Constitutive Activation of Src Kinase as a Mechanism of Acquired Resistance to Dasatinib in Triple Negative Breast Cancer.

Abstract Background: Dasatinib, a multi-target inhibitor of tyrosine kinases including Src, Bcr-Abl, PDGFR, and ephrin A receptor kinases, preferentially inhibits growth in triple negative breast cancer (TNBC) cell lines compared to other breast cancer subtypes. Previous studies have shown that dasatinib is a substrate for the drug efflux pumps, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). We sought to develop a model of acquired resistance to dasatinib to investigate potential mechanisms of resistance.Materials and Methods: Resistance to dasatinib in MDA-MB-231 was achieved by continuous exposure to incrementally increasing concentrations of dasatinib (200 - 500 nM). IC50 values and combination assays were measured using the acid phosphatase assay. Parental and resistant cells were treated with dasatinib (1 µM) for 2 hours and accumulation of dasatinib in cells was quantified by liquid chromatography–mass spectrometry (LC-MS). The effect of the P-gp/BCRP inhibitor, elacridar, on dasatinib sensitivity was also assessed. Src and phospho-Src (P-Src) levels were assessed by western blot.Results: The MDA-MB-231-Das cell line displayed significant resistance to dasatinib (IC50 > 5 µM) compared to the parental MDA-MB-231 cells (IC50 39.3 ± 14.2 nM). There was no significant difference in the doubling time of the parental and MDA-MB-231-Das cells (Table 1). In addition, the growth rate of the resistant cells did not significantly decrease in response to dasatinib treatment, in contrast to the parental cell line. No difference in accumulation of dasatinib was observed in the parental and resistant cells. Furthermore, inhibition of P-gp and BCRP with elacridar did not enhance response to dasatinib in either cell line. P-Src levels were not altered in the resistant cell line. However, P-Src levels did not decrease in response to dasatinib treatment in the MDA-MB-231-Das cells, whereas a dose dependent decrease in P-Src levels was observed for the parental cells.Discussion: Src kinase has been implicated in aberrant signalling through multiple receptors involved in tumorigenesis, and is a putative target for response/resistance to dasatinib. We developed a novel triple negative breast cancer model of acquired resistance to dasatinib, and examination of Src phosphorylation suggests that constitutive activation of Src is a potential mechanism of resistance.Table 1 Doubling time (± standard deviation (hrs)) and growth rate of MDA-MB-231 and MDA-MB-231-Das cells with and without dasatinib (D) treatment. ControlD 50 nMD 100 nMMDA-MB-231 (parental)17.6 ± 1.232.2 ± 3.3*46.8 ± 5.1*Growth rate (%)100.054.737.6MDA-MB-231-Das (Dasatinib-resistant)19.1 ± 2.421.0 ± 0.221.2 ± 3.1Growth rate (%)100.091.090.1  Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5066.