Molecular Mechanisms Of Growth Control And Carcinogenesis

Dental & Craniofacial Research

Investigators

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Abstract

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. [unreadable] [unreadable] Molecular dissection of the pathway linking growth factor receptors to the nucleus: their role in normal cell growth and cancer[unreadable] [unreadable] Galpha13-Rho signaling axis is required for SDF-1-induced migration through CXCR4: The CXC chemokine SDF-1 (also known as CXCL12) binds to CXCR4, a G protein coupled receptor (GPCR) that plays a critical role in many physiological processes that involve cell migration, ranging from stem cell homing, angiogenesis, to immune response. CXCR4 is also implicated in various pathological conditions, including metastasis and HIV infection. We took advantage of the potent chemotactic activity of SDF-1 in Jurkat T-cells to examine which G protein subunits contribute to CXCR4-mediated cell migration. Whereas Gi and Gbeta-gamma subunits are involved in Rac activation and cell migration, CXCR4 also stimulated Rho potently, but independently of Gi. Instead, we found that Galpha13 mediates the activation of Rho by CXCR4, and that both Galpha13 and Rho are required for directional cell migration in response to SDF-1. These findings identified the Galpha13-Rho signaling axis as a potential pharmacological target in many human diseases that involve the aberrant function of CXCR4. [unreadable] [unreadable] Rac inhibits thrombin-induced Rho activation: Evidence of a Pak-dependent GTPase crosstalk: The strict spatio-temporal control of Rho GTPases is critical for many cellular functions, including cell motility and growth. The prototypical Rho family GTPases, Rho, Rac, and Cdc42 regulate the activity of each other by a poorly understood mechanism. We found that constitutively active Rac inhibits stress fiber formation and Rho stimulation by thrombin, a GPCR agonist. Because a mutant Rac that does not activate Pak1 failed to inhibit Rho activation, we asked whether Pak1 could regulate GEFs for Rho. We found that Pak1 specifically associates with p115-RhoGEF, and knock down experiments revealed that p115-RhoGEF plays a major role in signaling from thrombin receptors to Rho. We provided evidence that Pak1 binds the DH-PH domain of p115-RhoGEF thereby disrupting receptor-dependent Rho signaling, thus providing a mechanism for cross-talk between these small-GTPases.[unreadable] [unreadable] [unreadable] Molecular basis of developmental and tumor-induced angiogenesis [unreadable] [unreadable] Molecular mechanisms by which Semaphorins and Plexins promote angiogenesis: We have shown that semaphorin 4D (Sema4D) elicits a pro-angiogenic phenotype in endothelial cells by promoting the activation of the PI3K-Akt signaling pathway through its receptor, Plexin-B1. By the use of a receptor chimeric approach, Plexin-B1 mutants, knock down strategies, and dominant negative inhibitors, we found that this response is dependent upon the activation of RhoA and its downstream target, Rho kinase (ROK). Furthermore, we observed that Plexin-B1 utilizes RhoA and ROK to activate an integrin-dependent signaling network that stimulates Pyk2, PI3K, Akt, and ERK, and endothelial motility. On the other hand, we found that Sema4D must be processed and released from its membrane bound form to act in a paracrine manner on endothelial cells. In collaboration with MCU, K. Holmbeck, and T. Bugge (PTRU), we used general and specific inhibitors of MMPs and knockout MEFs to demonstrate that Sema4D is a novel target for membrane type 1-MMP (MT1-MMP). MT1-MMP is not expressed in non-tumorigenic epithelial cells but present in head and neck squamous cell carcinoma (HNSCC) cells. By lentiviral-delivery of shRNAs, we showed that the knock down of MT1-MMP prevents the release of Sema4D into its soluble form from these cells, thereby inhibiting their pro-angiogenic responses in vitro and in vivo. These effects were rescued by re-expression of a catalytically active MT1-MMP, or a truncated form of Sema4D. The proteolytic cleavage of Sema4D may provide a novel molecular mechanism by which MT1-MMP controls tumor-induced angiogenesis.[unreadable] [unreadable] The molecular basis of VEGF-induced endothelial cell permeability: VEGF is a pleiotropic factor for endothelial cells that plays a central role in both vasculogenesis and angiogenesis. Deregulated VEGF expression also contributes to the aberrant growth of most solid tumors by promoting their neo-vascularization. In an effort aimed at exploring the mechanism by which VEGF stimulates the proliferation, migration, and survival of endothelial cells, we began investigating how VEGF induces vessel leakiness, its first described function. In a recent study, we identified a novel signaling mechanism, involving the sequential activation of Src, Vav2, Rac1, and Pak1, by which VEGF promotes the rapid removal of a key cell-cell adhesion molecule, VE-cadherin, from the cell surface, leading to the disassembly of endothelial cell junctions and enhanced permeability. These findings opened new therapeutic opportunities for the treatment of many human diseases that involve pathological vessel leakiness. [unreadable] [unreadable] AIDS-associated Kaposi s sarcoma: molecular mechanisms[unreadable] [unreadable] 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. In prior studies, we have developed a high throughput in vivo endothelial specific retroviral gene transfer system, and used it to express candidate KSHV oncogenes in mice. Surprisingly, among the many KSHV genes tested, only one gene, a constitutively active GPCR, vGPCR, was able to promote the development of visible vascular tumors that strikingly resembled human KS lesions. While investigating the nature of the mitogenic and survival pathways utilized by vGPCR to induce tumorigenesis, we observed that vGPCR stimulates the serine/threonine kinase Akt in vivo, and that the activation of Akt and its downstream target, mTOR, represents a hallmark of human KS. vGPCR promotes the activation of mTOR by causing the phosphorylation and inactivation of tuberin (TSC2), a tumor suppressor protein. Interestingly, pharmacologic inhibition of mTOR with rapamycin prevented vGPCR sarcomagenesis, while over-activation of this pathway by haploinsufficiency for TSC2 predisposes mice to the development of KS-like vascular sarcomas. These findings identified the Akt-mTOR pathway as a therapeutic target for KS and provided the rationale for the clinical evaluation of rapamycin and its analogs for the treatment of this AIDS-malignancy.[unreadable] [unreadable] A NF-kB gene expression signature contributes to KSHV vGPCR-induced direct and paracrine neoplasia: Using a full-genome microarray analysis we found that vGPCR promotes a dramatic change in gene expression in endothelial cells either by acting directly in vGPCR-expressing cells, or indirectly through the release of soluble factors. By using gene set enrichment analysis of the microarray data, we found that NF-kB signaling is potently triggered by vGPCR expression and in cells exposed to vGPCR-induced secretions, thus mimicking its paracrine effect. Indeed, we observed that vGPCR activates the NF-kB pathway effectively, and NF-kB activation is a prominent feature in both human and experimental KS. Of interest, constitutive NF-kB signaling is not sufficient to promote endothelial cell transformation. However, using genetic approaches to block NF-kB we found that this transcription factor is strictly required for vGPCR-induced direct and paracrine transformation. Taken together, these results strongly support the role of NF-kB and its regulated genes in KS pathogenesis.