The long-term objective of this application is to determine the molecular mechanism(s) of interleukin (IL)-8 in malignant melanoma pathogenesis. IL-8, a member of the CXC chemokine family is constitutively expressed in malignant melanoma and functions as an autocrine/paracrine growth, invasive and angiogenic factor. The effect of IL-8 is mediated through two high affinity receptors, CXCR1 and CXCR2, expressed on melanoma cells as well as endothelial cells. Binding of IL-8 to CXCR1 and CXCR2 can initiate diverse cellular responses, however, the precise role of activation of CXCR1 and/or CXCR2 by IL-8 in malignant melanoma is not known. Therefore, the specific objective of this application is to determine the distinct functional role of CXCR1 and CXCR2 in mediating IL-8-induced cellular responses that regulate melanoma growth, invasion, angiogenesis, and metastasis. We hypothesize that interaction of IL-8 with CXCR1 and CXCR2 activates different downstream signaling pathways and plays a diverse role in melanoma growth, invasion, angiogenesis, and metastasis. We will test this hypothesis and accomplish the objective of this application by: 1) Determining whether activation of CXCR1 and/or CXCR2 by IL-8 result in distinct or overlapping roles in regulating cellular phenotypes associated with melanoma growth, invasion and metastasis; 2) Examining the diverse roles of CXCR1 and CXCR2 in endothelial cells in an IL-8-induced angiogenic response in melanoma; and 3) Identifying the signal transduction mechanism(s) involved in IL-8-induced CXCR1- and CXCR2-dependent modulation of melanoma cell growth, survival and invasive response. We will examine the CXCR1 and CXCR2-dependent IL-8-mediated effects in vitro and in vivo. We will use specific targeting of CXCR1 and/or CXCR2 in vivo using small molecule antagonists and adenoviral-siRNA vectors in xenograft models and mCXCR2 knockout nude mice. Using pathways specific inhibitors, phospho-specific antibodies, immunoblotting and immunohistochemistry, we will examine the downstream signaling pathways following CXCR1 and/or CXCR2 activation in vitro and in vivo. These studies will identify distinct and/or overlapping roles for CXCR1 and CXCR2 activation and their downstream signaling pathways in mediating IL-8-induced responses in malignant melanoma growth, angiogenesis, and metastasis. We anticipate that the knowledge gained from these studies will identify new therapeutic targets for inhibiting the ligand binding and/or signal transduction events and the development of therapeutics for malignant melanoma.
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