Net1 Controls Src Activation to Regulate Breast Cancer Cell Motility and Invasion

The cytoplasmic tyrosine kinase Src supports many phenotypes in cancer cells, including proliferation, migration and invasion, survival, and metastasis. We have previously shown that Src promotes cytoplasmic localization of the RhoGEF Net1, where it stimulates RhoA activation, breast cancer cell motility, and extracellular matrix invasion. In the present work, we show that the Net1 expression in human breast tumors correlates with Src phosphorylation on its activating site Y419. We also show in human breast cancer cell lines that endogenous Net1 and Src interact, and that Net1 expression is required for full Src activation. Net1 must localize to the cytosol to promote Src activation, but surprisingly, the catalytic activity of Net1 toward Rho GTPases is not necessary for Src activation. Instead, Net1 requires interaction with the scaffolding protein Dlg1. Dlg1 knockdown prevents Src activation by Net1 and precludes interaction between Net1 and Src. Moreover, Net1 knockdown cooperates with small molecule inhibition of Src to inhibit breast cancer cell motility and extracellular matrix invasion. These data show a previously unrecognized relationship between Net1 and Src in human breast tumors and breast cancer cell lines, and suggest that therapeutic targeting of Net1 may be of benefit in breast cancers with elevated Src activity.