Abstract PD5-2: Preclinical assessment of HDAC inhibitor entinostat combined with all trans retinoic acid (ATRA) in aromatase inhibitor resistant breast cancer
Authors: Shah P , Sabnis G , Goloubeva O , Kazi A , Schech A , Gau Y , Chumsri S , Brodie A 
Published: August 2014

Treatment with aromatase inhibitors (AI’s) is highly effective against breast cancer in ER positive postmenopausal women. However, some patients eventually become resistant to AIs. Tumor initiating cells (TIC’s) represent a subpopulation of tumor cells, which show self-renewal capacity. We are focused towards discovering strategies to reduce the growth of breast cancer TICs, which may result in resistance. We have developed a xenograft model that mimics post-menopausal hormone responsive breast cancer. In this model, aromatase transfected human hormone sensitive MCF-7 cells (MCF-7Ca) are inoculated in ovariectomized athymic nude mice and allowed to grow in presence of D4A (aromatizable substrate of estrogen). Results obtained using this model have been confirmed by numerous clinical trials. Using this model, we have established that single agent AI is better than tamoxifen in controlling tumor growth. We also observed that although, AI letrozole provides a longer control over tumor growth, tumors eventually began to grow. In the current study, we investigated the effect of ATRA (All-trans Retinoic acid) (125μg/day, ip) and a histone deacetylase (HDAC) inhibitor entinostat (SNDX- 275) (50μg/day, po) with or without letrozole on letrozole resistant tumors in a xenograft model system. Ovariectomized athymic nude mice bearing xenografts of MCF-7Ca cells, were treated with letrozole till they became resistant (15 weeks). At this time, the mice were grouped to receive ATRA, entinostat plus ATRA or the combination of ATRA plus entinostat plus letrozole till week 23. The mice treated with entinostat plus ATRA letrozole showed a significant decrease in tumor growth rate compared to mice treated with single agents or entinostat plus ATRA (p&lt;0.0001, p = 0.02). On week 20 weeks, 2 mice from each treatment group were euthanized and tumors were harvested. The tumors were digested enzymatically with collagenase and hyaluronidase and freed of debris using centrifugation and filtration. Mammosphere forming ability of TICs in the tumor tissue was measured by seeding 10,000 viable cells from each treated tumors under non-adherent conditions to access the self-renewal capacity. Quantitative PCR analysis of tumors cells showed a significant downregulation of the known TIC molecular markers, BCRP, ALDH, BMI-1 and Nanog compared to letrozole treated tumors. Similar results were also obtained when LTLT-Ca (long term letrozole treated MCF-7Ca) cells treated with ATRA and entinostat in combination with letrozole and then seeded in non-adherent conditions. The combination of ATRA plus ENT plus letrozole significantly (p&lt;0.01) reduced number of mammospheres formed compared to single agents alone. We have shown previously, that LTLT-Ca cells have higher percentage of side population (cells expressing higher level of efflux pumps such as BCRP) compared to MCF-7Ca cells. The treatment of LTLT-Ca cells with the combination of ENT and ATRA with letrozole drastically reduced the percentage of side population. Overall, these studies indicate that the combination of ATRA, entinostat and letrozole is effective in reducing tumor recurrence in letrozole resistant tumors.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD5-2.