Although the prognosis for patients with early-stage breast cancer has improved, the therapeutic options for patients with locally advanced and metastatic disease are limited. To improve the treatment of these patients, the molecular mechanisms underlying breast cancer invasion and metastasis must be understood. In this study, we report that signaling through the G12 family of heterotrimeric G proteins (G␣12 and G␣13) promotes breast cancer cell invasion. Moreover, we demonstrate that inhibition of G12 signaling reduces the metastatic dissemination of breast cancer cells in vivo. Finally, we demonstrate that the expression of G␣12 is significantly up-regulated in the earliest stages of breast cancer, implying that amplification of G12 signaling may be an early event in breast cancer progression. Taken together, these observations identify the G12 family proteins as important regulators of breast cancer invasion and suggest that these proteins may be targeted to limit invasion-and metastasis-induced patient morbidity and mortality.cadherin ͉ Rho ͉ G protein-coupled receptor

The G12 subfamily of heterotrimeric guanine nucleotide-binding proteins consists of two a subunits, Ga12 and Ga13. These proteins mediate signalling via G protein-coupled receptors and have been implicated in various physiological and pathophysiological processes. A number of direct and indirect effectors of Ga12 and Ga13 have been identified that mediate, or have been proposed to mediate, the diverse cellular responses accompanying activation of G12 proteins. This review describes the signalling pathways and cellular events stimulated by G12 proteins, with a particular emphasis on processes that are important in regulating cell migration and invasion, and could potentially be involved in the pathophysiology of cancer metastasis. Experimental findings directly implicating G12 proteins in the spread of metastatic disease are also summarized, indicating the importance of targeted inhibition of G12 signalling as a potential therapeutic option for locally advanced and metastatic disease.

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.

Signaling through the heterotrimeric G protein, G12, via Rho induces a striking increase in breast cancer cell invasion. In this study, evidence is provided that the c-Jun NH2-terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively-active Gα12 or activation of G12 signaling by thrombin leads to increased JNK and c-Jun phosphorylation. Pharmacologic inhibition of JNK or knockdown of JNK expression by siRNA significantly decreases G12-induced JNK activation as well as the ability of breast cancer cells to invade a reconstituted basement membrane. Furthermore, expression of dominant-negative Rho or treatment of cells with an inhibitor of the Rho kinase, ROCK, reduces G12-induced JNK and c-Jun activation, and ROCK inhibitor treatment also inhibits G12-induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. Taken together, our data indicate that JNK activation is required for G12-induced invasion of breast cancer cells and that JNK is downstream of Rho and ROCK on this pathway. This study implicates a G12-stimulated mitogen-activated protein kinase cascade in cancer cell invasion, and supports a role for JNK in cancer progression.