Alteration of DNMT1/DNMT3A by eribulin elicits global DNA methylation changes with potential therapeutic implications for triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is an aggressive disease subtype with limited treatment options. Eribulin is a chemotherapeutic approved for the treatment of advanced breast cancer that has been shown to elicit epigenetic changes. We investigated the effect of eribulin treatment on genome-scale DNA methylation patterns in TNBC cells. Following repeated treatment, The results showed that eribulin-induced changes in DNA methylation patterns evident in persister cells. Eribulin also affected the binding of transcription factors to genomic ZEB1 binding sites and regulated several cellular pathways, including ERBB and VEGF signaling and cell adhesion. Eribulin also altered the expression of epigenetic modifiers including DNMT1, TET1, and DNMT3A/B in persister cells. Data from primary human TNBC tumors supported these findings: DNMT1 and DNMT3A levels were altered by eribulin treatment in human primary TNBC tumors. Our results suggest that eribulin modulates DNA methylation patterns in TNBC cells by altering the expression of epigenetic modifiers. These findings have clinical implications for using eribulin as a therapeutic agent.

Keywords: DNA methylation; DNMT1/DNMT3A; Epithelial-to-mesenchymal transition; mesenchymal-to-epithelial transition.

Figure 1.. Eribulin treatment alters DNA methylation patterns in TNBC cells.

(A) Schematic representation of multi-round drug treatment procedure that resulted in the generation of resistant clones (B) Principal Component Analysis (PCA) of most variable genes between MDA MB-231 parental, ERI1, ERI2, PAC1 and PAC2 from DNA methylation array. (C) (D)

Figure 2.. Additional eribulin treatment increases alterations in DNA methylation patterns.

(A) Volcano plot of dmCpGs associated with first eribulin treatment compared to the methylation status of parental cells determined by an epigenome-wide association study. (B) Volcano plots of dmCpGs associated with second eribulin treatment compared to the methylation status of parental cells determined by an epigenome-wide association study. Colored in red are considered to be differentially methylated at q-value < 0.05. (C) Comparison of number of dmCpGs based on treatment type and number of treatments. Colored in red are number of hypomethylated CpGs compared to the methylation status of parental cells. Colored in blue are number of hypermethylated CpGs compared to methylation status of parental cells. (D) Venn diagram of dmCpGs associated with the first treatment of eribulin and associated with the second treatment of eribulin. (E) Number of genes that have either decreased, unchanged, or increased proportion of CpGs that are differentially methylated with the second treatment of eribulin as compared to the proportion of CpGs that are differentially methylated with the first treatment of eribulin.

Figure 3.. Eribulin treatment associated with differentially methylated CpGs are enriched.

Enrichment of dmCpGs at different genomic contexts (A) in relation to the gene structure and (B) in relation to the CpG islands. Odds ratios were calculated by Fisher’s exact test. Blue points and confidence intervals indicate enrichment from dmCpGs associated with the first eribulin treatment. Yellow points and confidence intervals indicate enrichment from dmCpGs associated with the second eribulin treatment. Circular points indicate CpGs with increased methylation levels in treated cells. Triangular points indicate CpGs with decreased methylation levels in treated cells.

Figure 4.. Genes with differentially methylated CpGs are associated with key biological processes in the mesenchymal-to-epithelial transition.

(A) ERBB signaling pathway was significant associated with dmCpGs from the second eribulin treatment. (B) VEGF signaling pathway were associated with hypermethylated CpGs from the second eribulin treatment. (C) Heatmap depicting the levels of cytokines and chemokines determined through a multiplex cytokine assay. ZEB1 binding levels at (D) NKFB1, (E) NKFB2, and (F) VEGFA in untreated and eribulin treated cells.

Figure 5.. Differential methylation patterns are detected in Mesenchymal to Epithelial Transition-associated genes.

(A) Heatmap of the direction of methylation level change (beta coefficient from epigenome-wide association study) in CpGs located in MET-associated genes. Tiles with * indicate a significant difference in methylation levels compared to untreated cells. The vertical tracking bar indicates which MET-associated gene the CpG is located in. (B) Proportion of dmCpGs in different genomic regions of MET-associated genes by direction of methylation change and number of eribulin treatment. (C) Heatmap of the level of significance for transcription factor motifs associated with dmCpGs in ZEB1 binding sites.

Figure 6.. Eribulin treatment in primary human triple-negative breast cancers alters the expression of DNA methylation regulatory enzymes.

(A) Immunoblotting was performed to assess the protein levels of DNA methylation markers in the MDA MB-231 parental line and its resistant counterparts. (B, C) Representative images and quantification of DNMT1 and DNMT3A were obtained through immunohistochemistry (IHC) analysis of specimens treated with AC-T and ERI. Statistical analysis was conducted using the Wilcoxon-Mann-Whitney test.