Epigenetic mechanism of acquired doxorubcin resistance in breast cancer cells

Breast cancer is the second leading cause of cancer related death in Women globally. Chemotherapy resistance is a major limitation in clinical treatment of breast cancer. Acquired resistance against doxorubicin, which is the mainstay treatment for solid cancers including breast cancer, has frequently been reported. Clinical doxorubicin treatment schedule and background genetic status i.e. Estrogen Receptor (ER) status in different subtype of breast cancers in resistance development is not clear. Aberrant expression of genes leading to doxorubicin resistance has been well documented but the molecular mechanism leading to doxorubicin resistance-associated altered expression of genes is not clear. Accumulating evidence implicates the epigenetic changes as an important regulatory mechanism for aberrant expression of genes. Therefore, objective of this study was to develop clinically relevant acquired doxorubicin resistant in vitro breast cancer cell model and comprehensively evaluate the epigenetic aberrations, and identify the epigenetic mechanism underlying acquired doxorubicin resistance in breast cancer cells. Doxorubicin resistant cells were selected by exposing estrogen receptor (ER) positive MCF-7 and triple negative MDA-MB-231 breast cancer cell lines to clinically relevant doses of doxorubicin and clinically relevant treatment strategies for chronic period (18 months). Acquired resistance development was monitored and characterized by various parameters such as cytotoxicity, cell cycle/apoptosis, anchorage independent growth on soft agar, and cell migration potential, gene and protein expression, and evaluation for global and gene-specific promoter methylation aberrations. Results revealed intermittent but not continuous treatment resulted in acquired resistance. ER positive breast cancer subtypes developed more and rapid resistant than triple negative breast cancer cells. Acquired resistance was associated with acquisition of Epithelial to Mesenchymal transition (EMT), cancer stem cell (CSC)-like phenotype, increased tumorigenic and migration potential. Changes in expression of genes/proteins for cell survival, apoptosis, drug transport, DNA repair, EMT, stemness and metastasis further confirmed the acquisition of resistance to doxorubicin. Aberrant expression of epigenetic regulatory genes involved in DNA methylation (DNMT1, DNMT3a/3b, MBD4 and TET1), and histone modifications (HDAC1, HAT1 and HMT1) were also observed in doxorubicin resistant cells. Importantly, the increases in temporal changes in DNMTs and HMT1 expression during resistance acquisition were associated with increased levels of resistance. Significant elevation of global H3 acetylation including H3K9ac, H3K18ac, H3K27ac, and H3K56ac, and methylation including H3K4me3 and H3K27me3 were associated with the acquired resistant phenotype. Importantly, loss of MMR gene MSH2 expression associated with promoter hyper methylation, suggest the epigenetic inactivation of MMR-dependent apoptotic pathway as a potential mechanistic basis for acquired resistance in these cells. Pre-treatment with demethylating agent 5-Aza-deoxycytidine and HDAC inhibitor Trichostatin A significantly re-sensitized resistant cells to doxorubicin and further confirmed potential role of epigenetic changes in doxorubicin resistance development in breast cancer cells. In summary, result of this study for the first time suggests that acquisition of doxorubicin resistance in breast cancer cells involve epigenetic aberrations that are temporal and progressive. Therefore, these global and target-specific epigenetic alterations can potentially be used as biomarker for early detection as well as the targets for epigenetic therapy to re-sensitize the doxorubicin resistant breast cancer cells.