Melanoma Cell Surface Proteoglycans in Metastasis

黑色素瘤硫酸软骨素蛋白多糖是一种高水平表达的细胞表面蛋白多糖 在绝大多数人类黑色素瘤上，并与促进肿瘤黏附、迁移和 入侵。为了进一步研究MCSP在肿瘤进展中的作用，我们克隆并稳定表达了 两个MCSP阴性黑色素瘤细胞系中的MCSP核心蛋白。MCSP的表达促进了生长和 黑色素瘤细胞在异种移植模型中的成瘤及抗细胞外单抗的研究 MCSP结构域可抑制体内肿瘤的形成。MCSP的表达刺激整合素介导的信号 MCSP诱导的细胞扩散和粘着斑激酶增加所证明的信号转导 磷酸化。此外，MCSP的表达刺激酪氨酸磷酸化增强，并 ERK的磷酸化，这是独立于锚定的生长所必需的，而细胞质尾巴- 删除的MCSP无法支持非锚定生长或增强ERK激活。委员会成员 ERK/MAPK途径，包括BRAF(在这些细胞中突变为V600E活性形式)、pMEK和 PERK均以GST-MCSP胞浆尾部融合蛋白沉淀，而截短融合蛋白 缺少MCSP尾部的c-末端，这些分子就不能结合。这些结果表明，MCSP 作为ERK/MAPK通路成员的对接场所。我们假设MCSP作为一部小说发挥了作用 帮助组装和高效激活关键信号通路的跨膜支架蛋白 促进黑色素瘤的生长、存活和侵袭。目标1将研究MCSP在肿瘤生长和维持中的作用 使用siRNA抑制人黑色素瘤细胞中MCSP的表达。Aim#2将重点介绍 ERK/MAPK通路连接到MCSP的细胞质结构域。目标#3将定义 MCSP的胞外结构域需要激活对肿瘤生长至关重要的关键信号通路。 黑色素瘤是一种毁灭性的疾病，对目前的治疗几乎完全无效。自.以来 绝大多数黑色素瘤在原发肿瘤和转移灶中都表达MCSP，这表明 黑色素瘤细胞可能使用这种分子来获得相对于MCSP阴性细胞的竞争优势 恶性进展的多个阶段。这些研究的长期目标是确定 开发MCSP表达/功能的抑制剂作为治疗恶性黑色素瘤的新疗法。

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 maintanence 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 signalling pathways important for tumor growth. Melanoma is a devastating disesase 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.