ABSTRACT: Doxorubicin is considered to be the most effective chemotherapeutic drug used to treat breast cancer. Unfortunately, resistance to this drug is common, resulting in poor patient prognosis and survival. Dynamically reorganized chromatin allows rapid access of the gene regulatory machinery to open genomic regions facilitating subsequent gene expression through direct transcription factor (TF) activation and regulatory element binding. To better understand the regulatory network underlying doxorubicin resistance in breast cancer cells, we explored the systematic alterations of chromatin accessibility and gene expression by the assay for transposase-accessible chromatin using sequencing (ATAC-seq) in combination with RNA sequencing, followed by integrative analysis to identify potential regulators and their targets associated with differentially accessible regions (DARs) in doxorubicin-resistant MCF7 (MCF7-DR) cells. A total of 3963 differentially expressed genes (DEGs) related to doxorubicin resistance were identified, including dramatically up-regulated MT1E, GSTP1, LDHB, significantly down-regulated TFF1, UBB, DSCAM-AS1, and histone-modifying enzyme coding genes HDAC2, EZH2, PRMT5, etc. By integrating with transcriptomic datasets, we identified 18,228 DARs in MCF7-DR cells compared to control, which were positively (r = 0.6) correlated with their nearest DEGs. There were 11,686 increased chromatin-accessible regions, which were enriched in up-regulated genes related to diverse KEGG pathways, such as the cell cycle, regulation of actin cytoskeleton, signaling pathways of MAPK, PI3K/Akt and Hippo, which play essential roles in regulating cell apoptosis, proliferation, metabolism, and inflammatory responses. The 6,542 decreased chromatin-accessible regions were considered to be related to declined doxorubicin-associated biological processes, for instance, endocrine and insulin resistance, central carbon metabolism, signaling pathways of TGF-beta and P53. Analyses of the DAR sequences showed that, besides the AP-1 family, TEAD and FOX family TF DNA-binding motifs were highly enriched in hyper- and hypo-accessible regions respectively. Moreover, critical TFs and their potential targets associated with DARs were identified, such as TEAD1, RUNX1, GRHL2 and FOXA1. In addition, we put forward that the loss of FOXA1/GRHL2 collaboration might be responsible for acquired resistance to doxorubicin in breast cancer. These data provided clear insights and resources for an improved understanding of the non-genetic landscape of doxorubicin-resistant breast cancer cells based on chromatin accessibility and transcript levels, which allowed for detection of critical TFs, potential cis-regulatory elements, and therapeutic targets.
