Molecular Regulation of Human Melanoma Metastasis

描述(申请人提供)：本申请的长期目标是确定白介素8在恶性黑色素瘤发病机制中的分子机制(S)。IL-8是CXC趋化因子家族的一员，在恶性黑色素瘤中有结构性表达，具有自分泌/旁分泌生长、侵袭和血管生成的作用。IL-8的作用是通过表达在黑色素瘤细胞和内皮细胞上的两种高亲和力受体CXCR1和CXCR2来实现的。IL-8与CXCR1和CXCR2结合可启动多种细胞反应，但IL-8激活CXCR1和/或CXCR2在恶性黑色素瘤中的确切作用尚不清楚。因此，本应用的具体目的是确定CXCR1和CXCR2在介导IL-8诱导的调节黑色素瘤生长、侵袭、血管生成和转移的细胞反应中的不同功能作用。我们假设IL-8与CXCR1和CXCR2的相互作用激活了不同的下游信号通路，并在黑色素瘤的生长、侵袭、血管生成和转移中发挥了不同的作用。我们将检验这一假说，并实现这一应用的目标，方法是：1)确定IL-8激活CXCR1和/或CXCR2在调节与黑色素瘤生长、侵袭和转移相关的细胞表型方面是否起着截然不同或重叠的作用；2)检测内皮细胞中CXCR1和CXCR2在IL-8诱导的黑色素瘤血管生成反应中的不同角色；以及3)确定IL-8诱导的CXCR1和CXCR2依赖的黑色素瘤细胞生长、存活和侵袭反应的信号转导机制(S)。我们将在体外和体内检测依赖CXCR1和CXCR2的IL-8介导的作用。我们将使用小分子拮抗剂和腺病毒siRNA载体在异种移植模型和mCXCR2基因敲除裸鼠体内使用CXCR1和/或CXCR2的特异性靶向。利用通路特异性抑制物、磷酸化特异性抗体、免疫印迹和免疫组织化学方法，我们将在体外和体内研究CXCR1和/或CXCR2激活后的下游信号通路。这些研究将确定CXCR1和CXCR2激活及其下游信号通路在介导IL-8诱导的恶性黑色素瘤生长、血管生成和转移中的不同和/或重叠作用。我们预计，从这些研究中获得的知识将确定新的治疗靶点，以抑制配体结合和/或信号转导事件，并开发恶性黑色素瘤的治疗方法。

DESCRIPTION (provided by applicant): 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.