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Originally published In Press as doi:10.1074/jbc.M604376200 on June 20, 2006

J. Biol. Chem., Vol. 281, Issue 36, 26483-26490, September 8, 2006
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A Role for the G12 Family of Heterotrimeric G Proteins in Prostate Cancer Invasion*Formula

Patrick Kelly{ddagger}1, Laura N. Stemmle§, John F. Madden§, Timothy A. Fields§, Yehia Daaka, and Patrick J. Casey{ddagger}2

From the {ddagger}Department of Pharmacology and Cancer Biology and §Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 and the Department of Pathology, Medical College of Georgia, Augusta, Georgia 30912

Received for publication, May 8, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Many studies have suggested a role for the members of the G12 family of heterotrimeric G proteins (G{alpha}12 and G{alpha}13) 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 G{alpha}12 or G{alpha}13 in the PC3 and DU145 prostate cancer cell lines did not promote cancer cell growth. Instead, expression of the activated forms of G{alpha}12 or G{alpha}13 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It is estimated that over 230,000 new cases of prostate cancer will be diagnosed in the United States this year (1). 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 (1). 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)3 controls many physiologic and pathophysiologic processes (2, 3). However, it is only recently that the functional significance of GPCRs in prostate cancer invasion and metastasis has begun to be appreciated (4). GPCRs for thrombin (5), thromboxane A2 (6, 7), bradykinin (8), lysophosphatidic acid (9), and SDF-1 (10) 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 (5, 11, 12). Moreover, studies suggest that the activation of PAR-1 increases prostate cancer cell resistance to apoptotic stimuli (5), stimulates the expression of angiogenic factors (13), and promotes prostate cancer cell invasion (14, 15). 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 {alpha}-subunit and a beta{gamma}-subunit dimer. The {alpha}-subunits of heterotrimeric G proteins can be divided into four families based on sequence homology: Gs,Gi,Gq, and G12 (4, 16, 17). 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 (18, 19). These findings led to the hypothesis that GPCRs may signal through the G12 proteins to promote tumorigenesis and tumor cell growth (20). 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 (21-25). Since similar cellular movements underlie cancer cell invasion (26-29), 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 (30). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Reagents—Antibodies to RhoA, G{alpha}q,G{alpha}12, and G{alpha}13 and the blocking peptide for the G{alpha}12 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 G{alpha}12 and G{alpha}13 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 G{alpha}q 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 [GenBank] 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) (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. 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 G{alpha}q(Q209L), G{alpha}12(Q231L), G{alpha}13(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-XTM 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 G{alpha}12(Q231L), G{alpha}13(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 x 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) MatrigelTM. 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 CellstripperTM (Mediatech, Herndon, VA), and 5 x 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 [GenBank] , 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 x 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 x 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 beta-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).


Figure 1
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  		FIGURE 1.
Expression of the G{alpha}12 and G{alpha}13 proteins in an immortalized prostate epithelial cell line (PrEC (LHS)) compared with expression in the LNCaP, DU145, and PC3 cell lines. An equal number of actively proliferating cells (log phase) from each cell line were harvested and then lysed in equal volumes of radioimmune precipitation buffer. Equal volumes of each lysate (~40 µg of total protein) were then resolved by SDS-PAGE, and the expression of G{alpha}12 and G{alpha}13 were determined by immunoblot (IB). {alpha}-Tubulin was used as a loading control.

 


Figure 2
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  		FIGURE 2.
G{alpha}12 protein levels are up-regulated in carcinoma in situ and in invasive adenocarcinoma of the prostate. Sections of formalin-fixed paraffinembedded prostate tissue were stained for G{alpha}12 with anti-G12 antiserum (Santa Cruz Biotechnology) as described under "Experimental Procedures." Original images were taken with the x40 objective. A, benign prostate tissue. B, PIN. C, section containing both benign prostate epithelium and invasive adenocarcinoma of the prostate. In C, the arrowheads indicate cancer, and full arrows indicate benign epithelium.

 
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 G{alpha}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. Bound immune complex was visualized with diaminobenzidine (Innovex Biosci, Richmond, CA); hematoxylin counterstain (Fisher) was used. The G{alpha}12 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 (32). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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{alpha}12 and G{alpha}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. Interestingly, G{alpha}12 and G{alpha}13 expression was significantly higher in the more tumorigenic and invasive DU145 and PC3 (33) cell lines than in the less tumorigenic, noninvasive LNCaP cell line (33) or in the nontransformed prostate epithelial cell line (Fig. 1). This up-regulation appeared to be particularly pronounced for the G{alpha}12 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.

G{alpha}12 Is Up-regulated in Pathologic Specimens of Adenocarcinoma of the Prostate—To determine whether the in vitro findings that G{alpha}12 expression is elevated in aggressive prostate cancer cells extended to actual human tissues, we performed immunohistochemical analysis of G{alpha}12 expression in histopathologic specimens taken from patients with adenocarcinoma of the prostate. Anti-G{alpha}12-stained sections of prostate revealed that prostate cancer cells consistently expressed higher levels of G{alpha}12 protein compared with benign prostate epithelial cells within the same tissue section (Fig. 2). G{alpha}12 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-G{alpha}q antibody demonstrated that benign prostate epithelial cells and prostate cancer cells express similar levels of G{alpha}q (data not shown), suggesting that this elevation in expression is specific to G{alpha}12.

To broaden our analysis of G{alpha}12 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 G{alpha}12 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 G{alpha}12 staining on 13 samples from radical prostatectomy cancer specimens obtained from our institution (Table 1). Staining of these sections confirmed that G{alpha}12 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 G{alpha}12 expression increases soon after neoplastic transformation of the prostate and before the tumors become invasive.


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  		TABLE 1
Distribution of prostate surgical specimen field characteristics by G{alpha}12 staining

Sections from prostatectomy specimens (n – 13) were stained for G{alpha}12 as described under "Experimental Procedures." Areas of benign epithelium, PIN, and carcinoma in sequential (x20) microscopic fields were scored 0–3+ based on staining intensity. The number of microscopic fields containing each of the diagnoses and their respective mean scores are indicated below.

 
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 (18, 19, 34, 35). However, in breast cancer cells, G12 signaling did not appear to promote cell growth or tumorigenesis (30). 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 G{alpha}12 (G{alpha}12 Q231L) and G{alpha}13 (G{alpha}13 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 G{alpha}12 and G{alpha}13, preventing them from interacting with their downstream effectors (36, 37). 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 G{alpha}12 (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.


Figure 3
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  		FIGURE 3.
Expression of the activated form of G{alpha}12 (G{alpha} 12QL) or G{alpha}13 (G{alpha} 13QL) but not G{alpha}q (G{alpha} qQL) induces prostate cancer cell invasion in vitro. PC3 (A) and Du145 (B) cells were transduced with the indicated adenovirus, starved for 18 h, and then allowed to invade GFR Matrigel-coated transwell filters for 30 h. A and B, immunoblot analysis shows expression levels of G{alpha}12, G{alpha}13, and G{alpha}q in the PC3 cell line after infection with the indicated adenovirus as an example of protein expression. {alpha}-Tubulin was used as a loading control. A and B, graph shows-fold increase in invasion over GFP control. Experiments were performed in duplicate, and at least four fields were counted for each replicate. All experiments were performed at least three times. All results are presented as mean ± S.E. *, p < 0.05 as determined by paired Student's t test. IB, immunoblot.

 
G12 Signaling Promotes Prostate Cancer Cell Invasion—Since previous studies have suggested a role for the G12 proteins in cell migration (21-25) and cancer cell invasion (30), we next examined the role of G12 signaling in prostate cancer cell invasion. We found that expression of the activated forms of G{alpha}12 or G{alpha}13 in the PC3 (Fig. 3A) and DU145 (Fig. 3B) cell lines significantly increased the ability of these cells to invade a MatrigelTM 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 G{alpha}q (G{alpha}q Q209L) in the prostate cancer cells as a control. Expression of activated G{alpha}q 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 (38), PDZ-RhoGEF (39), and LARG (40). G{alpha}12 and G{alpha}13 bind to these RhoGEFs through an N-terminal RGS motif, recruiting them to the membrane, where they are able to promote Rho activation (38-40). Since many studies have demonstrated that the Rho family of proteins and their downstream effectors play a significant role in prostate cancer invasion (41), 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 of the activated forms of G{alpha}12 or G{alpha}13 induced a significant increase in the levels of GTP-bound RhoA in both the PC3 (Fig. 4A) and DU145 (Fig. 4B) cell lines. In contrast, expression of the activated form of G{alpha}q induced little or no change in the level of GTP bound RhoA. Thus, the G12 proteins are able to stimulate Rho activity in prostate cancer cells. Next, we tested the effects of inhibiting the Rho signaling pathway on G12-induced prostate cancer cell invasion. Treatment of the PC3 and DU145 cell lines with C3 toxin, a specific, irreversible inhibitor of the Rho GTPases, completely blocked G{alpha}12-stimulated invasion (Fig. 4, C and D). Treatment of the PC3 and DU145 cell lines with Y29632, a cell-permeable inhibitor of one of the principle downstream effectors of Rho, Rho kinase, also significantly reduced G12-induced invasion, albeit not to the same extent as treatment with C3 toxin (Fig. 4, C and D). Expression of a dominant negative form of Rho kinase (DN-ROK) in the PC3 and DU145 cell lines also blocked G12-induced invasion by about 50% (Fig. 4, C and D). Taken together, these results suggest that Rho activation is required for cellular invasion induced by G12 and that the effects of Rho are mediated at least in part through Rho kinase.


Figure 4
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  		FIGURE 4.
G12 signaling promotes prostate cancer cell invasion through the activation of Rho GTPases. Expression of G{alpha}12QL and G{alpha}13QL induces RhoA activation in PC3 (A) and DU145 cells (B). Cells were transduced with the indicated adenovirus and then starved for 18 h. Cells were lysed, and lysates were subjected to pull-down assays using a GST fusion of the activated RhoAbinding domain of rhotekin. Levels of precipitated RhoA were determined by immunoblot analysis (IB) using anti-RhoA antibody. Levels of total RhoA, G{alpha}12,G{alpha}13, and G{alpha}q were also determined as a control. All panels are representative of two or more separate experiments. Expression of G{alpha}12QL induces PC3 (C) and DU145 cell (D) invasion in through a Rho-dependent pathway. Cells were transduced with the indicated adenovirus, starved for 18 h, and then allowed to invade GFR Matrigel-coated transwell filters for 30 h. For experiments with C3 toxin, cells were treated with the recombinant toxin for 1 h prior to harvest. For experiments with Y29632, inhibitor (50µM) was added at the time of plating to both the upper and lower wells of the transwell apparatus. Experiments were performed in duplicate, and results are presented as -fold increase over that observed with GFP control. All experiments were performed at least three times. All results are presented as mean ± S.E. *, p < 0.01; **, p < 0.05 as determined by paired Student's t test.

 
G12-induced Inactivation of E-cadherin Is Not Sufficient to Promote Prostate Cancer Cell Invasion—Previous work in our laboratory has demonstrated that G12 proteins have the potential to promote cancer cell invasion in a Rho-independent manner (42). By binding to the cytoplasmic tail of E-cadherin (43), the G12 proteins are able to disrupt E-cadherin-mediated adhesion and allow cell migration (42). Hence, we sought to examine the potential contribution of G12-induced E-cadherin inactivation in G12-stimulated prostate cancer cell invasion. In order to study the role of the G12 to E-cadherin signaling axis in prostate cancer cell invasion, we utilized the {Delta}244-249 mutant of G{alpha}12. G{alpha}12({Delta}244-249) is a mutant form of G{alpha}12 that does not bind to the family of RhoGEFs described above but retains the ability to interact with the cytoplasmic tail of E-cadherin (32). As such, G{alpha}12({Delta}244-249) is functionally uncoupled from Rho signaling but is still able to inactivate E-cadherin-mediated adhesion (32).

As expected, expression of the activated form of G{alpha}12({Delta}244-249) in the E-cadherin-positive DU145 cell line did not cause an increase in the level of GTP RhoA, whereas expression of the activated forms of G{alpha}12 or G{alpha}13 induced a significant increase in the level of GTP RhoA, and the expression of the activated form of G{alpha}q induced a minor increase in the level of GTP RhoA (Fig. 5A).

To confirm that G12 was able to inactivate E-cadherin in a Rho-independent fashion, activated G{alpha}12 and activated G{alpha}12({Delta}244-249) were expressed in the DU145 cells, and the cadherin function was analyzed using a so-called "fast aggregation" assay (42, 44). In this assay, the cells were trypsinized in the presence of 10 mM Ca2+ to maintain cadherin integrity and then disassociated and allowed to aggregate for 1 h in the presence of 1 mM Ca2+. Aggregate size and number were then scored as a measure of cadherin function. Inclusion of the Ca2+ chelator EGTA in the assay completely disrupted aggregate formation (Fig. 5B), confirming that the cell-cell interactions observed were mediated by cadherins. Moreover, consistent with our previous findings (42), expression of either G{alpha}12 or G{alpha}12({Delta}244-249) significantly decreased the ability of the DU145 cells to aggregate (Fig. 5B), suggesting that G12 signaling is able to inactivate E-cadherin in this cell line. Finally, we examined whether G12-induced inactivation of E-cadherin was able to promote DU145 cell invasion independent of Rho activation. The activated forms G{alpha}12 and G{alpha}12({Delta}244-249) were expressed in the DU145 cells, and cellular invasion was assayed. Although both activated G{alpha}12 and G{alpha}12({Delta}244-249) were able to disrupt E-cadherin-mediated adhesion, only activated G{alpha}12 induced prostate cancer cell invasion. Thus, although the G12 to cadherin pathway appears to be intact in the DU145 cells, G12-induced inactivation of E-cadherin does appear to be sufficient to stimulate invasion of this cell type.

G12 Signaling Is Required for Thrombin- and Thromboxane A2-stimulated Invasion of Prostate Cancer Cells—As noted in the Introduction, studies have demonstrated that signaling through GPCRs for factors such as thrombin (15), thromboxane A2 (6), bradykinin (8), and lysophosphatidic acid (9) promotes prostate cancer cell invasion and metastasis. Interestingly, many of these GPCRs are known to couple to the G12 family of heterotrimeric G proteins (45). Thus, we decided to determine whether G12 signaling via Rho is involved in the invasion-promoting activity of these GPCRs. First, we confirmed that GPCR signaling does indeed elicit Rho activation in prostate cancer cells through a G12-dependent pathway. Treatment of the PC3 (Fig. 6A) or the DU145 (Fig. 6B) cell lines with thrombin resulted in a significant increase in the level of activated RhoA. When G12 signaling was inhibited using the p115-RGS, this activation was blocked. Since the thrombin receptor, PAR-1, is also known to couple to the Gq family of heterotrimeric G proteins, we inhibited Gq signaling using RGS2 as a control. Inhibition of Gq with RGS2 did not affect thrombininduced RhoA activation (Fig. 6, A and B). To confirm the specificity of this approach, we also analyzed thrombin-induced Ca2+ transients in the PC3 and DU145 cell lines. As expected, expression of the p115-RGS had no effect on thrombin-induced Ca2+ fluxes, whereas expression of RGS2 significantly impaired thrombin-induced Ca2+ flux (data not shown). As an additional control, we expressed a disabled form of p115-RGS, p115-RGS (E29K). This point mutant of the p115-RGS neither binds to nor functions as a GTPase-activating protein for the G12 proteins and thus does not affect G12 signaling (46). As shown in Fig. 6, expression of this protein did not influence thrombin-induced RhoA activation, indicating that the inhibitory effects of p115-RGS are the result of its ability to bind to and inactivate the G12 proteins.


Figure 5
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  		FIGURE 5.
G12-induced inactivation of E-cadherin is not sufficient to promote prostate cancer cell invasion. A, expression of the activated form of G{alpha}12QL({Delta}244-249) does not induce Rho activation in the DU145 cell line. DU145 cells were transduced with the indicated adenovirus and then starved for 18 h. Cells were lysed, and lysates were subjected to pull-down assays using a GST fusion of the activated RhoA-binding domain of rhotekin. Levels of precipitated RhoA were determined by immunoblot analysis (IB) using anti-RhoA antibody. Levels of total RhoA, G{alpha}12,G{alpha}13, and G{alpha}q were also determined as a control. B, expression of G{alpha}12QL and G{alpha}12QL({Delta}244-249) inhibits calcium-dependent aggregation of DU145 cells. DU145 cells were transduced with the indicated adenovirus, starved for 18 h, and then subjected to a 1-h "fast aggregation" assay (see "Experimental Procedures" for details). The percentage of cell aggregation was determined by counting the number of cells in each field in aggregates larger than 10 cells and dividing this by the total number cells in each field. Data shown are representative of results obtained in three separate experiments. C, expression of the activated form of G{alpha}12QL({Delta}244-249) does not induce invasion in the DU145 cell line. Du145 cells were transduced with the indicated adenovirus, starved for 18 h, and then allowed to invade GFR Matrigel-coated transwell filters for 30 h. The graph shows -fold increase in invasion over GFP control. Experiments were performed in duplicate, and at least four fields were counted for each replicate. All experiments were performed at least three times. All results are presented as mean ± S.E. *, p < 0.01 as determined by paired Student's t test.

 
Having confirmed that thrombin activates Rho in a G12-dependent fashion in prostate cancer cells, we next analyzed the effects of inhibiting G12 signaling on thrombin-induced prostate cancer cell invasion. Stimulation of the PC3 cell line with thrombin induced a 3-fold increase in invasion through a Matrigel layer (Fig. 6C). Strikingly, inhibition of G12 signaling with p115-RGS completely blocked the effects of thrombin, whereas expression of RGS2 or p115-RGS (E29K) had no effect (Fig. 6C). To establish whether these results were specific to thrombin signaling, we repeated these experiments using the thromboxane A2 receptor agonist U46619. [GenBank] Similar to thrombin, stimulation of the PC3 cells with U46619 [GenBank] enhanced PC3 cell invasion, and this effect was blocked by expression of p115-RGS but not by expression of RGS2 or of p115-RGS (E29K) (Fig. 6D). Finally, to determine if this effect represents a more generalized prostate cancer phenomenon or is specific to the PC3 cell line, we repeated these experiments using the DU145 cell line. As in the case of the PC3 cell line, stimulation of the DU145 cells with thrombin induced Matrigel invasion, and this effect was blocked by expression of p115-RGS but not by expression of RGS2 or of p115-RGS (E29K) (Fig. 6E). Taken together, these data provide convincing evidence that G12 signaling plays an important role in promoting prostate cancer invasion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
When prostate cancer is confined to the prostate gland, the majority of cases can be treated with surgery and/or radiation therapy. However, once the cancer has spread outside the prostate, either by invading the surrounding tissues directly or by metastasizing to distant sites within the body, the therapeutic options become limited. As such, understanding the molecular mechanisms underlying prostate cancer progression from a localized growth to a systemic disease is critical. Here, we show that the G12 proteins are up-regulated in localized prostate cancer. Further, we demonstrate that signaling by G12 proteins through the RhoA family of GTPases is a potent stimulator of prostate cancer cell invasion, suggesting that the G12 proteins may play a central role in prostate cancer progression.

Immunohistochemical staining of benign prostate and prostate cancer specimens revealed that the expression of G{alpha}12 is elevated in both prostate intraepithelial neoplasia and invasive prostate cancer, when compared with benign prostate epithelial cells. Taken together with the findings that the expression levels of both G{alpha}12 and G{alpha}13 are higher in aggressive prostate cancer cell lines compared with nontransformed prostate epithelial cell lines, these data suggest that the G12 proteins are up-regulated during prostate carcinogenesis. Interestingly, it appears that G{alpha}12 is up-regulated at similar points in both breast (30) and prostate cancer development. In addition, it appears that this increase in G{alpha}12 and G{alpha}13 expression seen in prostate cancer parallels the increase in expression previously reported for the Rho GTPases (47, 48) and several of the GPCRs known to promote prostate cancer cell invasion and metastasis (5, 7, 8, 49). As such, it appears that as prostate cancer progresses, the G12 signaling pathway may be up-regulated as an invasion-promoting signaling unit.


Figure 6
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[in this window]
[in a new window]
  		FIGURE 6.
G12 signaling is required for GPCR-stimulated invasion of prostate cancer cells. Thrombin-stimulated RhoA activation in PC3 cells (A) and DU145 cells (B) is inhibited by expression of p115-RGS but not by expression of RGS2 or expression of a GAP-deficient form of the p115-RGS, p115-RGS (E29K). Cells were transduced with the indicated adenovirus, starved for 18 h, and then stimulated with thrombin (1 unit/ml) or a vehicle control for 5 min. Cells were lysed, and lysates were subjected to pull-down assays using a GST fusion of the activated RhoA-binding domain of rhotekin. Levels of precipitated RhoA were determined by immunoblot analysis (IB) using anti-RhoA antibody. Levels of total RhoA, Myc-p115-RGS, and RGS2 were also determined as a control. All panels are representative of two or more separate experiments. C-E, expression of p115-RGS to inhibit G12 signaling blocks thrombin (C)- and thromboxane A2 (U46619) (D)-induced invasion of the PC3 cell line and thrombin-induced invasion of the DU145 cell line (E), whereas expression of RGS2 to inhibit Gq signaling or p115-RGS (E29K) has no effect. Cells were transduced with the indicated adenovirus, starved for 12 h, and then allowed to invade GFR Matrigel-coated transwell filters for 36 h in the presence of 1 unit/ml thrombin (C and E), 100 nM U46619 (D), or vehicle control. Experiments were performed in triplicate and results are presented as fold increase over vehicle-treated GFP control. All results are presented as mean ± S.E. All experiments were performed at least three times.

 
This study also establishes the G12 proteins as potent stimulators of prostate cancer cell invasion. Although much of the work to date on the role of the G12 family of proteins in cancer has focused on the ability of these proteins to promote cell growth and transformation (18, 19, 34, 35), work in other systems has implicated a role for these proteins in cell migration. In addition to the study noted above demonstrating a role for G12 proteins in breast cancer metastasis (30), reports have appeared suggesting a role for these proteins in neutrophil (25) and fibroblast migration (50). Further, studies on the role of G12 signaling in development have suggested that genetic ablation of the G12 proteins results in impaired cell migration (21-24). Interestingly, some of these studies that were conducted in Drosophila and zebrafish, revealed that signaling through the G12 family is required for the cell shape changes and movements required for normal gastrulation. Since many studies have demonstrated close parallels between gastrulation and cancer invasion (26-29), our findings that the G12 proteins are critical regulators of cancer invasion may represent the latest example of cancer usurping a normal biologic process to grow and metastasize.

Most of the studies demonstrating a role for G12 signaling in cell migration have focused on the ability of G{alpha}12 and G{alpha}13 to stimulate Rho. Moreover, a number of previous reports demonstrate that increased RhoA/C activity can promote invasion of prostate (48, 51, 52) and other cancers (41, 53-55). Thus, it was not surprising that G12-induced Rho activation was strictly required for G12-induced prostate cancer cell invasion. Since the invasion-promoting effects of the Rho pathway are pleiotropic and include such elements as activation of transcription factors, control of actinomysin contractility and cell polarity, modulation of cell adhesion, and induction of the epithelial to mesenchymal transition (41), it will now be interesting to determine which of these effects are important in G12-induced invasion.

Previous studies from our laboratory have provided evidence that G12 signaling has the potential to promote cell migration in a Rho-independent manner. We have previously demonstrated that G{alpha}12 and G{alpha}13 are able to inactivate E-cadherin through a direct interaction with its cytoplasmic tail and promote cell migration in E-cadherin-positive cells (42, 43). Therefore, we were somewhat surprised that G12-induced inactivation of E-cadherin was not sufficient to promote invasion of the DU145 cell line. Nevertheless, this result is consistent with a previous report demonstrating that inactivation of E-cadherin on DU145 cells using an antibody to block E-cadherin function resulted in only a minimal increase in cell migration (56). As such, the increase in cell motility resulting from the inactivation of E-cadherin by activated G{alpha}12({Delta}244-249) may not have been sufficient to produce a detectable change in DU145 cell invasion. Alternatively, these data may simply indicate that E-cadherin function has little involvement in prostate cancer cell invasion.

We recently reported that the G12 proteins are up-regulated in breast cancer and that G12 signaling promotes breast cancer metastasis by stimulating breast cancer cell invasion (30). Together with the finding reported here that G12 proteins are up-regulated in prostate cancer and that signaling through this pathway promotes prostate cancer cell invasion, a view is emerging that the principle role of G12 signaling in these common human cancers is to stimulate invasion and metastasis and not tumor growth as was previously suggested. Further, from a clinical perspective, these studies suggest that targeted inhibition of G12 signaling may provide effective therapies to slow invasion and reduce the morbidity and mortality associated with these and possibly other cancers.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants CA100869 (to P. J. C.) and AG17952 and DK60917 (to Y. D.), Department of Defense Grant DAMD17-03-1-0691 (to T. A. F.), and the Morris Cancer Center (to P. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2. Back

1 Supported by the Duke University Medical School Alumni Scholarship. Back

2 To whom correspondence should be addressed: Box 3813, Duke University Medical Center, Durham, NC 27710-3813. Tel.: 919-613-8613; Fax: 919-613-8642; E-mail: casey006{at}mc.duke.edu.

3 The 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. Back


   ACKNOWLEDGMENTS
 
We thank C. Demarco for generating the Myc-p115-RGS(E29K) mutant and K. Young and E. Kasbohm for technical assistance.



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