articleEGFR / Met association regulates EGFR TKI resistance in breast cancer

Breast cancers show a lack of response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), despite 30% of tumors expressing EGFR. The mechanism of this resistance is unknown; however, we have recently shown that Met kinase activity compensates for loss of EGFR kinase activity in cell culture models. Met has been implicated in the pathogenesis of breast tumors and therefore may cooperate with EGFR for tumor growth. Here we have found that EGFR phosphorylation and cell proliferation is in part regulated by Met expression. In addition, we found that Met constitutive phosphorylation occurred independent of the Met ligand hepatocyte growth factor (HGF). Ligand-independent Met phosphorylation is mediated by Met amplification, mutation, or overexpression and by Met interaction with other cell surface molecules. In SUM229 breast cancer cells, we found that Met was not amplified or mutated, however it was overexpressed. Met overexpression did not directly correlate with ligand-independent Met phosphorylation as the SUM229 cell line was the only Met expressing breast cancer line with constitutive Met phosphorylation. Interestingly, Met expression did correlate with EGFR expression and we identified an EGFR/Met complex via co-immunoprecipitation. However, we only observed Met constitutive phosphorylation when c-Src also was part of this complex. Ligand-independent phosphorylation of Met was decreased by down regulating EGFR expression or by inhibiting c-Src kinase activity. Lastly, inhibiting EGFR and Met kinase activities resulted in a synergistic decrease in cell proliferation, supporting the idea that EGFR and Met functionally, as well as physically interact in breast cancer cells to regulate response to EGFR inhibitors. Introduction Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor shown to be mechanistically involved in cell growth and survival (reviewed in [1]). Ligand activation of EGFR results in homoand hetero-dimerization with other members of the EGFR family of receptor (reviewed in [1]). This dimerization enables EGFR to autophosphorylate, resulting in the recruitment of signaling proteins to the receptor (reviewed in [1]). Approximately 30% of human breast tumors overexpress EGFR, and this overexpression correlates with a loss of estrogen responsiveness and a poor prognosis [2-5]. Despite strong correlative evidence from human breast tumors, transgenic mouse models have clearly demonstrated that overexpression of the EGFR alone is insufficient for tumor formation [6]. EGFR tyrosine kinase inhibitors (TKIs) are in clinical use in lung and pancreatic cancers, but have yet to demonstrate efficacy in breast cancer. We and others have recently identified the receptor tyrosine kinase Met as a key regulator of EGFR tyrosine kinase inhibitor resistance in cancer [7,8]. Met also is overexpressed in breast cancer cells and human breast tumors and its expression correlates with EGFR expression in basal type breast cancers [9-11]. Met or hepatocyte growth factor receptor is characterized as a receptor tyrosine kinase [12]. However, unlike EGFR, there are two broad mechanisms of Met activation: ligand-dependent and ligand-independent. In the mammary gland, ligand-dependent activation of Met involves the paracrine production of HGF by stromal cells, including fibroblasts [13]. Ligand-independent activation of Met has been shown to occur through a number of mechanisms, including mutation of Met, constitutive dimerization of Met associated with overexpression, pathway activation via hypoxic conditions, transactivation by other membrane proteins (including EGFR), and loss of negative regulators [14]. * Correspondence: boernerj@karmanos.org 1 Karmanos Cancer Institute at Wayne State University, John R. St, Detroit, MI