Molecular mechanism of cell invasion

描述(申请人提供)：人类肿瘤细胞通过质膜突起侵入细胞外基质，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及其蛋白复合体对齿足类起作用。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内陷相关蛋白在侵袭和转移表型发育中的调节作用。恶性的人类黑色素瘤和卵巢癌细胞具有明确的侵袭特征；它们为在肿瘤微环境中生存和随后的自发转移做好了准备。第一个特定目标是分析这些与内陷相关的蛋白和整合素在肿瘤微环境中对细胞存活的作用，以及在肿瘤细胞侵袭和转移中的作用。将通过RNA干扰(RNAi)敲除侵袭性肿瘤细胞中的mRNAs来破坏与内陷相关的黏附和蛋白分解活性，从而确定内陷蛋白的特殊重要性。第二个目的是从酶的活性结构域的表达以及多肽和抗体抑制剂的鉴定来评估Seprase的激活机制。第三个目标是确定卵巢癌从原发肿瘤向腹水发展过程中的重要分子。将从卵巢癌患者的原发肿瘤和腹水中分离出肿瘤细胞，利用DNA微阵列和实时荧光定量RT-PCR检测其基因表达谱，并对其体外侵袭能力和体内肿瘤生长转移潜能进行验证。侵袭性肿瘤细胞中上调的特定基因将使用RNAi敲除方法进行验证。第四个目的是通过对免疫亲和纯化的内毒素复合体进行蛋白质组学分析，寻找seprase和MT1-MMPs的天然底物。最后，利用一种新的动物模型系统，将识别与抗肿瘤和抗转移控制有关的由多相诱导的免疫成分。因此，这项建议将分析侵袭和转移表型的分子基础，最终目标是确定内向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.