An enteropathogenic microbial toxin modulates the breast cancer epigenome resulting in concurrent silencing of tumor suppressor genes.

Abstract

Background: Recent studies have determined a close association between host microbiota and breast cancer initiation, growth and therapeutic outcomes. We previously uncovered how enterotoxigenic Bacteroides fragilis (ETBF), a gut microbe present in malignant breast tissue, aids breast cancer development and metastatic progression via activating multiple oncogenic pathways and modulating breast tumor microenvironment. Brief exposure to ETBF-secreted toxin, BFT (Bacteroides fragilis toxin), imparts long-term oncogenic, pro-stemness and pro-metastasis memory in breast cancer cells indicating the involvement of epigenetic alterations hence, we aimed to investigate the potential involvement of epigenetics in biological impact of ETBF in breast cancer.

Methods: RNA sequencing and Infinium methylation EPIC microarray were performed in mammary tumors developed from the BFT-exposed breast cancer cells. Alterations in the expression of tumor suppressor genes (TSGs) were assessed using RT-PCR, ICC and IHC. Demethylation agent and HDAC inhibitor were utilized to rescue the expression of BFT-mediated hypermethylated TSGs, and examine the migration/invasion potential of BFT-exposed breast cancer cells.

Results: EPIC methylation microarray analysis of BFT-exposed mammary tumors uncovered 64 differentially methylated significant CpG sites whose analysis led to the identification of 26 hypomethylated and 156 hypermethylated genes. Of the hypermethylated genes, also showing reduced expression in RNA-seq in BFT-exposed group, we identified five important TSGs, namely, NF2, RSK3, FAT4, DCN and DOK2. Survival analysis revealed that decreased expression of these TSGs associated with worse prognosis. While BFT exposure reduced the expression of these TSGs, treatment with Azacytidine or/and Trichostatin A resulted in rescue of the TSGs from BFT-induced repression while mitigating BFT-driven migration and invasion of breast cancer cells.

Conclusions: Collectively, BFT exposure epigenetically modifies the expression of TSGs and impacts migration/invasion potential of breast cancer cells, and treatment with demethylation agent(s) and HDAC inhibitors effectively diminishes the functional impact of BFT.