Many studies have suggested a role for the members of the G 12 family of heterotrimeric G proteins (G␣ 12 and G␣ 13 ) in oncogenesis and tumor cell growth. However, few studies have examined G 12 signaling in actual human cancers. In this study, we examined the role of G 12 signaling in prostate cancer. We found that expression of the G 12 proteins is significantly elevated in prostate cancer. Interestingly, expression of the activated forms of G␣ 12 or G␣ 13 in the PC3 and DU145 prostate cancer cell lines did not promote cancer cell growth. Instead, expression of the activated forms of G␣ 12 or G␣ 13 in these cell lines induced cell invasion through the activation of the RhoA family of G proteins. Furthermore, inhibition of G 12 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 G 12 signaling with p115-RGS in these cell lines blocked thrombin-and thromboxane A2-stimulated cell invasion. These observations identify the G 12 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.

The heterotrimeric G protein G 12 has been implicated in such cellular regulatory processes as cytoskeletal rearrangement, cell-cell adhesion, and oncogenic transformation. Although the activated ␣-subunit of G 12 has been shown to interact directly with a number of protein effectors, the roles of many of these protein-protein interactions in G 12 -mediated cell physiology are poorly understood. To begin dissecting the specific cellular pathways engaged upon G 12 activation, we produced a series of substitution mutants in the regions of G␣ 12 predicted to play a role in effector binding. Here we report the identification and characterization of an altered form of G␣ 12 that is functionally uncoupled from signaling through the monomeric G protein Rho, a protein known to propagate several G␣ 12 -mediated signals. This mutant of G␣ 12 fails to bind the Rho-specific guanine nucleotide exchange factors p115RhoGEF and LARG (leukemia-associated RhoGEF), fails to stimulate Rho-dependent transcriptional activation, and fails to trigger activation of RhoA and the Rho-mediated cellular responses of cell rounding and c-jun N-terminal kinase activation. Importantly, this mutant of G␣ 12 retains coupling to the effector protein E-cadherin, as evidenced by its ability both to bind E-cadherin in vitro and to disrupt E-cadherin-mediated cell-cell adhesion. Furthermore, this mutant retains the ability to trigger ␤-catenin release from the cytoplasmic domain of cadherin. This identification of a variant of G␣ 12 that is selectively uncoupled from one signaling pathway while retaining signaling capacity through a separate pathway will facilitate investigations into the mechanisms through which G 12 proteins mediate diverse biological responses.

Wnt proteins constitute a family of secreted signaling molecules that regulate highly conserved pathways essential for development and, when aberrantly activated, drive oncogenesis in a number of human cancers. A key feature of the most widely studied Wnt signaling cascade is the stabilization of cytosolic β-catenin, resulting in β-catenin nuclear translocation and transcriptional activation of multiple target genes. In addition to this canonical, β-catenin-dependent pathway, Wnt3A has also been shown to stimulate RhoA GTPase. While the importance of activated Rho to non-canonical Wnt signaling is well appreciated, the potential contribution of Wnt3A–stimulated RhoA to canonical β-catenin-dependent transcription has not been examined and is the focus of this study. We find that activated Rho is required for Wnt3A–stimulated osteoblastic differentiation in C3H10T1/2 mesenchymal stem cells, a biological phenomenon mediated by stabilized β–catenin. Using expression microarrays and real-time RT-PCR analysis, we show that Wnt3A–stimulated transcription of a subset of target genes is Rho-dependent, indicating that full induction of these Wnt targets requires both β-catenin and Rho activation. Significantly, neither β–catenin stabilization nor nuclear translocation stimulated by Wnt3A is affected by inhibition or activation of RhoA. These findings identify Rho activation as a critical element of the canonical Wnt3A–stimulated, β–catenin-dependent transcriptional program.