Constitutive c-Met phosphorylation mediates growth in the absence of EGFR kinase activity in breast cancer cells dependent on EGFR expression for growth.

Abstract

Abstract Abstract #4059 Breast cancers are not responsive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) even though 30% of breast cancers overexpress the EGFR. We have observed that EGFR remains tyrosine phosphorylated in breast cancer cells that do not require EGFR kinase activity for growth, yet express high levels of EGFR. In one such cell line, SUM229, we found that this EGFR kinase-independent EGFR tyrosine phosphorylation encompasses all of the tyrosine phosphorylation sites of the EGFR for which antibodies are available, with the exception of tyrosine 1148. Using shRNA lentiviral constructs targeting EGFR, we also found that EGFR expression is required for the growth of the SUM229 cells, even though the kinase activity is not required. In addition, we found that this aberrant tyrosine phosphorylation and growth in the absence of EGFR kinase activity is mediated in part by c-Src and c-Met kinase activity. Interestingly, c-Met is constitutively phosphorylated in the SUM229 cells. This study describes the mechanism of c-Met constitutive activity and defines the Met-mediated signaling pathways to growth and survival in the absence of EGFR kinase activity. Specifically, through sequence analysis of mRNA from SUM229 cells, c-Met is not mutated, confirming the lack of identification of c-Met mutations in breast cancer. In addition, using publically available databases and our own immunoblotting experiments, c-Met is neither amplified nor overexpressed at the mRNA or protein level in the SUM229 cells. Similarly, ligand-mediated regulators of c-Met phosphorylation, including HGF, HGFAC, HAI-1, and HA1-2 are not overexpressed at the mRNA level and HGF protein expression and secretion are comparable to levels seen in breast cancer cells without c-Met constitutive phosphorylation. However, c-Met localization to the cell surface appears to be elevated in the SUM229 cells and c-Met co-immunoprecipitates with EGFR under these conditions. Taken together, these data suggest that c-Met and EGFR association at the cell surface may be mediating c-Met constitutive phosphorylation in the absence of EGFR kinase activity. With respect to the signaling pathways that mediate EGFR kinase-independent, yet c-Met kinase-dependent growth in SUM229 cells we have found that the phosphorylation of Akt, MAPK, p38MAPK, and JNK1/2 are unaffected by inhibition of both EGFR and c-Met kinase activities. Interestingly, the phosphorylation of Jak2, PLCg, and Gab1 are decreased when EGFR and c-Met kinases are inhibited. These data suggest that pathways signaling through Jak2, PLCg, and/or Gab1 may be mediating growth in the absence of EGFR kinase activity. In preliminary data, we know that these pathways do not involve PKC, Akt, or PI3-Kinase. Therefore, current investigations are focused on the STAT family of proteins as well as the Rho/Rac/Cdc42 mediated signaling pathways. Taken together, these data suggest that c-Met mediates a unique signaling pathway in SUM229 cells that stimulates both growth and survival in the absence of EGFR kinase activity. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4059.