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. Molecular dissection of the pathway linking growth factor receptors to the nucleus: their role in normal cell growth and cancer. Small GTPases of the Rho family, including Rho, Rac, and Cdc42, play a central role linking G protein coupled receptors (GPCRs) and polypeptide growth factor receptors to the nuclear expression of growth promoting genes, such as c-jun and c-fos. Prior work conducted in our laboratory indicated that the pathway by which GPCRs stimulate Rho involves the activation of alpha subunits of the G12/13 and Gq family of heterotrimeric G proteins, and that Galpha12/13 in turn stimulate Rho through the direct interaction with a group of Rho guanine nucleotide exchange factors (GEFs) that include PDZ-RhoGEF (PRG), LARG, and p115-RhoGEF (p115). These GEFs are characterized by the presence of a RGS domain that binds Galpha12/13, thus providing a molecular bridge for the activation of Rho by Galpha12/13. As part of an effort aimed at elucidating how Galpha12/13 stimulate RhoGEFs, we have addressed recently the structural requirements for Galpha13-dependent stimulation of Rho by engineering chimeric G proteins using Galphai2, which does not activate RGS-RhoGEFs, as the backbone in which Galpha13 sequences were swapped or mutated. The emerging results revealed that Galpha13 depends on most of its GTPase domain and its membrane localization for the effective coupling of Galpha13 to the RGS-containing family of RhoGEFs, and ultimately to stimulate Rho and cell growth. Because small GTP-binding proteins of the Ras and Rho family are essential components of biochemical pathways transducing proliferative signals from the vast majority of growth factor receptors, as part of our program we have investigated the nature of the genes regulated by these GTPases and their contribution to cell transformation. In a recent study, we have compared the transcriptional targets of Ras and Rho GTPases, and observed that osteopontin (OPN) exhibits the best correlation between cell transformation and up-regulation of gene expression. OPN is a secreted adhesive glycoportein with diverse functions, which has been implicated in cancer development, progression and metastasis. Focusing on the H-ras oncogene, we have found that ras and OPN acted synergistically to induce foci formation, and that the transforming, invasive and migratory activity of ras was reduced by short interfering OPN RNAi (siRNA). Together, the available data support the existence of an unexpected aberrant Ras-OPN autocrine loop, and suggest that OPN overexpression may represent a key molecular event in cancer progression and metastasis, particularly in a large number of human neoplasias that harbor activating ras mutations. GPCRs and polypeptide growth factors promote the re-initiation of DNA-synthesis through multiple intracellular signaling pathways that converge in the nucleus to control the expression of growth-promoting genes. Among them, the AP-1 family of transcription factors, including c-Fos and c-Jun family members, plays a key role. In a prior study, we have observed that PDGF stimulates the ERK-mediated phosphorylation of multiple residues in the carboxyl-terminal transactivation domain (TAD) of c-Fos, and that this phosphorylation is required to stimulate c-Fos- and AP-1-dependent transcription. However, how MAPK-dependent phosphorylation regulates c-Fos function is still unknown. In this reporting period, we have found that the prolyl-isomerase Pin1 binds c-Fos through specific phosphorylated residues within the c-Fos TAD, and that this interaction results in an enhanced transcriptional response of c-Fos to growth factors. These findings suggest that c-Fos represents a novel target for the isomerizing activity of Pin1, and support a role for Pin1 in the mechanism by which c-Fos regulates AP-1-dependent gene transcription upon phosphorylation by MAPKs. Molecular basis of developmental and tumor-induced angiogenesis. Tumor growth and metastasis requires the induction of angiogenesis, the growth and remodeling of new blood vessels from a pre-existing vascular network, to ensure the delivery of oxygen and nutrients to rapidly dividing transformed cells. Using a yeast two-hybrid approach to screen for molecules interacting with the PDZ-domain of PRG, we have previously identified the cytoplasmic tail of PlexinB2 as a candidate interacting molecule. Plexins are transmembrane receptors that transduce attractive and repulsive signals mediated by semaphorins during the development of the central nervous system. However, we have recently observed that Plexin B1 is highly expressed in endothelial cells, and that its ligand, Semaphorin 4D (Sema4D), is expressed in certain tumor cells, including squamous carcinomas. Indeed, we have recently shown that Sema4D can promote a potent chemotactic and pro-angiogenic response through Plexin-B1 in endothelial cells, and that this response requires the activation of the kinase Akt in a PI3K-dependent manner by Sema4D. The molecular dissection of the underlying mechanism revealed that binding of Sema4D to Plexin B1 results in the recruitment of a multimeric signaling complex that includes PYK2, Src, and PI3K to Plexin-B1, and the activation of Akt. Together, these recent findings support the emerging notion that plexins of the B family promote angiogenesis by initiating a complex array of signaling events. AIDS-associated Kaposi?s sarcoma: molecular mechanisms. Among the AIDS-associated malignancies, Kaposi?s sarcoma (KS) is the most common cancer arising in HIV-infected patients. The Human herpesvirus 8 (HHV-8) or KS associated herpesvirus (KSHV) is the infectious cause of KS. Oral transmission plays a key role in the spread of KS. As the most frequent oral neoplasm in AIDS patients, KS is an infectious disease of paramount concern for oral health. Among the cytokines elaborated by the KS spindle cell, interleukin-6 (IL-6) is unique in that it has been implicated in the pathogenesis of all KSHV-associated malignancies. KSHV encodes its own IL-6 homologue (vIL-6), which is highly expressed in certain B-cell lymphomas caused by KSHV. However, vIL-6 is not detected in KS lesions. KS may, instead, rely mainly on its cellular host to produce this critical growth factor. Indeed, cellular IL-6 serves as an autocrine growth factor for AIDS-KS cells, and an IL-6 promoter polymorphism resulting in enhanced IL-6 production is associated with an increased risk for development of KS in men infected with HIV, thus suggesting that cellular IL-6 substitutes for its viral homologue in KS. Because endothelial cells transformed by the KSHV encoded GPCR (vGPCR) can promote the growth of bystander endothelial cells through a paracrine mechanism, we decided to explore the contribution of vGPCR to the upregulation of IL-6 secretion in KS. In this reporting period, we have observed that endothelial cells expressing vGPCR secrete elevated levels of IL-6, and that this effect was dependent on the activation of transcription from a kappa B responsive element within the IL-6 promoter. Furthermore, we observed that the small GTP-binding protein Rac1 mediates the activation of NFkappaB and IL-6 transcription and secretion by vGPCR. Together, these results implicated Rac1 in the initiation and progression of KS, probably through the stimulation of NFkappaB and IL6 secretion, and thus identify this small GTPase and its downstream effectors as novel molecular targets in the development.
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