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 Gαs), 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 Gαq 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 Gαq 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. Hippo-Independent activation of YAP by the GNAQ uveal melanoma oncogene through a Trio-regulated Rho GTPase signaling circuitry. Uveal melanoma is the most frequent ocular malignancy in adults, for which no effective systemic therapies are currently available. Recent findings revealed that activating mutations in GNAQ and GNA11, encoding members of the Gαq family of G protein αsubunits, drive uveal melanoma oncogenesis. Approximately 6% of skin melanomas also express GNAQ oncogenes, hence rendering these melanomas insensitive to BRAF inhibitors in the clinic. However, the molecular events underlying these GNAQ-driven malignancies are not yet defined, thus limiting the ability to develop cancer-targeted therapies. Recently, we focused on the transcriptional co-activator YAP, a critical component of the Hippo signaling pathway that controls organ size. We found that Gαq stimulates YAP through a novel Trio-Rho/Rac signaling circuitry promoting actin polymerization, independently of PLCβand the canonical Hippo pathway. Furthermore, we show that Gαq promotes the YAP-dependent growth of uveal melanoma cells, thereby identifying YAP as a novel therapeutic target in uveal melanoma, the first described GNAQ/GNA11-initiated human malignancy. This cancer vulnerability can now be exploited for the development of new precision molecular therapies for GNAQ-driven human malignancies. 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 Activation of YAP/TAZ by KSHV vGPCR in sarcomagenesis: Building on our prior studies on uveal melanoma and transforming GPCRs, we have recently contributed to the discovery that vGPCR activates the YAP/TAZ transcription co-activators, which are inhibited by the Hippo tumor suppressor pathway. Down-regulation of YAP/TAZ blocks vGPCR-induced cell proliferation and tumorigenesis. Furthermore, we found that YAP/TAZ are activated in Kaposis sarcoma and correlates with KSHV markers. Our observations indicate that YAP/TAZ mediate the oncogenic function of vGPCR in Kaposis sarcoma tumorigenesis.
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