Reduced representation bisulfite sequencing (RRBS) of xenografts of mouse breast cancer cell line treated with C29, 5-aza-2'-deoxycytidine or a combination of both

Inhibition of estrogen related receptor alpha (ERRa) with C29 decreases global levels of DNA methylation and sensitizes breast cancer cells to the cytostatic effect of the DNMT inhibitor 5-aza-2'-deoxycytidine. We performed RRBS to understand the mechanisms behind the tumor suppression effect observed upon cotreatment with C29 and 5-azadC . We observed distinct patterns of differentially methylated regions in promoters for each condition and discovered that IRF4 was hypomethylated specifically in the condition with drug combination. Further analysis revealed that IRF4 was re-expressed in this context and was invovled in the tumor suppression response. NIC5231 mouse breast cancer cell lines were injected in mammary fat pad of NSG mice. Then, primary tumors were treated with either C29 (10 mg/kg) and/or 5-azadC (1 mg/kg), or vehicle control (5 mice per group). When the largest tumor had reached the maximum tumor size limit (1.5 mm3), DNA was extracted and sent to Diagenode for RRBS analysis (2 replicates consisting of a pool of DNA from 2 mice per condition).