Invasion of tumor cells through basement membranes and interstitial tissues allows tumors to metastasize and colonize new sites in the body. Recent studies indicate that tyrosine phosphorylation of membrane-bound proteins is critically important in invasion tumor cells. These studies show that tyrosine kinases and their membrane-bound substrates are highly elevated in association with invadopodia, the cellular protrusions at the leading edge of invasive cells. Formation of invadopodia and subsequent cell invasion were blocked by genistein, an inhibitor of tyrosine kinases. An in vitro model has been developed to study cell membrane-associated invasion. Cells are cultured on fluorescently labeled substratum so as to detect sites of matrix degradation colocalizing with invadopodia. A novel subcellular fractionation scheme was used to isolate invadopodia from an invasive, hormone-independent breast cancer cell line, MDA-MB231. Two major tyrosine phosphorylated proteins were detected, p19O and p1 30, that may represent the major tyrosine phosphorylated proteins that are visualized at the tips of invadopodia using immunofluorescence microscopy. Cortactin is another substrate for tyrosine phosphorylation in transformed, highly motile cells and it localizes to invadopodia associated with sites of degradation. ErbB- 2 and estrogen receptor (ER) are important target molecules for both prognosis and therapeutic intervention strategies since elevated levels of ER occur in more than 60% of human breast cancers and overexpression of erbB-2 is detected in approximately 30% of breast cancers. In invasive breast carcinoma, erbB-2 overexpression correlates with poor prognosis, and breast tumors positive for both erbB-2 and ER appear to be resistant to antiestrogen therapy. Gp30, identified as a ligand for erbB-2, may activate breast tumor cell progression to the estrogen independent phenotype. Preliminary data show that cortactin expression is upregulated in gp30 transfected breast cancer cells and the gp30 ligand stimulates matrix degradation and the formation of invadopodia characterized by high levels of tyrosine phosphorylated proteins and cortactin localization in an erbB-2 overexpressing cell line. We propose to 1) Determine the upregulation of membrane-associated degradation by the erbB-2 ligand, gp30, 2) Determine the regulation of cortactin by gp30 and the role of gp30 in stimulating membrane -associated invasion, and 3) Identify and characterize a major membrane-associated, tyrosine phosphorylated p19O in isolated invadopodia fractions from MDA-MB-231, hormone-independent breast cancer cells, and generate monoclonal antibodies against p 190.
