Abstract 772: Notch1 affects ABCC1-mediated drug efflux and drug sensitivity in MCF7 breast cancer cells

Abstract

Abstract Development of multidrug resistance is a major impediment to the successful treatment of cancer. Although many cellular mechanisms are involved in multidrug resistant cancers, overexpression of ATP-binding cassette (ABC) membrane transporters represents one of the main determinants of the multidrug resistance phenotype. We have previously demonstrated a positive correlation of expression of intracellular Notch1 (NIC) and the ABCC1 transporter; we found that NIC contributes to multidrug resistance by upregulation of ABCC1 (S Cho, M Lu et al, PNAS, in press, 2011). In order to determine whether there is a functional relationship between the two proteins, we performed drug efflux experiments. We treated drug-sensitive MCF-7 and multidrug resistant (MDR) MCF-7/VP breast cancer cells with the anticancer drug doxorubicin and measured the amount of drug efflux by flow cytometry over time. The MDR MCF-7/VP cells, which have high levels of activated Notch1 and ABCC1 proteins, exhibited increased doxorubicin efflux, compared to the drug-sensitive MCF-7 cells. Since γ-secretase (GS) is required for the processing of Notch into its active form, NIC, we blocked GS activity using a GS inhibitor, DAPT, in MCF-7/VP cells and assayed drug efflux from them. We found that inhibition of Notch signaling by DAPT blocks ABCC1 mediated drug efflux. To better understand the connection between Notch, ABCC1, and drug efflux, we compared drug efflux in MCF-7 cells stably overexpressing NIC (ICN1-c4, which also overexpress ABCC1), and the “empty vector” control MCF-7 cells (pcDNA3). As expected, we saw increased drug efflux in the ICN1-c4 cells compared to MCF7/pcDNA3 cells. To establish whether the observed drug efflux is ABCC1-specific, we treated ICN1-c4 cells with either the ABCC1-specific inhibitors reversan or probenecid, or with an ABCC1 siRNA to knockdown ABCC1 expression, and then examined these cells for changes in drug efflux. We found that inhibition of ABCC1, by reducing ABCC1 activity or levels in the presence of activated Notch1, blocks drug efflux from ICN1-c4 cells. In addition, we also found that the ABCC1 inhibitor reversan sensitizes ICN1-c4 cells to doxorubicin, and knockdown of ABCC1 reverses the resistance of ICN1-c4 cells to doxorubicin. These results strongly support the idea that Notch1 exerts its effect on ABCC1-mediated drug efflux to a large extent through its induction of ABCC1. Overall, our results reveal a novel functional relationship between expression of Notch and ABCC1. (Supported in part by CA40570 [to WTB] and in part by UIC.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 772. doi:1538-7445.AM2012-772