Alteration of DNA methyltransferases by eribulin elicits broad DNA methylation changes with potential therapeutic implications for triple-negative breast cancer

Abstract

Background: Triple-negative breast cancer (TNBC) is an aggressive disease with limited treatment options. Eribulin, a chemotherapeutic drug, induces epigenetic changes in cancer cells, suggesting a unique mechanism of action. Materials & methods: MDA-MB 231 cells were treated with eribulin and paclitaxel, and the samples from 53 patients treated with neoadjuvant eribulin were compared with those from 14 patients who received the standard-of-care treatment using immunohistochemistry. Results: Eribulin treatment caused significant DNA methylation changes in drug-tolerant persister TNBC cells, and it also elicited changes in the expression levels of epigenetic modifiers (DNMT1, TET1, DNMT3A/B) in vitro and in primary TNBC tumors. Conclusion: These findings provide new insights into eribulin's mechanism of action and potential biomarkers for predicting TNBC treatment response.

Keywords: DNA methylation; breast cancer; chemotherapy; epigenetic; epithelial-to-mesenchymal transition.

Figure 1.. Eribulin treatment alters DNA methylation patterns in triple-negative breast cancer cells.

(A & B) Schematic and brightfield representation of multi-round drug treatment procedure that resulted in the generation of resistant clones. (C) Principal component analysis of the most variable genes between MDA MB-231 parental, ERI1, ERI2, PAC1 and PAC2 from DNA methylation array. (D) Heat map of unsupervised hierarchical clustering of methylation β-values of the top 5% most variable CpGs (34,525 CpGs). Horizontal tracking bars indicate the treatment status. (E) Methylation dysregulation index of eribulin-treated and paclitaxel-treated cells at each treatment point. ERI: Eribulin; MDI: Methylation dysregulation index; PAC: Paclitaxel.

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

(A) Volcano plot of dmCpGs associated with first eribulin treatment compared with the methylation status of parental cells determined by an epigenome-wide association study. (B) Volcano plots of dmCpGs associated with second eribulin treatment compared with the methylation status of parental cells determined by an epigenome-wide association study. Those colored in red are considered to be differentially methylated at a q-value < 0.05. (C) Comparison of number of dmCpGs based on treatment type and number of treatments. Those colored in red are the number of hypomethylated CpGs compared with the methylation status of parental cells. Colored in blue are the number of hypermethylated CpGs compared with 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 with the proportion of CpGs that are differentially methylated with the first treatment of eribulin. dmCpG: Differentially methylated CpG.

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

Enrichment of differentially methylated CpG sites (dmCpGs) in various genomic contexts: (A) according to gene structure and (B) in association with 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.. Differential methylation patterns are detected in mesenchymal-to-epithelial transition-associated genes.

(A & B) RT-PCR and immunoblot analysis of epithelial-to-mesenchymal transition markers in cells treated with eribulin and paclitaxel. (C) Heat map of the direction of methylation level change (β coefficient from epigenome-wide association study) in CpGs located in mesenchymal-to-epithelial transition (MET)-associated genes. Tiles with * indicate a significant difference in methylation levels compared with untreated cells. The vertical tracking bar indicates which MET-associated gene the CpG is located in. (D) Proportion of dmCpGs in different genomic regions of MET-associated genes by direction of methylation change and number of eribulin treatments. (E) Heat map of the level of significance for transcription factor motifs associated with dmCpGs in ZEB1 binding sites. *p < 0.05; **p < 0.001; ***p < 0.0005; ****p < 0.0001; t-test.

Figure 5.. Genes with differentially methylated CpGs are associated with key biological processes in the mesenchymal-to-epithelial transition.

(A) The VEGF signaling pathway was associated with hypermethylated CpGs from the second eribulin treatment. (B) RT-PCR analysis was conducted on genes within pathways that were differentially methylated. (C–E) ZEB1 localization at genomic loci genes was evaluated by CUT&RUN signal track analysis across all conditions. (F) Heat map depicting the levels of cytokines and chemokines determined through a multiplex cytokine assay. *p < 0.05; **p < 0.001; ***p < 0.0005; ****p < 0.0001; t-test.

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

(A) Quantitative RT-PCR was performed to assess the expression levels of DNA methylation markers in the MDA MB-231 parental line and its resistant counterparts. (B) Immunoblotting was performed to assess the protein levels of DNA methylation markers in the MDA MB-231 parental line and its resistant counterparts. (C & D) Representative images and quantification of DNMT1 and DNMT3A were obtained through immunohistochemical analysis of specimens treated with AC-T and ERI. Statistical analysis was conducted using the t-test. AC-T: Adriamycin/cyclophosphamide followed by paclitaxel; ERI: Eribulin.