Identification of chromatin accessibility domains in human breast cancer stem cells

Abstract

Epithelial-to-mesenchymal transition (EMT) is physiological in embryogenesis and wound healing but also associated with the formation of cancer stem cells (CSCs). Many EMT signaling pathways are implicated in CSC formation, but the precise underlying mechanisms of CSC formation remain elusive. We have previously demonstrated that PKC is critical for EMT induction and CSC formation in inducible breast EMT/CSC models. Here, we used formaldehyde-assisted isolation of regulatory elements-sequencing (FAIRE-seq) to investigate DNA accessibility changes after PKC activation and determine how they influence EMT and CSC formation. During EMT, DNA accessibility principally increased in regions distant from transcription start sites, low in CpG content, and enriched with chromatin enhancer marks. ChIP-sequencing revealed that a subset of these regions changed from poised to active enhancers upon stimulation, with some even more acteylated in CSCs. While regions with increased accessibility were enriched for FOX, AP-1, TEAD, and TFAP2 motifs, those containing FOX and AP-1 motif were associated with increased expression of CSC-associated genes, while those with TFAP2 were associated with genes with increased expression in non-CSCs. Silencing of 2 members of the FOX family, FOXN2 and FOXQ1, repressed CSCs and the mesenchymal phenotype and inhibited the CSC gene signature. These novel, PKC-induced DNA accessibility regions help explain how the epigenomic plasticity of cells undergoing EMT leads to CSC gene activation.

Novel mechanistic targets of forkhead box Q1 transcription factor in human breast cancer cells

“…We have shown previously that the promotion of stem‐like phenotype in FoxQ1 overexpressing SUM159 cell line is associated with suppression of DACH1 and ZEB1 expression and upregulation of mRNA levels of MYC and TWIST2 10 and RNA‐seq results from the present study were consistent with these published observations (Figure S1C). At the same, some published changes in genes downstream of FoxQ1 7,9,14 were not validated in the RNA‐seq data from the present study, including PLD1, SNAI1, CTNNB1, JUP, S100A4, DCN, PDGFRB, CADM3, COL1A1, COL6A1, DSG2 , and TWIST1 (Figure S2A‐C). Two possibilities exist to explain the inconsistencies between published data and RNA‐seq results.…”

“…Published studies have revealed multiple downstream targets of FoxQ1 in breast cancer 7‐10,14,15 . For example, Zhang et al 7 showed downregulation of CDH1 in highly metastatic breast cancer cell lines (eg, MDA‐MB‐435, MDA‐MB‐231, SUM149, etc) in comparison with breast cancer cell lines with low metastatic capacity (eg, MCF‐7, MDA‐MB‐361, BT20, etc) as well as upregulation of mesenchymal markers such as N‐cadherin (CDH2) and fibronectin 1 (FN1) and downregulation of CDH1 in FoxQ1 overexpressing HMLE cells when compared with EV cells.…”

“…Two possibilities exist to explain the inconsistencies between published data and RNA‐seq results. The RNA‐seq results were obtained using SUM159 cells and this cell line was not used in other published studies 7,9,14 except for our own published study 10 . Secondly, the possibility of regulatory functions of other transcription factors for these genes cannot be excluded.…”

Novel mechanistic targets of Forkhead box Q1 transcription factor in human breast cancer cells

“…7-9,11). A-D, RNA-seq analysis of genes not consistent with the published literatures (7,9,11). Results shown are mean ± S.D.…”

“…Published studies have revealed multiple downstream targets of FoxQ1 in breast cancer (7)(8)(9)(10)(11)(12). For example, Zhang et al (7) showed downregulation of CDH1 (Ecadherin ) but upregulation of mesenchymal markers including CTNNA1 (catenin alpha 1), CDH2 (N-cadherin), and FN1 (fibronectin 1) in highly metastatic breast cancer cell lines (e.g., MDA-MB-435, MDA-MB-231, SUM149, etc.)…”