter cultures from a transformed bacterial colony were grown for 16 h and then used to inoculate 500 ml of LB and grown at 37 °C for 2-3 h until the optical density reached 0.5-0.6. At this point, the cells were induced with 0.5 mm isopropyl-d-thiogalactopyranoside (Sigma), and cultures were grown for an additional 2.5 h at 37 °C. The cells were harvested by centrifugation for 15 min at 6,000 × g at 4 °C, and the resulting pellet was resuspended in 2.5 ml of buffer A (2.3 m sucrose, 50 mm Tris-HCl, pH 7.7, 1 mm EDTA, and Complete Mini, EDTA-free protease inhibitor mixture tablets (Roche Applied Science)) followed by dilution with 10 ml of buffer B (50 mm Tris-HCl, pH 7.7, 10 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, and a 1:500 protease inhibitor mix). The cells were then passed three times at 10,000 p.s.i. through a microfluidizer (Microfluidics Corp., Newton, MA). The lysates were cleared by centrifugation at 30,000 × g for 30 min, and the resulting supernatant was incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) equilibrated in buffer B for 2 h at 4 °C with continuous rocking. Finally, the beads were washed three times in Buffer B. The C3 toxin was cleaved from the GST domain by gently rocking the beads overnight at 4 °C with 10 units of thrombin (Enzyme Research Laboratories, South Bend, IN) in 50 mm Tris-HCl, pH 7.7, 14 mm β-mercaptoethanol, 150 mm NaCl, and 2.5 mm CaCl2. The cleaved product was then dialyzed into phosphate-buffered saline, visualized by SDS-PAGE with Coomassie Blue staining, and stored in aliquots at -80 °C. Protein concentration was determined by Bradford assay (Bio-Rad). Immunohistochemistry—Institutional Review Board-approved prostate samples were from Ardais Co. (Lexington, MA). The tissue microarray (catalog no. BR801) was from US Biomax (Rockville, MD). Following paraffin removal and quenching of endogenous peroxidase, 5-μm sections were steamed in 10 mm citrate, pH 6.0, for 15 min in a steamer (catalog no. HS900; Black & Decker) and then incubated with Background Buster ® (Innovex Biosci, Richmond, CA) for 30 min. Sections were then incubated with Gα12 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. Bound immune complex was visualized with diaminobenzidine (Innovex Biosci, Richmond, CA); hematoxylin counterstain (Fisher) was used. The Gα12 staining was graded 0-3+ based on intensity by two independent board-certified pathologists (T. A. F. and J. F. M.), and data were analyzed using one-way analysis of variance and Dunn's multiple comparison test in Prism version 4.0c (GraphPad, San Diego, CA). Miscellaneous Methods—The levels of activated Rho were determined using pull-down assays with a GST fusion of the RhoA-binding domain of rhotekin as previously described (32Meigs T.E. Juneja J. DeMarco C.T. Stemmle L.N. Kaplan D.D. Casey P.J. J. Biol. Chem. 2005; 280: 18049-18055Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Protein concentration was determined by a Bio-Rad protein assay. Western blotting was performed using the Odyssey System (LICOR, Lincoln, Nebraska) according to the manufacturer's instructions. 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 Gα12 and Gα13 in an immortalized prostate epithelial cell line (PrEC LHS) (31Berger R. Febbo P.G. Majumder P.K. Zhao J.J. Mukherjee S. Signoretti S. Campbell K.T. Sellers W.R. Roberts T.M. Loda M. Golub T.R. Hahn W.C. Cancer Res. 2004; 64: 8867-8875Crossref PubMed Scopus (154) Google Scholar) and in the three commonly used prostate cancer cell lines: LNCaP, DU145, and PC3. Interestingly, Gα12 and Gα13 expression was significantly higher in the more tumorigenic and invasive DU145 and PC3 (33Webber M.M. Bello D. Quader S. Prostate. 1997; 30: 136-142Crossref PubMed Scopus (49) Google Scholar) cell lines than in the less tumorigenic, noninvasive LNCaP cell line (33Webber M.M. Bello D. Quader S. Prostate. 1997; 30: 136-142Crossref PubMed Scopus (49) Google Scholar) or in the nontransformed prostate epithelial cell line (Fig. 1). This up-regulation appeared to be particularly pronounced for the Gα12 protein (Fig. 1). These data provided the initial evidence that increased G12 signaling may be associated with increased tumorigenicity and/or invasiveness of these two prostate cancer cell lines. Gα12 Is Up-regulated in Pathologic Specimens of Adenocarcinoma of the Prostate—To determine whether the in vitro findings that Gα12 expression is elevated in aggressive prostate cancer cells extended to actual human tissues, we performed immunohistochemical analysis of Gα12 expression in histopathologic specimens taken from patients with adenocarcinoma of the prostate. Anti-Gα12-stained sections of prostate revealed that prostate cancer cells consistently expressed higher levels of Gα12 protein compared with benign prostate epithelial cells within the same tissue section (Fig. 2). Gα12 staining could be completely blocked by preincubation of the antibody with its blocking peptide, demonstrating antibody specificity (supplemental Fig. 1). Further, staining of these same sections with an anti-Gαq antibody demonstrated that benign prostate epithelial cells and prostate cancer cells express similar levels of Gαq (data not shown), suggesting that this elevation in expression is specific to Gα12. To broaden our analysis of Gα12 expression in prostate cancer, a tissue microarray of 16 examples of normal prostate and 73 examples of invasive carcinoma with matched benign tissue was examined by immunohistochemistry, and the results were graded 0-3+ based on staining intensity. This analysis (not shown) demonstrated that Gα12 expression is significantly increased in invasive carcinoma of the prostate; the staining intensity of normal prostate epithelium was 0.2 ± 0.1; the staining intensity for invasive prostate cancer was 1.6 ± 0.1. Since prostate intraepithelial neoplasms (PINs) were not adequately represented in the commercial tissue microarrays, we also performed the Gα12 staining on 13 samples from radical prostatectomy cancer specimens obtained from our institution (Table 1). Staining of these sections confirmed that Gα12 expression is increased in PIN as well as invasive carcinoma of the prostate; in this analysis, the staining intensity of normal prostate epithelium was 0.67 ± 0.05, the staining intensity of PIN was 2.2 ± 0.08, and the staining intensity of invasive prostate cancer was 2.4 ± 0.05. Together, these data support the conclusion that Gα12 expression increases soon after neoplastic transformation of the prostate and before the tumors become invasive.TABLE 1Distribution of prostate surgical specimen field characteristics by Gα12 stainingSpecimennStaining intensityAverage ± S.E.0123Benign prostate epithelium243126793170.67 ± 0.80HG-PIN12642246532.19 ± 0.83ap < 0.0001 for HG-PIN versus benign prostate epithelium.Invasive CA236328781262.39 ± 0.74bp < 0.0001 for invasive cancer versus benign prostate epithelium.a p < 0.0001 for HG-PIN versus benign prostate epithelium.b p < 0.0001 for invasive cancer versus benign prostate epithelium. Open table in a new tab G12 Signaling Does Not Promote Prostate Cancer Cell Growth or Tumorigenesis—Previous studies have suggested that G12 signaling is able to promote fibroblast growth and tumorigenesis (18Chan A.M. Fleming T.P. McGovern E.S. Chedid M. Miki T. Aaronson S.A. Mol. Cell Biol. 1993; 13: 762-768Crossref PubMed Scopus (145) Google Scholar, 19Xu N. Bradley L. Ambdukar I. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6741-6745Crossref PubMed Scopus (174) Google Scholar, 34Jiang H. Wu D. Simon M.I. FEBS Lett. 1993; 330: 319-322Crossref PubMed Scopus (101) Google Scholar, 35Voyno-Yasenetskaya T.A. Pace A.M. Bourne H.R. Oncogene. 1994; 9: 2559-2565PubMed Google Scholar). However, in breast cancer cells, G12 signaling did not appear to promote cell growth or tumorigenesis (30Kelly P. Moeller B.J. Juneja J. Booden M.A. Der C.J. Daaka Y. Dewhirst M.W. Fields T.A. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8173-8178Crossref PubMed Scopus (132) Google Scholar). In order to determine the biologic significance of the G12 proteins in prostate cancer, we first examined the effects of modulating the G12 pathway on prostate cancer cell growth and tumorigenesis. In order to drive G12 signaling in the DU145 and PC3 cell lines, we used adenovirus to express the activated forms of Gα12 (Gα12 Q231L) and Gα13 (Gα13 Q226L). Expression of these activated variants had no effect on in vitro prostate cancer cell growth (supplemental Fig. 2, A and B). In addition, in order to inhibit G12 signaling, we used adenovirus to express the RGS domain of the p115-Rho-specific guanine nucleotide exchange factor (RhoGEF) (p115-RGS). This domain selectively binds Gα12 and Gα13, preventing them from interacting with their downstream effectors (36Shi C.S. Sinnarajah S. Cho H. Kozasa T. Kehrl J.H. J. Biol. Chem. 2000; 275: 24470-24476Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 37Martin C.B. Mahon G.M. Klinger M.B. Kay R.J. Symons M. Der C.J. Whitehead I.P. Oncogene. 2001; 20: 1953-1963Crossref PubMed Scopus (109) Google Scholar). Expression of this inhibitor of G12 signaling also had no effect on prostate cancer cell growth (supplemental Fig. 2, A and B). To examine the effects of G12 signaling on prostate cancer cell tumorigenesis, recombinant retroviruses were used to stably express either Gα12 (Q231L) or the p115-RGS in the PC3 cell line. Interestingly, expression of neither of these proteins had any effect on the growth of PC3 cells in soft agar (supplemental Fig. 2C) or when implanted into the flanks of immunocompromised mice (data not shown). Taken together, these results suggest that G12 signaling does not affect prostate cancer cell growth or tumorigenesis. G12 Signaling Promotes Prostate Cancer Cell Invasion—Since previous studies have suggested a role for the G12 proteins in cell migration (21Parks S. Wieschaus E. Cell. 1991; 64: 447-458Abstract Full Text PDF PubMed Scopus (247) Google Scholar, 22Lin F. Sepich D.S. Chen S. Topczewski J. Yin C. Solnica-Krezel L. Hamm H. J. Cell Biol. 2005; 169: 777-787Crossref PubMed Scopus (89) Google Scholar, 23Offermanns S. Mancino V. Revel J.P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (294) Google Scholar, 24Ruppel K.M. Willison D. Kataoka H. Wang A. Zheng Y.W. Cornelissen I. Yin L. Xu S.M. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8281-8286Crossref PubMed Scopus (79) Google Scholar, 25Xu J. Wang F. Van Keymeulen A. Herzmark P. Straight A. Kelly K. Takuwa Y. Sugimoto N. Mitchison T. Bourne H.R. Cell. 2003; 114: 201-214Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar) and cancer cell invasion (30Kelly P. Moeller B.J. Juneja J. Booden M.A. Der C.J. Daaka Y. Dewhirst M.W. Fields T.A. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8173-8178Crossref PubMed Scopus (132) Google Scholar), we next examined the role of G12 signaling in prostate cancer cell invasion. We found that expression of the activated forms of Gα12 or Gα13 in the PC3 (Fig. 3A) and DU145 (Fig. 3B) cell lines significantly increased the ability of these cells to invade a Matrigel™ barrier reconstituted in a transwell migration chamber. Since most receptors that couple to the G12 family of heterotrimeric G proteins also couple to the Gq family, we expressed the activated form of Gαq (Gαq Q209L) in the prostate cancer cells as a control. Expression of activated Gαq failed to promote cellular invasion in either prostate cancer line (Fig. 3, A and B), suggesting that this ability to promote prostate cancer invasion is specific to the G12 family. G12 Signaling Promotes Prostate Cancer Cell Invasion through a Rho-dependent Pathway—The best characterized downstream effectors of the G12 family of heterotrimeric G proteins are members of the RhoA family of monomeric GTPases. The G12 proteins stimulate Rho activity principally through the direct interaction with a family of RhoGEFs that includes p115-RhoGEF (38Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (675) Google Scholar), PDZ-RhoGEF (39Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar), and LARG (40Fukuhara S. Chikumi H. Gutkind J.S. FEBS Lett. 2000; 485: 183-188Crossref PubMed Scopus (211) Google Scholar). Gα12 and Gα13 bind to these RhoGEFs through an N-terminal RGS motif, recruiting them to the membrane, where they are able to promote Rho activation (38Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (675) Google Scholar, 39Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 40Fukuhara S. Chikumi H. Gutkind J.S. FEBS Lett. 2000; 485: 183-188Crossref PubMed Scopus (211) Google Scholar). Since many studies have demonstrated that the Rho family of proteins and their downstream effectors play a significant role in prostate cancer invasion (41Sahai E. Marshall C.J. Nat. Rev. Cancer. 2002; 2: 133-142Crossref PubMed Scopus (1223) Google Scholar), we examined the role of Rho signaling in G12-induced prostate cancer invasion. First, we confirmed that G12 signaling is able to activate Rho in prostate cancer cells. Expression