Abstract 3783: Molecular targeting of sphingosine kinase increases the efficacy of hormonal therapy for breast cancer by blocking the catabolic function of autophagy

Abstract

Abstract In our past studies, we have implicated autophagy as a mechanism of cell survival that contributes to the development of antiestrogen resistance in breast cancer cells (Autophagy. 2009 Apr; 5(3): 400-3). The breast cancer model system used to establish this link between autophagy and antiestrogen resistance was the estrogen-receptor positive MCF-7 cell line. We subjected this cell line to a step-wise selection in increasing concentrations of 4-hydroxytamoxifen (active metabolite of tamoxifen) in the absence of clonal selection and established the antiestrogen resistant TR5 cell line. Analysis of antiestrogen resistant TR5 cells showed induction of ceramide levels during the drug selection that correlated with autophagy induction. Thus, we have begun to characterize the role of key enzymes of the ceramide pathway in pro-survival autophagy relative to the development of antiestrogen resistance. In this study, we show that the inhibition of sphingosine kinase activity effectively kills antiestrogen-sensitive MCF-7 cells and antiestrogen-resistant TR5 cells. Specifically, blockade of spingosine kinase action in MCF-7 and TR5 cells reduced cell number, increased the percentage of cells in the population that showed uptake of trypan blue, induced caspase-9 activation and increased the cleavage of poly-ADP-ribose polymerase and lamin A. Thus, blockade of sphingosine kinase resulted in a robust induction of apoptotic cell death. SIP, the product of sphingosine kinase 1, did not appear to block the death-promoting action of sphingosine kinase blockade. Thus, the pro-survival action(s) of SIP did not compensate for loss of sphingosine kinase action in hormonally-treated breast cancer cells. Importantly, blockade of sphingosine kinase in both MCF-7 and TR5 cells blocked autophagic flux with a marked accumulation of the autophagy proteins LC3 and p62 which are normally turned-over in active autolysosomes. Blockade of autophagic flux was determined using the long-lived protein turnover assay. Although autophagic flux was impaired, autophagosomes were readily seen in the cytoplasm of cells and the lipidated LC3 protein was localized within the membrane of these autophagosomes. Further, studies utilizing electron microscopy to examine cells treated with sphingosine kinase inhibitors did not readily show impaired autophagosome-lysosome fusion. These studies provide strong evidence that sphingosine kinase activity is required for an autolysosomal function downstream of autophagosome-to-lysosome fusion. These studies also support the approach of targeting sphingosine kinase (1 and/or 2) as an effective means to block pro-survival autophagy in breast cancer cells undergoing antiestrogen therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3783. doi:10.1158/1538-7445.AM2011-3783