Dysregulation of DNA methylation is an established feature of breast cancers. DNA demethylating therapies like decitabine are proposed for the treatment of triple-negative breast cancers (TNBCs) and indicators of response need to be identified. For this purpose, we characterized the effects of decitabine in a panel of 10 breast cancer cell lines and observed a range of sensitivity to decitabine that was not subtype-specific. Knockdown of potential key effectors demonstrated the requirement of deoxycytidine kinase (DCK) for decitabine response in breast cancer cells. In treatment-naive breast tumors, DCK was higher in TNBCs, and DCK levels were sustained or increased post chemotherapy treatment. This suggests that limited DCK levels will not be a barrier to response in TNBC patients treated with decitabine as a second line treatment or in a clinical trial. Methylome analysis revealed that genome-wide, region-specific, tumor suppressor gene-specific methylation, and decitabine-induced demethylation did not predict response to decitabine. Gene set enrichment analysis (GSEA) of transcriptome data demonstrated that decitabine induced genes within apoptosis, cell cycle, stress, and immune pathways Induced genes included those characterized by the viral mimicry response; however, knockdown of key effectors of the pathway did not affect decitabine sensitivity suggesting that breast cancer growth suppression by decitabine is independent of viral mimicry. Finally, taxolresistant breast cancer cells expressing high levels of multidrug resistance transporter ABCB1 remained sensitive to decitabine, suggesting that the drug could be used as second-line treatment for chemoresistant patients.

<p>Genes up-regulated by decitabine treatment in MDA-MB-468, MDA-MB-231, or SUM159 with hypermethylated promoters (TSS200)</p>

<p>Gene Set Enrichment Analysis for genes upregulated by decitabine treatment in MDA-MB-468, MDA-MB-231, or SUM159</p>

<p>HM450 DNA methylation B-values for genes up- or down-regulated by decitabine in MDA-MB-468, MDA-MB-231, or SUM159</p>

<p>Additional figures, tables, and methods</p>