Regulation of actin polymerization during tumor cell invasion

Abstract

Tumor metastasis, the spread of cancer from a primary site to a distant organ, is the leading cause of mortality among cancer patients. For a carcinoma cell to metastasize, it must first successfully invade the basement membrane underlying the epithelium and intravasate through the basement membrane of the endothelium into the bloodstream. It is thought that invasive tumor cells use subcellular structures, known as invadopodia, actin-rich membrane protrusions that focally degrade the extracellular matrix (ECM) using matrix metalloproteinase (MMP) activity, to degrade through basement membranes during invasion and intravasation. However, the mechanisms regulating invadopodium assembly and maturation are not well understood. Based on previous studies showing that cortactin function is critical for the matrix degradation activity of invadopodia, I hypothesized that cortactin's protein domains and tyrosine phosphorylation have distinct roles during invadopodium maturation. Using a combination of microscopy and biochemical techniques, I have dissected the stages of invadopodium assembly and maturation in mammary carcinoma cells and show that invadopodia use cortactin phosphorylation as a master switch during maturation. In particular, cortactin phosphorylation was found to regulate cofilin and Arp2/3 complex-dependent actin polymerization. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments to create barbed ends in invadopodia to support Arp2/3-dependent actin polymerization. Cortactin phosphorylation is also important for the recruitment of Nck1 to invadopodia through a direct phosphotyrosine:SH2-domain interaction leading to N-WASp-dependent Arp2/3 activation. Specifically, tyrosine phosphorylation sites 421 and 466, but not 482, are required for Nck1 recruitment and actin polymerization in invadopodia. Interestingly, Nck1 binds with a slight preference in vitro and has an increased FRET interaction in invadopodia with phospho-tyrosine 466 compared to 421 demonstrating SH2-domain specificity for cortactin tyrosine phosphorylation sites. Following barbed end formation, cortactin is dephosphorylated which blocks cofilin severing activity thereby stabilizing invadopodia. These findings identify novel mechanisms for actin polymerization in the invadopodia of metastatic carcinoma cells and define four distinct stages of invadopodium assembly and maturation consisting of invadopodium precursor formation, actin polymerization, stabilization, and matrix degradation. Invadopodia are thought to be required for tumor metastasis and the novel interactions and signaling mechanisms identified in these studies have potential to contribute to the development of new therapeutic drug targets to block tumor metastasis.