PD08-08: Preclinical Efficacy of the Combination of Met and Src Family Kinase Inhibitors in Triple-Negative Breast Cancer.

Abstract

Abstract Background: Epidermal growth factor receptor (EGFR), although overexpressed in almost 60% of all triple-negative breast cancers, has yet to be identified as an effective therapeutic target for the treatment of the disease. We have previously shown in cell culture models of triple-negative breast cancer that primary resistance to EGFR tyrosine kinase inhibitors through ligand-independent phosphorylation can be overcome by co-inhibiting EGFR with either Met or Src family kinases (SFKs). We also demonstrated that inhibiting Met kinase activity reduced EGFR and c-Src phosphorylation and inhibiting SFK reduced EGFR phosphorylation. Taken together, these data suggest that EGFR, Met and SFKs regulate the activity of each other through transphosphorylation. This process of transphosphorylation, or crosstalk, allows for the continued activation of key signaling proteins required for proliferation and transformation. Here we demonstrate that inhibiting both Met and SFKs decreases cell viability and eliminates the need for inhibition of EGFR tyrosine kinase activity, as the autophosphorylation sites of EGFR were lost with the combination treatment. Methods: Seven triple-negative (SUM102, SUM149, SUM229, SUM1315, BT20, BT549, MDA-MB-231), two HER2+ (SUM190 and SKBr3), and two ER/PR+ (MCF7 and T47D) breast cancer cell lines were used to test the efficacy of the combination of Met and SFK inhibitors, Arq-197 and dasatinib, respectively. Cell viability after drug treatment was assessed by MTT assays. IC50 values were calculated and drug synergy calculations were performed. Transphosphorylation was measured by immunoblotting using phospho-specific antibodies. Apoptosis was characterized by immunoblotting. Results: All seven triple-negative breast cancer cell lines demonstrated decreased cell viability with the combination of Arq-197 and dasatinib when compared with either drug alone. In contrast, none of the HER2+ or ER/PR+ breast cancer cell lines had any additional loss of viability with the combination treatment. We found that the interactions between Arq-197 and dasatinib were synergistic in several of the breast cancer cell lines. Specifically, in one such cell line (BT20), the combinatorial index values were less than 1.0. Interestingly, in this cell line, the tyrosine phosphorylation levels on residues 1068 and 992 of EGFR were significantly altered with the combination treatment and activation of apoptotic proteins, including PARP, occurred. Conclusions: These data demonstrate that two tyrosine kinases, known to be downstream of EGFR activation, interact to promote resistance to EGFR inhibitors. However, inhibiting both Met and SFKs was sufficient to decrease cell viability and stimulate apoptosis in triple-negative breast cancer cells. Further studies to validate the relevance of these findings are ongoing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-08.