The ability of cancer cells to detach from their tumor of origin and ?invade? into surrounding tissue marks a turning point in cancer progression. Invadopodia and microvesicles are cytoskeleton-rich membrane structures via which cells can proteolyze the extracellular environment to facilitate invasion, and their regulation is a focus of this dissertation. Robust motility is also integral for tumor cell invasion, and we describe how signaling of the small GTPase ARF6 coordinates these activities.
This dissertation describes how alterations in extracellular matrix compliance guide the formation of microvesicles and invadopodia to facilitate matrix proteolysis. The data presented here indicates that formation of these structures is mutually exclusive, regulated by antagonistic signaling between the small GTPases Rac1 and RhoA. In addition, we have described how Rho signaling regulates myosin light chain phosphatase during myosin contractility, required for release of microvesicles from the tumor cell surface.
Critical to ARF6-mediated regulation of tumor cell invasion are the activities of the GTPase activating proteins (GAPs) and exchange factors (GEFs) that control its nucleotide status. Activation of ARF6 downstream of Wnt5a stimulates invasive ability via the Wnt/β-catenin canonical pathway, and is opposed by signaling through SLIT2-ROBO1, which stimulates ARF6 GAPs. During epithelial cell migration, the actin-binding protein CD2AP is capable of downregulating ARF6 activity by recruiting and stabilizing the GAPs ArfGAP3 and ASAP at the cell surface.
To further delineate the contribution of ARF6 to cell invasion, we developed new reagents that target its downregulation, including a lentiviral vector for ARF6 silencing. This vector has demonstrated very effective knockdown of ARF6 levels, however, additional work will be necessary to address off-target effects of the virus before it can be utilized for studies of tumor cell invasion. We also tested the hypothesis that Src-mediated phosphorylation of the ARF6 GAP ACAP2 will result in increased ARF6-GTP levels during tumor cell invasion, and have created plasmids for the expression of ACAP2 phosphorylation mutants for these studies. Finally, we describe the use of a tumor cell spheroid assay for assessing invasive behavior, which sheds light on the negative impact that constitutive ARF6 activation has on the development of metastatic lesions.