Podosomes are actin-rich adhesive structures found in transformed cells that are implicated in cancer cell invasion and metastasis. Cortactin is an Arp2/3 activating and actin-binding protein initially identified as a major substrate in v-Src transformed cells, and is a core podosome component. Cortactin has recently been implicated in podosome formation and function in smooth muscle cells. However, a complete understanding of cortactin’s role in podosome formation during Src-induced transformation has not been reported. To determine the role of cortactin in podosome formation in response to Src transformation, we introduced a GFP-tagged temperature sensitive mutant of v-Src (tsLa29-GFP) into Src-deficient (SYF) fibroblasts. The optimum timing for podosome formation when cells expressing tsLa-29-GFP were switched from the non-permissive temperature (41°C) to the permissive temperature (35°C) was 2 h as determined by confocal fluoresence microscopy and rhodamine phalloidin labeling. Inhibition of cortactin expression by siRNA substantially diminished the ability of tsLA29-GFP to induce podosome formation at the permissive temperature. Pretreatment of SYF cells expressing tsLA29-GFP for 1 h with the Src/Abl small molecule inhibitor AZD0530 also ablated podosome formation at the permissive temperature. Collectively, these findings indicate that cortactin is required for Src-mediated podosome formation and that downregulation of Src activity correlates with inhibited podosome formation. We are currently investigating the role of Src-mediated cortactin phosphorylation in podosome assembly, extracellular matrix degradation and invasion.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]