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 i s 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. ? ?
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