Molecular mechanism of cell invasion

描述（由申请人提供）：人肿瘤细胞利用质膜突起侵入细胞外基质，侵入伪足接触并溶解基质。一种瞬时表达的II型跨膜丝氨酸蛋白酶seprase及其蛋白复合物在侵袭伪足上发挥作用。本研究的目的是在蛋白质相互作用和基因表达水平上研究这些侵袭足相关蛋白在侵袭和转移表型发展中的调控。恶性人类黑色素瘤和卵巢癌细胞具有可定义的侵袭伪足特征;它们为在肿瘤微环境中存活和随后的自发转移做好准备。第一个具体目标集中于分析这些侵袭足相关蛋白酶和整合素在肿瘤微环境中细胞存活以及肿瘤细胞侵袭和转移中的作用。侵袭性伪足蛋白的具体重要性将使用侵袭性肿瘤细胞中mRNA的RNA干扰（RNAi）敲低来破坏侵袭性伪足相关的粘附和蛋白水解活性来定义。第二个目的是评估seprase激活机制的背景下，表达的活性结构域的酶和识别肽和抗体抑制剂。第三个目标是鉴定在卵巢癌从原发肿瘤到腹水的进展中重要的分子。将从卵巢癌患者的原发肿瘤和腹水中分离肿瘤细胞;使用DNA微阵列和实时RT-PCR确定其基因表达谱;将证明其体外细胞侵袭性和体内肿瘤生长和转移潜力。将使用RNAi敲低方法验证在侵袭性肿瘤细胞中上调的特定基因。第四个目的是通过对免疫亲和纯化的侵袭伪足复合物进行蛋白质组学分析来发现seprase和MT 1-MMP的天然底物。最后，使用新的动物模型系统，将鉴定与抗肿瘤和抗转移控制有关的seprase诱导的免疫组分。因此，该建议将分析侵袭和转移表型的分子基础，最终目标是确定侵袭伪足蛋白作为转移性疾病的治疗和诊断靶点。

DESCRIPTION (provided by applicant): Human tumor cells invade the extracellular matrix using plasma membrane protrusions, invadopodia, that contact and dissolve the matrix. A transiently expressed, type II transmembrane serine protease, seprase, and its protein complexes function on invadopodia. The goal of this proposal is to investigate the regulation of these invadopodia-associated proteins in development of the invasive and metastatic phenotypes at the levels of protein interaction and gene expression. Malignant human melanoma and ovarian carcinoma cells have definable invadopodia profiles; they are primed for survival in the tumor microenvironment and for subsequent spontaneous metastasis. First Specific Aim focuses on the analysis of the role of these invadopodia-associated proteases and integrins in cell survival in the tumor microenvironment, and in tumor cell invasion and metastasis. The specific importance of invadopodia proteins will be defined using RNA interference (RNAi) knockdown of mRNAs in invasive tumor cells to destroy the invadopodia-associated adhesive and proteolytic activities. Second Aim assesses the mechanism of seprase activation in the context of expression of active domains of the enzyme and identification of peptide and antibody inhibitors. Third Aim will identify molecules important in progression of ovarian cancer from primary tumor to ascites. Tumor cells will be isolated from primary tumor and ascites of patients with ovarian cancer; their gene expression profiles will be determined using DNA microarray and real-time RT-PCR; their cell invasiveness in vitro and tumor growth and metastasis potential in vivo will be demonstrated. Specific genes upregulated in invasive tumor cells will be validated using the RNAi knockdown approach. Fourth Aim is to discover natural substrates for seprase and MT1-MMP by proteomic analysis on immuno-affinity-purified invadopodia complexes. Finally, using a novel animal model system, seprase-induced immune components implicated in the anti-tumor and anti-metastasis control will be identified. Thus, this proposal will analyze molecular basis of the invasive and metastatic phenotypes, with the eventual goal of determining invadopodia proteins as therapeutic and diagnostic targets for metastatic disease.