The objective of this proposal is to study the mechanisms of localized matrix degradation by invadopodia and to determine their significance for breast cancer invasion and metastasis. Invadopodia are long membrane protrusions of invasive cells, they contact and degrade the cellular matrix.
Our aims are to: 1) demonstrate the role of c-Src and its substrate cortactin in localized matrix degradation mediated by invadopodia, 2) use stable transfectants of dominant negative c-Src and cortactin to test the role of these molecules in invasion and metastasis in the nude mouse model, and 3) determine the relevance of invadopodia to malignant breast cancer in humans.
In Aim 1, mutants of c-Src tyrosine kinase and its substrate cortactin will be introduced in breast cancer cells. Transfection of constitutively activated c-Src kinase promotes the formation of invadopodia, and conversely, transfection of the inactive c-Src kinase inhibits invadopodia. A requirement for phosphorylation of cortactin on tyrosine will be tested by transfecting cells with point and deletion mutants of cortactin and determining dominant negative effects upon upon invadopodia formation. c-Src variants with altered SH2 domains will be transfected into invasive cells to test the requirement for c-Src SH2/cortactin association during invadopodia formation. Phosphopeptide inhibitors of c-Src SH2 domain will be tested for their ability to block c-Src/cortactin association.
In Aim 2, when c-Src or cortactin variants that block invadopodia are identified; the cell lines expressing these variants will be grown as xenographed tumors in the nude mouse to measure tumor growth and to visualize invadopodia. Tumor cells will be injected intracardially to measure the incidence of metastases to the lung and bone to test the hypothesis that when invadopodia are inhibited, metastasis is suppressed. Finally, in Aim 3, invadopodia will be identified in snap frozen and archival human breast tumors using combinations of anti-invadopodia antibodies that have been characterized in isolated cells (Aim 1) and in xenographed tumors (Aim 2). In conclusion, the results of these studies will provide the basis for understanding the signal transduction pathways that promote tumor cell invasion and their relevance for aggressive breast cancer growth in humans. Model systems for testing inhibitors of invadopodia will be developed, and they will be extremely valuable for future studies on invasion and metastasis.
