Cdc42-interacting protein 4 is a Src substrate that regulates invadopodia and invasiveness of breast tumors by promoting MT1-MMP endocytosis

Invadopodia are actin-rich membrane protrusions that promote extracellular matrix degradation and invasiveness of tumor cells. Src protein-tyrosine kinase is a potent inducer of invadopodia and tumor metastases. Cdc42-interacting protein 4 (CIP4) adaptor protein interacts with actin regulatory proteins and regulates endocytosis. Here, we show that CIP4 is a Src substrate that localizes to invadopodia in MDA-MB-231 breast tumor cells expressing activated Src (MDA-SrcYF). To probe the function of CIP4 in invadopodia, we established stable CIP4 knockdown in MDA-SrcYF cell lines by RNA interference. Compared with control cells, CIP4 knockdown cells degrade more extracellular matrix (ECM), have increased numbers of mature invadopodia and are more invasive through matrigel. Similar results are observed with knockdown of CIP4 in EGF-treated MDA-MB-231 cells. This inhibitory role of CIP4 is explained by our finding that CIP4 limits surface expression of transmembrane type I matrix metalloprotease (MT1-MMP), by promoting MT1-MMP internalization. Ectopic expression of CIP4 reduces ECM digestion by MDA-SrcYF cells, and this activity is enhanced by mutation of the major Src phosphorylation site in CIP4 (Y471). Overall, our results identify CIP4 as a suppressor of Src-induced invadopodia and invasion in breast tumor cells by promoting endocytosis of MT1-MMP.