Melanoma Cell Surface Proteoglycans in Metastasis

描述（由申请人提供）：黑色素瘤硫酸软骨素蛋白多糖（MCSP）是一种在绝大多数人类黑色素瘤中高水平表达的细胞表面蛋白多糖，与促进肿瘤粘附、迁移和侵袭有关。为了进一步表征MCSP在肿瘤进展中的作用，我们在两个MCSP阴性的黑色素瘤细胞系中克隆并稳定表达了MCSP核心蛋白。在异种移植瘤模型中，MCSP的表达促进了黑色素瘤细胞的生长和肿瘤形成，针对MCSP细胞外结构域的单克隆抗体在体内抑制了肿瘤的形成。MCSP的表达刺激整合素介导的信号转导，MCSP诱导细胞扩散和局灶黏附激酶磷酸化的增加证明了这一点。此外，MCSP的表达促进酪氨酸磷酸化和ERK磷酸化的增强，这是锚定非依赖性生长所必需的，而细胞质尾部缺失的MCSP不能支持锚定非依赖性生长或增强ERK激活。ERK/MAPK通路的成员，包括BRAF（在这些细胞中突变为V600E活性形式），pMEK和pERK都与GST-MCSP细胞质尾部融合蛋白沉淀，而缺乏MCSP尾部c末端的截断融合蛋白无法结合这些分子。这些结果表明MCSP作为ERK/MAPK通路成员的对接位点。我们假设MCSP作为一种新的跨膜支架蛋白，帮助组装和有效激活关键信号通路，促进黑色素瘤的生长、存活和侵袭。Aim #1将利用siRNA抑制MCSP在人黑色素瘤细胞中的表达，研究MCSP在肿瘤生长和维持中的作用。目标2将重点关注ERK/MAPK通路的成员如何与MCSP的细胞质结构域连接。Aim #3将定义MCSP细胞外结构域的结构特征，这些结构域是激活对肿瘤生长至关重要的关键信号通路所必需的。黑色素瘤是一种毁灭性的疾病，对目前的治疗几乎完全没有反应。由于绝大多数黑色素瘤在原发肿瘤和转移性病变中都表达MCSP，这表明黑色素瘤细胞可能在恶性进展的多个阶段利用MCSP分子获得相对于MCSP阴性细胞的竞争优势。这些研究的长期目标是确定利用MCSP表达/功能抑制剂作为治疗恶性黑色素瘤的新疗法的潜力。

DESCRIPTION (provided by applicant): Melanoma chondroitin sulfate proteoglycan (MCSP) is a cell surface proteoglycan expressed at high levels on the vast majority of human melanomas and is implicated in promoting tumor adhesion, migration and invasion. To further characterize MCSP in tumor progression we have cloned and stably expressed the MCSP core protein in two MCSP-negative melanoma cell lines. MCSP expression enhanced growth and tumor formation of melanoma cells in xenograft models, and monoclonal antibodies against the extracellular domain of MCSP inhibited tumor formation in vivo. Expression of MCSP stimulates integrin-mediated signal transduction as evidenced by MCSP-induced increases in cell spreading and focal adhesion kinase phosphorylation. Furthermore, expression of MCSP stimulates enhanced tyrosine phosphorylation and the phosphorylation of ERK, which is required for anchorage-independent growth, while a cytoplasmic tail deleted MCSP failed to support anchorage-independent growth or enhance ERK activation. Members of the ERK/MAPK pathway, including BRAF (which is mutated to the V600E active form in these cells), pMEK and pERK all precipitated with a GST-MCSP cytoplasmic tail fusion protein, while a truncated fusion protein lacking the c-terminal end of the MCSP tail failed to bind these molecules. These results indicate that MCSP acts as a docking site for ERK/MAPK pathway members. We hypothesize that MCSP functions as a novel transmembrane scaffold protein that helps assemble and efficiently activate key signaling pathways to promote melanoma growth, survival and invasion. Aim #1 will study MCSP in tumor growth and maintenance using siRNA to inhibit MCSP expression in human melanoma cells. Aim #2 will focus on how members of the ERK/MAPK pathway link to the cytoplasmic domain of MCSP. Aim #3 will define structural features of the extracellular domain of MCSP required for activation of key signaling pathways important for tumor growth. Melanoma is a devastating disease that is almost completely nonresponsive to current therapies. Since the vast majority of melanomas express MCSP in both primary tumors and in metastatic lesions, this suggests that melanoma cells may use this molecule to attain a competitive advantage over MCSP-negative cells at multiple stages of malignant progression. The long term goal of these studies is to determine the potential to exploit inhibitors of MCSP expression/function as novel therapies in the treatment of malignant melanoma.