Abstract 237: Chloroquine inhibits cancer stem cells in triple negative breast cancer via regulation of DNA methylation.

Abstract

Abstract Post-treatment enrichment of the tumor initiating CD44+/CD24- breast cancer stem cell (CSC) population is believed to be responsible for breast tumor recurrence and metastasis. Among breast cancer subtypes, triple negative breast cancer (TNBC) particularly is known to have an abundant CSC population, and is characterized by frequent metastatic recurrence. Chloroquine (CQ), an anti-malarial drug, is a lysotropic reagent that inhibits autophagy. Recently, CQ has been shown to reduce the CSC population in various cancers and has been tested in clinical studies. Concurrently, CQ was identified as a potential CSC inhibitor discovered from gene expression signatures of the CD44+/CD24- CSC population. However, little, aside from inhibition of autophagy, is known about the working mechanism of chloroquine in reducing CSCs, particularly in TNBCs. Based on recent recommendations that low doses of CQ be used to limit toxicities in the heart and retina, we investigated how low doses of CQ enhance the antitumor effects of paclitaxel (PTX) and reduce the CSC population. CSC population changes and apoptosis were analyzed using flow cytometry analysis (CSC: CD44+/CD24-; apoptosis: annexin V) and western blot analysis for cleaved caspase 3, and CSC function was measured by mammosphere formation efficiency in SUM159PT, MDA-MB-231, MDA-MB-468, and Hs578T TNBC cell lines. We observed enhanced cytotoxic effects and significant reduction of the CSC population by combined treatment of PTX and CQ compared to single treatment of either PTX (5nM) or CQ (1 or 5 uM) in SUM159, Hs578T, and MDA-MB-231 cells. The enhanced cytotoxicity by co-treatment of CQ and PTX correlated well with inhibition of autophagy, as indicated by cleavage of LC3B, increased expression of p62, and accumulation of autophagosomes. Moreover, the combined treatment inhibited PTX induced STAT3 activation in CSCs and epigenetically regulated gene expression critical in maintenance of CSCs via repression of DNMT1 expression. Finally, we observed enhanced therapeutic efficacy in vivo with the combination of CQ (10-20 mg/kg, daily) and PTX (30 mg/kg, two times per week) when compared to either CQ or PTX alone (p< 0.05). Herein, we demonstrate effective reduction of the CD44+/CD24- CSC population by combined CQ and PTX treatment through autophagy inhibition. Moreover, we found that the low-dose combined treatment of CQ with PTX was able to regulate gene expression by altering DNA methylation, subsequently reducing CSCs in the TNBC cancer cells. Thus, a low dose treatment of CQ along with chemotherapy may be effective in treating TNBC patients, lend further support for in-depth studies on the mechanism of CQ among the subgroups of TNBC. Citation Format: Dong Soon Choi, Elvin Blanco, Sergio M. Granados-Principal, Bhuvanesh Dave, Melissa Landis, Helen Wong, Jenny Chang. Chloroquine inhibits cancer stem cells in triple negative breast cancer via regulation of DNA methylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 237. doi:10.1158/1538-7445.AM2013-237