50% Effort. Molecular dissection of the pathway linking growth factor receptors to the nucleus: their role in normal cell growth and cancer. The emerging mutational landscape of G-proteins and G-protein coupled receptors in cancer. The presence of genetic alterations in G proteins and GPCRs were initially restricted to only few neoplastic lesions in endocrine tumors. However, we have recently reported the widespread presence and high frequency of mutations in GPCRs and G proteins in most tumor types. Specifically, in a recent in depth analysis of the human oncogenome we have found that a striking 4.2% of all tumor sequences exhibit activating mutations in GNAS (encoding Gs), including thyroid and pituitary tumors, as well as colon cancer, hepatocellular carcinoma, and parathyroid, ovarian, endometrial, biliary tract, and pancreatic tumors. Mutually exclusive activating mutations in GNAQ or GNA11 (encoding Gq family members) occur in 5.6% of tumors, including >66% of ocular melanomas, thus providing a clear example of a human malignancy that is initiated by gain of function mutations in Gq and G11 proteins. GNAQ and GNA11 mutations are also found in tumors arising from the meninges (59%), in most blue nevi of the skin (83%), and in a subset of cutaneous melanomas (6%). Surprisingly, nearly 20% of human cancers harbor mutations in GPCRs, including frequent mutations in thyroid-stimulating hormone receptor (TSHR), smoothened (SMO), glutamate receptors (GRMs), members of the adhesion family of GPCRs, and receptors for lysophosphatidic acid (LPA) and sphingosine 1 phosphate (S1P). Overall, as GPCRs are the target of >25% drugs in the market, we expect that this information can be exploited for the development of novel strategies targeting GPCRs, G proteins, or their aberrant signaling circuitry for cancer prevention and treatment. A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase network mediating GPCR-initiated mitogenic signaling and cancer growth. Multiple mitogens stimulate Gq-coupled GPCRs, and this receptor family contributes to cancer growth in many human malignancies. We have used Gq-coupled receptors activated solely by synthetic ligands (RASSLs) to build, and hence understand, GPCR-regulated signaling networks in normal and cancer cells. Gq-RASSL transduces potent mitogenic signals and is transforming if persistently activated, a process that requires the expression of c-Jun and c-Fos AP-1 family members. To investigate how GPCRs regulate AP-1-dependent gene transcription, we performed a genome-wide high-throughput RNAi screen in Drosophila S2 cells expressing Gq-GPCR and an AP-1 reporter system. Interestingly, we found that Rho family GTPases, specifically Rho and Rac, and their downstream effectors, such as Pak and Jun N-terminal kinase (JNK) were integral to AP-1 activation. While molecules linking GPCRs to the hydrolysis of phosphatidylinositol and PKC activation were dispensable, we found that Trio, a Rho guanine nucleotide exchange factor (GEF) that binds directly to Gq, is essential for AP-1 activation by Gq-coupled GPCRs. Remarkably, Trio is essential for the activation of JNK and p38 MAPKs, c-Jun and c-Fos expression, AP1 activation, and cell proliferation and transformation in mammalian cells. Furthermore, many cancers, including head and neck cancer (HNSCC) overexpress Trio due to genetic amplification. Trio downregulation prevents the mitogenic response to Gq-coupled receptor agonists in normal and cancer cells, and halts the growth of uveal melanoma cells harboring Gq mutations. These findings indicate that the growth promoting activity of Gq-linked GPCRs involves the activation of AP-1 by a Rho-GTPase network through Trio, and that this process is governed by highly specific protein-protein interactions and phosphorylation events rather than by diffusible second messengers. 30% Effort. Molecular basis of developmental and tumor-induced angiogenesis. Molecular mechanisms by which Semaphorins and Plexins control angiogenesis and lymphangiogenesis: Semaphorin 3E (Sema3E) and its receptor Plexin-D1 control the patterning of the developing vasculature. However, it was not known whether Sema3E-Plexin-D1 signals in adult and in pathological angiogenesis. We have recently observed that Sema3E behaves as a potent natural anti-lymphangiogenic molecule in a number of in vivo models of developmental and tumor-induced lymphangiogenesis. In particular, we observed that Sema3E provokes the rapid retraction of lymphatic endothelial cells, and diminish the pro-angiogenic activity of VEGF-C and S1P in vitro and in vivo, and prevents the pro-lymphangiogenic effect of HNSCC cells when grown in mouse xenografts. We are now investigating the underlying molecular mechanisms by which Sema3E acts in lymphatic endothelial cells. We have also focused in the possibility that another semaphorin, Sema3F, may represent an anti-lymphangiogenic metastasis suppressor gene, given that one of its co-receptors, NRP2, is expressed primarily in lymphatic endothelial cells, and that Sema3F gene loss is a frequent event in advanced HNSCC lesions. 20% effort. AIDS-associated Kaposis sarcoma: molecular mechanisms. Dephosphorylated 4EBP disrupts paracrine transformation by the KSHV vGPCR oncogene upon mTOR inhibition: Early work from our group led to the identification of the Akt/mTOR pathway as a critical signaling axis contributing to KSHV-induced cancer progression, and treatment of KS patients with rapamycin provided the first evidence of the antineoplastic activity of mTOR inhibitors in humans. Thus, the study of KS may provide a unique opportunity to dissect the contribution of specific mTOR substrates to cancer development. We focused on a direct target of mTOR, 4EBP1/2/3 (4EBP), which inhibits the translation of eukaryotic initiation factor 4E (4E)-bound mRNAs. 4EBP phosphorylation by mTOR relieves its inhibitory activity, hence resulting in increased 4E-dependent mRNA translation. We developed a paracrine transformation model, recapitulating the cellular composition of KS lesions, in which vGPCR-expressing cells promote the rapid proliferation of endothelial cells expressing KSHV-latent genes by the release of growth factors. Using this model, we showed that the accumulation of dephosphorylated 4EBP in response to rapamycin or by the expression of an mTOR-insensitive mutant of 4EBP1 is sufficient to disrupt the 4E function downstream of mTOR thereby halting KS development. These findings may provide a preclinical platform and the rationale for the development of novel mTOR inhibiting agents that may selectively disrupt the mTOR-4EBP interaction for the treatment of KS and other tumor lesions exhibiting hyperactive mTOR pathway The IKK kinase is critical for vGPCR-induced tumorigenesis. In previous studies we have shown that vGPCR stimulates the expression of pro-angiogenic cytokines by the activation of NFB. We have recently observed that this process involves the increased activity of IKK, an IKK-related kinase, and that IKK is critically required for vGPCR tumorigenesis. Loss of IKK in genetically engineered mice effectively abrogated NFB activation and tumorigenesis triggered by vGPCR. Collectively, our findings uncover the critical role of IKK in promoting inflammation and tumorigenesis induced by a viral GPCR.
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