Transcriptional control of epithelial to mesenchymal transition by regulatory factors and epigenetic mechanisms

The World Health Organization (WHO) states cancer to be a leading cause of death worldwide accounting for 7.6 million deaths (around 13% of all deaths) and is projected rising to over 11 million in 2030. This is an alarming call to researchers for putting more effort into the analysis of the underlying patho-mechanisms. In a very simplified manner, cancer represents the destruction of healthy tissues and organs by uncontrolled cell proliferation and subsequent formation of a tumor. One key feature of solid tumors that marks the mostly deadly feature of the disease is the acquisition of the potential to invade into the surrounding tissue and form secondary tumors at distant sites, a process called ‘metastasis’. To gain migratory and invasive properties, cancer cells undergo epithelial to mesenchymal transition (EMT) where epithelial cells lose epithelial properties, e.g. their polarized organization and cell-cell junctions, and thus undergo changes in cytoskeleton organization and cell shape and acquire mesenchymal characteristics. Importantly, besides the formation of metastatic lesions, EMT is also involved during development as well as wound healing.
To gain insights into the complex process of EMT and to identify new potential markers for ongoing metastasis, we established different in vitro EMT model systems. Global expression profiling during TGF-β-induced EMT revealed genome-wide transcriptome reprogramming during EMT and identified Krupple-like factor 4 (Klf4) and the SRY-Related HMG-Box Gene4 (Sox4) as one of the key transcription factors that were modulated and may possibly contribute to transcriptional changes during EMT.
We investigated the role of Klf4 and Sox4 during EMT by employing two different in vitro systems of EMT, using normal murine mammary gland (NMuMG) and Polyoma middle T- breast cancer (Py2T) cells, which undergo a progressive EMT upon transforming growth factor (TGF-β) treatment. We further validated the role of Sox4 in breast cancer carcinogenesis in vivo by orthotropic injection of Sox4-depleted cells into the mammary fat pad of nude mice. In addition, we also investigated whether such TGF-β-induced EMT accompanies epigenetic reprogramming and revealed how Polycomb group (PcG) complex-mediated H3K27me3 modification modulates transcription of key genes underlying this process, thereby regulating EMT.
Klf4 is a zinc-finger protein, known to be abnormally expressed in various cancers. It can act as a tumor suppressor or as an oncogene in context dependent manner in different carcinomas. Klf4 is downregulated during TGF-β-induced EMT. Our data reveal a tumor suppressor role for Klf4 in breast carcinogenesis. Klf4 is essential for the maintenance of an epithelial phenotype during EMT, and forced expression of Klf4 leads to blockage of epithelial differentiation. Furthermore, Klf4 is inhibitory to EMT-driven cell migration and also behaves as a survival factor during TGF-β-induced EMT. Genome-wide location analysis by next generation ChIP-seq analysis revealed that Klf4 directly occupies the promoter of many key EMT genes such as N-cadherin, Vimentin, β-catenin and Mapk8. Moreover, one of these Klf4 targets, Mapk8, encoding Jnk1, is upregulated during EMT and a double-knockdown of Klf4 and Jnk1 is able to overcome Klf4 knockdown-induced EMT, migration and apoptosis. These observations underscore a role of Klf4 during EMT by targeting and regulating crucial EMT genes.
Sox4 is also known to be deregulated in many cancers. Sox4 is upregulated during TGF-β-induced EMT. We show that Sox4 is required for maintaining mesenchymal identity and depletion of Sox4 prevents TGF-β-induced EMT. Sox4 reduction further impairs the migratory capacity of cells. Moreover, Sox4 provides a survival advantage to cells during breast carcinogenesis. In addition, Sox4 contributes towards TGF-β-induced tumorigenicity and metastatic spread. Gene expression profiling after Sox4 depletion in complementation with Chromatin immunoprecipitation analysis revealed many key EMT genes such as Spred1, Edn1, Palld, Cyr61, Ereg, Areg and Yap1 which are directly targeted by Sox4 for transcriptional regulation. Furthermore, Sox4 also controls many genes which are shown to regulate various other features of EMT as well as cancer development such as angiogenesis, adhesion, migration, morphogenesis, cell cycle and cytoskeleton re-modeling. Ezh2, a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), has been also found to be transcriptionally regulated by Sox4. To delineate the role of Ezh2 during EMT, a loss of function approach has been used to demonstrate that Ezh2 is required for proper acquisition of EMT and EMT-driven processes such as migration and apoptosis. Taken together, our data provides a role of Sox4 during EMT via transcriptional regulation of key genes, including the Polycomb component, Ezh2.
We also studied the role of two prominent epigenetic modifications- DNA methylation and histone 3 lysine 27 tri-methylation (H3K27me3) during TGF-β-induced EMT in a mammary epithelial cell line. Our data revealed no evidence of a reprogramming of DNA methylation during this process. To assess the role of H3K27me3 during EMT, we performed chromatin immunoprecipitation using H3K27me3-specific antibodies followed by next-generation sequencing (ChIP-seq) on 6 different stages of EMT progression. This analysis revealed that many key EMT genes are regulated by H3K27me3 mark including Mcam, Pdgfrb and Itga5 which are repressed by this mark in epithelial cells and loose it during EMT as they get activated conversely, Cdh1, Ocln and Cdx2 gain this mark during EMT and get repressed in mesenchymal cells. We further illustrated that the coordinated activities of Ezh1 and Ezh2 are required for H3K27me3-mediated repression of the gene expression and their co-depletion de-represses target genes and blocks EMT. This study provides novel insights into the important regulatory role of the Polycomb machinery during EMT.
In summary, our findings demonstrate how transcription factors, such as Klf4 and Sox4 and the epigenetic machinery, such as PcG proteins, regulate EMT by directly contributing to the transcriptional reprogramming underlying this process.