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

␣ subunits. p115 RhoGEF stimulates the intrinsic GTP hydrolysis activity of G␣12/13 subunits and acts as an effector for G13-coupled receptors by linking receptor activation to RhoA activation. The presence of RGS box and Dbl homology domains within LARG suggests this protein may also function as a GAP toward specific G␣ subunits and couple G␣ activation to RhoA-mediating signaling pathways. Unlike the RGS box of p115 RhoGEF, the RGS box of LARG interacts not only with G␣12 and G␣13 but also with G␣q. In cellular coimmunoprecipitation studies, the LARG RGS box formed stable complexes with the transition state mimetic forms of G␣q, G␣12, and G␣13. Expression of the LARG RGS box diminished the transforming activity of oncogenic G protein-coupled receptors (Mas, G2A, and m1-muscarinic cholinergic) coupled to G␣q and G␣13. Activated G␣q, as well as G␣12 and G␣13, cooperated with LARG and caused synergistic activation of RhoA, suggesting that all three G␣ subunits stimulate LARG-mediated activation of RhoA. Our findings suggest that the RhoA exchange factor LARG, unlike the related p115 RhoGEF and PDZ-RhoGEF proteins, can serve as an effector for Gq-coupled receptors, mediating their functional linkage to RhoA-dependent signaling pathways.

Increased expression of protease-activated receptor 1 (PAR1), a G protein-coupled receptor for thrombin, has previously been correlated with breast carcinoma cell invasion. PAR1 is irreversibly proteolytically activated, internalized, and sorted directly to lysosomes, a critical process for the termination of signaling. We determined that activated PAR1 trafficking is severely altered in metastatic breast carcinoma cells but not in nonmetastatic or normal breast epithelial cells. Consequently, the proteolytically activated receptor is not sorted to lysosomes and degraded. Altered trafficking of proteolytically activated PAR1 caused sustained activation of phosphoinositide hydrolysis and extracellular signal-regulated kinase signaling, even after thrombin withdrawal, and enhanced cellular invasion. Thus, our results reveal that a novel alteration in trafficking of activated PAR1 causes persistent signaling and, in addition to other processes and proteins, contributes to breast carcinoma cell invasion.The genetic changes that initiate tumor cell metastasis and invasion are unclear. The procoagulant activities of tumor cells and surrounding stromal cells that lead to thrombin generation and fibrin deposition appear to provide an important influence on tumor cell invasion and metastasis (3). Although a link between coagulation factors and tumor progression and metastasis has been firmly established, the molecular basis remains poorly understood (7, 26).Thrombin, the main effector protease of the coagulation cascade, is generated by the actions of tissue factor and other coagulation factors. The first description of thrombin-induced murine tumor cell metastasis was provided by Nierodzik and colleagues (23,24). Subsequent studies determined that tissue factor, like thrombin, is highly expressed in carcinoma cells and contributes to metastasis (20,21). In addition, the anticoagulants heparin and warfarin have been shown to significantly decrease experimentally induced pulmonary metastasis in vivo (37). A similar decrease in tumor cell metastasis in vivo was observed with hirudin, a specific inhibitor of thrombin (9). Fibrin, generated by the action of thrombin on fibrinogen, is a significant component of the tumor microenvironment and has been strongly associated with tumor progression and metastasis (7). A recent genetic study revealed that, in fibrinogendeficient mice, the development of both spontaneous and experimental lung metastases was substantially diminished (27). However, hirudin further diminished the metastatic potential of tumor cells in fibrinogen-deficient mice. Thus, while fibrinogen is one critical component of metastasis, thrombin appears to contribute to tumor cell dissemination through at least one fibrinogen-independent mechanism.

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) was originally identified as a fusion partner with mixed-lineage leukemia in a patient with acute myeloid leukemia. LARG possesses a tandem Dbl homology and pleckstrin homology domain structure and, consequently, may function as an activator of Rho GTPases. In this study, we demonstrate that LARG is a functional Dbl protein. Expression of LARG in cells caused activation of the serum response factor, a known downstream target of Rho-mediated signaling pathways. Transient overexpression of LARG did not activate the extracellular signal-regulated kinase or c-Jun NH 2 -terminal kinase mitogen-activated protein kinase cascade, suggesting LARG is not an activator of Ras, Rac, or Cdc42. We performed in vitro exchange assays where the isolated Dbl homology (DH) or DH/pleckstrin homology domains of LARG functioned as a strong activator of RhoA, but exhibited no activity toward Rac1 or Cdc42. We found that LARG could complex with RhoA, but not Rac or Cdc42, in vitro, and that expression of LARG caused an increase in the levels of the activated GTP-bound form of RhoA, but not Rac1 or Cdc42, in vivo. Thus, we conclude that LARG is a RhoA-specific guanine nucleotide exchange factor. Finally, like activated RhoA, we determined that LARG cooperated with activated Raf-1 to transform NIH3T3 cells. These data demonstrate that LARG is the first functional Dbl protein mutated in cancer and indicate LARG-mediated activation of RhoA may play a role in the development of human leukemias.

Vav2, like all Dbl family proteins, possesses tandem Dbl homology (DH) and pleckstrin homology (PH) domains and functions as a guanine nucleotide exchange factor for Rho family GTPases. Whereas the PH domain is a critical positive regulator of DH domain function for a majority of Dbl family proteins, the PH domains of the related Vav and Vav3 proteins are dispensable for DH domain activity. Instead, Vav proteins contain a cysteine-rich domain (CRD) critical for DH domain function. We evaluated the contribution of the PH domain and the CRD to Vav2 guanine nucleotide exchange, signaling, and transforming activity. Unexpectedly, we found that mutations of the PH domain impaired Vav2 signaling, transforming activity, and membrane association. However, these mutations do not influence exchange activity on Rac and only slightly affect exchange on RhoA and Cdc42. We also found that the CRD was critical for the exchange activity in vitro and contributed to Vav2 membrane localization. Finally, we found that phosphoinositol 3-kinase activation synergistically enhanced Vav2 transforming and signaling activity by stimulating exchange activity but not membrane association. In conclusion, the PH domain and CRD are mechanistically distinct, positive modulators of Vav2 DH domain function in vivo.Rho family proteins are members of the Ras superfamily of small GTPases. Currently, 18 mammalian Rho family proteins have been identified, with Rac1, Cdc42, and RhoA being the best characterized (37, 45). Rho family GTPases are guanine nucleotide binding proteins that function as molecular switches that cycle between active GTP-bound and inactive GDP-bound states. Dbl family proteins serve as guanine nucleotide exchange factors (GEFs), which accelerate the intrinsic GDP/ GTP exchange activity of Rho GTPases to cause formation of the active GTP-bound protein (8, 39). The activated Rho GTPases then interact with a wide spectrum of downstream effector proteins to mediate cellular activities that include regulation of actin cytoskeletal organization, gene expression, and cellular proliferation (3). A second class of regulatory proteins, Rho family-specific GTPase-activating proteins, stimulate intrinsic GTPase activity to return these small GTPases to their inactive GDP-bound state and to terminate downstream signaling (37).Vav proteins (Vav, Vav2, and Vav3) are mammalian members of the Dbl family of proteins (7). All Vav proteins have similar structural organizations. Like all Dbl family proteins, Vav proteins possess a Dbl homology (DH) domain followed by a COOH-terminal pleckstrin homology (PH) domain (8,39). Previous studies indicate that the DH domain interacts directly with Rho family GTPases to catalyze GDP release (8, 39). The Vav DH domains exhibit broad GTPase specificity and serve as GEFs for multiple Rho GTPases (RhoA, RhoG, Rac1, and Cdc42), although different studies have reached contrasting conclusions regarding the specific GTPases targeted by Vav (1,12,17,23,28,35,43).The invariant topography of DH and PH domains (DH/PH dom...