Recent efforts have been undertaken to identify trans
-regulatory factors that trigger epigenetic silencing in cancer cells. Deregulated signaling related to TGF-β and estrogen has been implicated in this initiation process during the differentiation of breast progenitor cells (35
). In this study, we suggest that activated PI3K/AKT signaling is also a trigger of epigenetic silencing. This oncogenic signaling was previously found to be activated in mammary epithelial cells chronically exposed to inflammatory microenvironments and could induce epigenetic silencing of a tumor suppressor gene, CST6, in vitro
). We further demonstrate that activation of this oncogenic signaling pathway causes global alterations of H3K27me3, thereby leading to epigenetic silencing of multiple loci. This new line of evidence reveals an interesting mechanism that oncogenic signaling can aberrantly transform differentiated epithelial cells through its epigenetic influence in the human breast.
In analogous to embryonic stem cells, differentiation-control genes in breast progenitor cells also possess a “bivalent” feature, consisting of an active mark, H3K4me2, and a repressive mark, H3K27me3 (5
). Co-existence of these histone marks is to maintain progenitor cells in a “ready state” for lineage-specific development (12
). In terminally differentiated cells, the bivalent loci retain either activating or repressive marks that are essential to specify specific lineage functions. This epigenetic programming, however, can be redirected by oncogenic signaling (38
). Interestingly, as demonstrated in this study, we uncovered that activated PI3K/AKT signaling can drive a subset of downstream loci to undergo epigenetic silencing in breast epithelial cells. Indeed, 28% (n=138) of the 488 genes identified through the integrative screening of ChIP-seq and expression microarray data encode putative cellular functions required for normal differentiation (Supplementary Fig S3 and Table S3
). In our experimental model, these genes become transcriptionally silenced and display the repressive H3K27me3 mark in breast epithelial cells.
One of interesting findings in our study is that many of loci loss H3K27me3 but gain DNA methylation in cancer cells. These data seem to support the occurrence of an epigenetic switch between H3K27me3 mark and DNA methylation during tumor progression (15
). This mutually exclusive silencing pattern also indicates that both repressive marks actually have an independent role in the maintenance of gene silencing in breast cancer cells. In this regard, Cha et al.
) reported that AKT kinases can phosphorylate EZH2 and impair its trimethylation activity, resulting in a depletion of H3K27me3 in some cancer cell lines, such as MDA-MB-453 and T-47D. Interestingly, this finding is consistent with our present results that show loss of H3K27me3 and compensatory gain of DNA hypermethylation at same loci in those two cancer cell lines. Meanwhile, our preliminary data indicate that PI3K inhibitor LY294002 can dramatically decrease levels of DNA methyltransferase 3 in multiple breast cancer lines (data not shown). Another study has also suggested that activated PI3K/AKT signaling greatly stabilizes DNA methyltransferase 1 (DNMT1) by attenuating the ubiquitin-proteosome reaction in cancer cells (41
). It is also worthy of note that one recent study demonstrated that estrogen receptor signaling can regulate methylation of H3K27 through the PI3K/AKT pathway (42
While more than 20 PI3K-targeted inhibitors have recently been introduced in clinical trials, more work still needs to be done to improve treatment effectiveness in patients (32
). One new strategy is to combine these inhibitors with epigenetic drugs, which have also been used as single agents for myelodysplastic syndrome and other malignancies (45
). As we demonstrated in this study, these combined approaches show promising results in breast cancer treatment. Furthermore, these combined agents can be administered in low-dose ranges (i.e., nanomolar concentrations), which are capable of evoking long-term reactivation of tumor suppressor genes and promote cellular reprogramming. Emerging evidence has shown that this low-dose treatment strategy is sufficient to suppress hematopoietic malignancies and at the same time avoids cytotoxicity (47
). Although this low-dose treatment was not used in this study, our future work will focus on identifying optimal doses in nanomolar concentrations that will not result in acute cytotoxic effects but rather induce sustained anti-tumor responses in vitro
and in xenograft models.
Whereas PI3K/AKT signaling is well known to contribute multi-oncogenic functions, our present study has uncovered its unique role in epigenetic silencing. These data also support the notion that polycomb- and DNA hypermethylation-based mechanisms can compensate each other for the maintenance of gene silencing under different genetic backgrounds. As oncogenic signaling cascades can perturb epigenetic balance in cancer genomes, our experimental evidence therefore provides support for combinatorial PI3K/AKT-targeted and epigenetic therapies in future cancer treatment.