Complications from metastasis are the major cause of death in breast cancer patients. Metastatic carcinoma cells must initially invade through the basement membrane, a network of extracellular matrix proteins that support the overlaying epithelium (1
). Once they have escaped the primary tumor, cancer cells must also degrade the vascular sub-endothelial basement membrane to gain entry into the bloodstream. Directed invasion of cancer cells through the ECM and their intravasation into the bloodstream is mediated by chemoattractants such as EGF, which are produced by other cell types including tumor-associated macrophages (2
). It is believed that invasive cancer cells penetrate these barriers by forming specialized F-actin rich protrusions called invadopodia that localize matrix degrading activity to cell-substrate contact points (4
Invadopodia proceed through several different stages during their maturation into functional structures. First, small punctate clusters, or invadopodium precursors, uniquely containing actin and the actin polymerization regulators cortactin, N-WASp, and cofilin are formed (9
). These precursors can have two fates: they can disappear or become stabilized and mature into functional invadopodia. Stabilization and maturation requires cortactin tyrosine phosphorylation, which leads to generation of free actin barbed ends in invadopodia and increased invadopodial lifetime (9
). The final maturation stage involves acquisition of matrix degrading ability through delivery of matrix metalloproteinases such as MT1-MMP into invadopodia (9
). Understanding the mechanisms that govern invadopodium formation and function is an essential step in the prevention of invasion and metastasis.
The cortactin gene (CTTN
), located in chromosome 11q13, is amplified in various human carcinomas and is usually correlated with poor patient prognosis (15
). Cortactin localizes to invadopodia in invasive breast cancer cells, where it regulates both their formation and function (10
). Cortactin tyrosine phosphorylation is essential for generation of free actin barbed ends required for actin polymerization in invadopodia (9
) and for efficient ECM degradation (17
) and metastasis in vivo
). Cortactin phosphorylation promotes release of cofilin from cortactin, leading to free actin barbed end generation (9
), and also enables binding of proteins such as Nck1, leading to enhancement of N-WASp-mediated Arp2/3-dependent actin polymerization (9
). Combination of both pathways is important for actin polymerization in invadopodia, which is believed to mediate invadopodium-dependent protrusion into the ECM. Cortactin phosphorylation is therefore a critical regulator of invadopodial function and tumor metastasis, but how this phosphorylation is achieved specifically in invadopodia has not been established.
We show here that Arg and Src, but not Abl, localize to invadopodia of mammary carcinoma cells, where they specifically mediate cortactin tyrosine phosphorylation. Arg is not required for initial invadopodium precursor formation, but is critical for regulating invadopodial activation in response to EGF by promoting the generation of free actin barbed ends. Both Src and Arg localize to invadopodia and are essential for their activation and function. Interestingly, Arg overexpression in Src knockdown cells can partially rescue invadopodial activation and function, while Src overexpression cannot compensate for loss of Arg function. These experiments demonstrate an EGFR-Src-Arg-cortactin pathway mediates functional maturation of invadopodia and breast cancer cell invasion, and identify Arg as a novel mediator of invadopodia function in breast cancer cells.