Certain alterations of proteins involved in mitogenic signaling are known to exert profound effects on cellular behavior, including malignant transformation. Our overall objective is to explore the molecular bases of cancer, approaching this problem through the study of normal and aberrant functioning of molecules that participate in the transduction of proliferative signals. G protein-coupled receptors (GPCRs) signal through Gq and G13-initiated MAPK cascades regulating c-Jun expression to induce cell transformation: Whereas the ability of GPCRs to stimulate normal and aberrant cell growth has been intensely investigated, the precise molecular mechanisms underlying their transforming potential are still not fully understood. Taking advantage of the potent mitogenic effect of thrombin and the focus-forming activity of one of its receptors, PAR-1, we have recently studied how this receptor, which is coupled to Galphai, Galphaq/11, and Galpha12/13, transduces signals from the membrane to the nucleus to initiate transcriptional events involved in cellular transformation. Using endogenous and transfected thrombin receptors, the ectopic expression of GPCRs coupled to Galphaq and Galphai, as well as chimeric G protein alpha subunits and murine fibroblasts knock out for Galphaq/11 and Galpha12/13, we found that although coupling to Galpahi is sufficient to induce ERK activation, the ability to stimulate Galphaq/11 and/or Galpha12/13 is necessary to induce c-jun expression and cellular transformation. Furthermore, we have observed that Galphaq/11 and Galpha12/13 can initiate the activation of MAPK cascades, including JNK, p38, and ERK5, which in turn regulate transcription factors that control the expression from the c-jun promoter, and that c-Jun and the kinases regulating its expression are integral components of the transforming pathway initiated by PAR-1. These results suggest that the ability to stimulate c-jun expression can distinguish transforming from not transforming GPCRs. They also help explain how mitogens acting on GPCRs stimulate the expression of growth promoting genes. The small GTP-binding protein RhoA regulates c-Jun by a ROCK-JNK signaling axis. Rho GTPases, including RhoA, Rac1 and Cdc42, regulate gene expression in the nucleus in addition to their best known function in cytoskeletal control. The nature of the genes that are regulated by Ras and Rho GTPases was investigated using cDNA microarrays comprising 19,117 unique elements. By this approach, we found that these GTPases stimulate unique as well as common sets of genes, thus providing key information to begin unraveling the complexity of the molecular mechanisms by which they transform cells. In this regard, we have previously shown that RhoA stimulates the expression of c-jun, and that c-Jun is required for the RhoA-induced focus formation. In search for the mechanism by which RhoA stimulates c-jun, we found that RhoA signals to the nucleus by a biochemical route that bifurcates at the level of a kinase known as ROCK to initiate two independent pathways, one controlling the actin cytoskeleton and c-fos expression, and another leading to the activation of a novel MAPK cascade stimulating JNK. The latter results in the activation of transcription factors pre-bound to the c-jun promoter and the expression of c-Jun. Ultimately, these divergent pathways acting downstream from ROCK converge in the nucleus to control the levels and transcriptional activity of AP1 complexes, thus coordinating RhoA-induced cytoskeletal changes with the transcriptional activation of genetic programs involved in key cellular processes, including cell invasion and normal and cancerous cell growth. LARG and PDZ-RhoGEF link Plexin B to Rho: A potential role in axon guidance and tumor-induced angiogenesis: Plexins are members of a novel family of transmembrane receptors that transduce attractive and repulsive signals mediated by the axon-guiding molecules semaphorins. Although emerging evidence implicates Rho GTPases in these biological events, Plexins lack any known catalytic activity in their conserved cytoplasmic tails, and how they transduce signals from semaphorins to RhoA is still unknown. Using a yeast two-hybrid screen for molecules interacting with the PDZ-domain of PDZ-RhoGEF (PRG), we identified the cytoplasmic tail of PlexinB2 as a candidate interacting molecule. Indeed, we found that Plexin B2 can associate directly with both PRG and LARG, and that this physical interaction was mediated by a PDZ-binding motif found in Plexins of the B family. Ligand-induced dimerization of Plexin B is sufficient to stimulate endogenous RhoA potently, and that LARG and PRG mediate this effect, and also cell rounding and neurite retraction when endogenous Plexin B1 was stimulated by semaphorin 4D in neuronal-derived cells. To explore the biological relevance of these findings, we have recently generated PRG and LARG knock out mice. Available information suggests that the function of these Rho GEFs is partially overlapping, and that their genetic deletion results in behavioral defects consistent with their role in axon guidance. Interestingly, we have also found that Plexin B1 is highly expressed in endothelial cells, and that its ligand, semaphorin 4D, is expressed in certain tumor cells. Whether Plexin B, LARG, and PRG play a role in developmental and tumor-induced angiogenesis is under current investigation. Endothelial-specific retroviral transduction of Kaposi?s Sarcoma Virus oncogenes reveals a key role for the vGPCR in the initiation of Sarcomagenesis: Kaposi's Sarcoma (KS) is the most common cancer arising in HIV-infected patients and the most frequent oral neoplasm in immunosuppressed patients. KS has also emerged as one of the most prevalent cancers among children and adult men in the developing world. The Kaposi's Sarcoma Associated Herpesvirus (KSHV; HHV-8) has been recently identified as the infectious cause of Kaposi?s Sarcoma. Of interest, compelling evidence now supports a critical role for the oral cavity as the primary source of infectious HHV-8 in both immunocompetent and immunosuppressed patients. Indeed, mucosal shedding of this virus may represent the principal mode of viral transmission. The molecular characterization of the KSHV genome has revealed the presence of numerous potential oncogenes. To begin addressing their contribution to the development of the endothelial cell-derived KS tumor, we developed a novel transgenic animal model expressing the avian retroviral receptor, TVA, under the TIE2 promoter. This enabled endothelial cell-specific infection in vivo using avian leukosis virus expressing candidate KSHV oncogenes, including Kaposin, vFlip, vGPCR, vIRF-1, vCyclin, and vBCL-2. Remarkably, retroviral transduction of one gene, vGPCR, was sufficient to induce angioproliferative tumors that strikingly resemble human KS. Furthermore, by the use of immortalized murine endothelial cells expressing key latent KSHV genes, we obtained evidence that cells expressing vGPCR cooperate with cells expressing latent genes to promote tumorigenesis. Collectively, these results implicate vGPCR in both the initiation and promotion of Kaposi's sarcomagenesis. vGPCR is a member of the CXC chemokine GPCR family that, unlike its cellular homologues, exhibits ligand-independent activities. Which mitogenic and survival pathways are utilized by vGPCR to induce tumorigenesis is under current investigation, as they may represent suitable targets for the development of pathogenesis-based therapies against KSHV.
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