UC San Diego

Invasive cancer cells gain the ability to degrade and migrate through the extracellular matrix (ECM). The invasiveness of these cells can be correlated with the presence of dynamic membrane structures called invadopodia or podosomes. Invadopodia are actin-rich membrane protrusions that display focal proteolytic activity towards the ECM. We identified a new protein that is closely related to the critical invadopodia protein, Tks5/ FISH, which we named Tks4. We hypothesized that Tks4 is also a key component in the formation of invadopodia. The Tks4 protein contains an amino-terminal Phox homology domain, four SH3 domains, multiple proline-rich motifs, and several Src phosphorylation sites. We found that Tks4 is localized to invadopodia in a variety of cell types, including Src-transformed fibroblasts and melanoma cells. Using Src-transformed fibroblasts as a model system, we found that Tks4 is required for both the formation and function of invadopodia. Depletion of Tks4 resulted in the formation of a "pre-invadopodia" structure, where many of the required structural and accessory proteins are appropriately localized. However, these pre-invadopodia structures failed to degrade the ECM, in large part because the matrix-degrading proteinase MT1-MMP was not properly localized and/or activated. We next showed that Tks4 is a critical component in melanoma cell invasion and metastasis. Knockdown of Tks4 in human melanoma cells resulted in decreased degradation of extracellular matrices and decreased proliferation in 3-dimensional (3D) collagen-I matrix, as well as a significant reduction of metastases using an in vivo experimental lung metastasis assay. This decreased invasive capacity of Tks4-knockdown melanoma cells correlates with the decreased surface expression of MT1-MMP. We found that Tks4 regulates the cell surface localization of MT1-MMP via an RxxxxK motif in its cytoplasmic tail. Based on these results, we propose a model in which Tks4 regulates MT1-MMP localization, and therefore the function of invadopodia. A deeper understanding of the mechanisms regulating invadopodia formation and controlling the activity of MT1- MMP may provide novel targets for the development of anti- cancer therapeutics