Abstract 3726: Combinations of HDAC inhibitor, chemotherapeutic agent and retinoic acid induce growth arrest, differentiation and tumor regression in preclinical models of breast cancer.
Authors: Merino Vanessa F. , Nguyen Nguyen , Sadik Helen , Cho Sean , Zhou Xian Chong , Chen Qian , Pan Duojia , Sukumar Saraswati 
Published: August 2014

Abstract
The histone deacetylase inhibitor (HDACi), entinostat, is being actively explored as a new-generation epigenetic drug which can lead to the change in the expression status of genes/pathways, but has low efficacy in cancer monotherapy. All-trans retinoic acid (ATRA) induces the differentiation of various types of stem cells. Data from cell culture and xenograft models from our lab showed that a combination of entinostat (MS-275), doxorubicin and ATRA effectively decreased tumor size in three breast cancer cell line xenograft models. Here, we sought to further investigate the mechanism of action of the triple drug combination in cancer cells; in particular, its effect on the breast cancer stem cell population. We performed a comprehensive genome wide analysis of gene expression of MDA-MB-231 breast cancer cells treated with ATRA, MS-275 and doxorubicin as monotherapies and as combination therapies. We saw that the drug-response gene profile of ATRA is very similar to DMSO (vehicle)-treated cells. Accordingly, the addition of ATRA (A) to MS-275 (MA), Dox (AD) and MS-275/Dox (MAD) displayed minimal changes in the gene expression profile of each of the other treatments. Addition of Dox to MS-275 (MD), on the other hand, potentiated the “reprograming” effect of MS-275 and affected the expression of many antitumor genes known to be related to cell cycle and growth arrest. It also altered expression of genes involved in development and inflammation. The most differentially expressed genes, validated by qPCR, were novel genes from the cancer/testis antigens and tripartite motif (TRIM) family of proteins. Interestingly, in MDA-MB-231and SUM149 cells, even the addition of low doses of doxorubicin (12.5 nM) to MS-275 increased 2 and 2.6 fold the G2 cell cycle arrest in comparison to Dox and MS-275, respectively. Despite the gene expression pattern similarity between MS-275/Dox (MD) and MS-275/Dox/ATRA (MAD) groups, we saw that MAD was more effective in inducing cell death and apoptosis in vitro and in vivo. The epithelium specific ETS transcription factor-1 (ESE-1) was differentially regulated between MAD and MD and is, in fact, part of the MS-275/ATRA (MA) signature. Using limiting dilution transplantation assays in mammary fat pads of immunodeficient mice we observed that MAD treatment in vivo most effectively targeted breast cancer stem cells (BCSC) compared to any other combination of drugs. The cancer stem cell frequency of the cells isolated from MAD treated mice was 1 in 236,570. The second most effective treatment for BCSC was MA (1 in 150,721), followed by ATRA&gt; MS-275&gt;MD&gt;Dox&gt;DMSO&gt;AD. In conclusion, the reprogramming events initiated by HDACi and retinoid sensitize the cells to low doses of doxorubicin. The combination therapy may have a significant effect in decreasing breast tumor growth and recurrence.
Citation Format: Vanessa F. Merino, Nguyen Nguyen, Helen Sadik, Sean Cho, Xian Chong Zhou, Qian Chen, Duojia Pan, Saraswati Sukumar. Combinations of HDAC inhibitor, chemotherapeutic agent and retinoic acid induce growth arrest, differentiation and tumor regression in preclinical models of breast cancer. [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 3726. doi:10.1158/1538-7445.AM2013-3726