To delineate the upstream signaling mechanisms responsible for the maintenance of aberrant promoter DNA methylation patterns during breast cancer progression, we utilized a previously described breast cancer cell line model system. We found that the mesenchymal-like MIII cells, compared to its precursor H-Ras transformed epithelial MII cells (21
), harbor hyperactive TGFβ signaling and exhibit an EMT phenotype. Moreover, highly invasive properties of the MIII cells suggesting a pro-metastatic role was substantiated by differential expression of several genes in MIII compared to the MII cells sharing a similar expression pattern with a subset of genes previously identified as mediators of breast cancer metastasis to the lung (34
) (Figure S9
). Overall, these results indicate that the MCF10A-based breast cancer cell line model system is an attractive and highly relevant model to study the molecular mechanisms responsible for epigenetic regulation of EMT during transition from in situ
to invasive breast carcinoma.
By employing gene expression profiling and by examining the epigenetic regulation of differentially expressed genes in this breast cancer model system, we found that there was DNA hypermethylation-mediated silencing of genes involved in cell adhesion and tight junction formation, including CDH1, CGN
as well as the epithelial protease KLK10/NES1
in Basal-B-like breast cancer cells that have undergone EMT. These observations are also consistent with a recent report showing that suppression of CDH1
expression during sustained EMT is mediated by the establishment of promoter DNA hypermethylation (5
Furthermore, our studies demonstrate that overactive TGFβ signaling events, mediated by an autocrine feedback loop which maintains high TGFβ1 levels in the microenvironment, are responsible for sustaining the altered epigenome and the invasive properties of breast cancer cells. Moreover, our studies provide direct evidence for the involvement of intact hyperactive TGFβ-TGFβR-Smad2 signaling axis in orchestrating a specific DNA methylation pattern that favors EMT and the invasive behavior of breast cancer cells. Several observations support this conclusion. First, disruption of TGFβ signaling by either Smad7 overexpression or SMAD2
, but not SMAD4
, knock-down in the MIII cells reversed the EMT phenotype and caused re-establishment of the epithelial morphology. Second, the observed mesenchymal to epithelial transition was accompanied by the upregulation of transcripts for the CDH1
gene, encoding a key cell-cell adhesion molecule and negative regulator of WNT signaling cascade (35
), the tight junction genes CLDN4
as well as the protease KLK10/NES1
. CDH1 levels have been directly correlated with epithelial phenotype and metastatic properties of cancer cells (36
), while the KLK10/NES1 protease was shown to be specifically expressed in epithelial cells and suppress breast tumor growth in vivo
). Finally, significant decreases in promoter DNA methylation of the critical target genes upon TGFβ-TGFβR-Smad2 signaling disruption strongly support a direct involvement of this axis in modulating the functionality of the DNA methylation machinery to maintain the epigenetically silenced state.
Despite the identification of putative DNA demethylase enzymes and evidence for the involvement of a DNA repair pathway in this process (38
), the existence of active DNA demethylation mechanisms in mammals has been elusive (39
). Our data favors the alternate mechanism which proposes that suppression of the maintenance DNA methyltransferase, DNMT1, results in passive DNA demethylation (40
). We found that binding of DNMT1 to CDH1, CLDN4, CGN
promoters was significantly suppressed upon SMAD2
knock-down (), while DNMT1 and DNMT3B protein levels remain unaffected (Figure S6
). Therefore, we propose that reduced DNMT1 binding activity upon disruption of TGFβ-Smad signaling could result in loss of DNA methylation maintenance and passive demethylation of newly synthesized DNA (). The passive demethylation in the absence of intact Smad2, but not Smad4, suggests that Smad2 may play a role in loading DNMT1 onto specific gene promoters to modulate DNA methylation when TGFβ signaling becomes overactive. Alternatively, Smad2 may interact with other factors to transcriptionally regulate target genes or control DNMT1 activity via
post-translational modifications. Finally, it is also likely that DNMT1 binding is regulated by remodeling of localized chromatin in response to TGFβ signaling-mediated effects during breast cancer progression.
In summary, our data suggests that increased TGF β levels in the breast tumor microenvironment promote hyperactive Smad signaling to enable the acquisition of EMT-like properties. Furthermore, we propose that overactive TGFβ cascades play a major role in the “epigenetic memory” and maintenance of epithelial gene-specific silencing during EMT mediated by unique DNA methylation patterns (). To our knowledge, this is the first report to provide conclusive evidence that the reversal of the DNA hypermethylation status of gene promoters occurs as a result of a signaling pathway perturbation, in this case the TGFβ/Smad cascade. By extension, our study provides a framework for uncovering genes that are coordinately regulated by epigenetic mechanisms in response to specific signaling events commonly deregulated during cancer progression. Finally, our findings provide additional credence to the idea that inhibition of TGFβ-TGFβR-Smad2 signaling axis may be a useful therapeutic strategy to target breast cancer progression.