Transcriptional and post-transcriptional mechanisms regulating epithelial to mesenchymal transition in breast cancer

Abstract

The world health organization (WHO) declares cancer as one of the leading causes of mortality worldwide and reported 14 million new cases and 8.2 million cancer-associated deaths in 2012. The term cancer summarizes a broad spectrum of diseases reflecting the common feature of uncontrolled cell proliferation and systemic dissemination of tumor cells. Systemic dissemination of cancer cells requires in principle the invasion of tumor cells into the body’s circulation and their outgrowth at a distant site. In breast cancer, which is one of the top five diagnosed cancers among women, as in most cancer types metastatic outgrowth is the leading cause of death. Epithelial to mesenchymal transition (EMT) is an essential developmental process and comprises the gradual remodeling of epithelial cell architecture and functional capabilities. More precisely, cells lose epithelial cell characteristics like strong cell-cell junctions and an apical-basal cell polarity, which retain cells in a functional epithelial layer. During EMT, cells convert to a low proliferation state and acquire a spindle-like cell shape enabling single cell migration, invasion and increased cell survival. The aberrant activation of EMT promotes breast tumor cell invasion and dissemination, furthermore, its reverse process, mesenchymal to epithelial transition (MET), is believed to support metastatic outgrowth. Hence, we need to better understand the underlying molecular mechanisms controlling the dynamic nature of cell (de)differentiation and its consequences during malignant tumor progression. In the past years, intensive research has demonstrated that EMT/MET plasticity and its functional implications can be orchestrated by interconnected molecular networks consisting of transcription factors, epigenetic regulators, splicing factors and non-coding RNAs, which can be activated by a plethora of extracellular signals. However, we are just at the beginning to understand the role and regulation of such factors during EMT. Therefore, during my studies I aimed to identify critical players, in particular transcription factors and miRNAs implicated and conserved during normal and cancer-associated cell dedifferentiation and characterized their contribution to cancer progression in vitro and in vivo. We established different in vitro EMT systems to examine the stepwise morphological transition of epithelial mouse mammary cells by transforming growth factor β (TGFβ), a potent EMT inducing cytokine. Subsequent global gene expression profiling of various cell dedifferentiation states allowed us to monitor the transcriptomic alterations in a time-resolved manner. In combination with a bioinformatic analysis for DNA-binding motifs, we identified the transcription factor Tead2 as a potential EMT regulator. Tead2 is a transcriptional effector of the Hippo pathway, which tightly controls cell proliferation and organ growth. Upon EMT induction, the nuclear levels of Tead2 increase, which upon direct binding induces a predominantly nuclear localization of its cofactors Yap and Taz. Furthermsore, Tead2 is required during EMT and promotes tumor cell migration, invasion and lung colonization in vivo. Genome-wide chromatin immunoprecipitation/next generation sequencing in combination with gene expression profiling revealed the direct transcriptional targets of Tead2 during EMT in epithelial tumor cells. Among other EMT-relevant genes, we identified Zyxin an Actin remodeling and focal adhesion component important for Tead2-induced cell migration and invasion. Aside from transcriptional control non-coding RNAs can regulate EMT/MET processes. Analyzing global transcriptomic alterations of different cell dedifferentiation states by deep sequencing analysis, we identified a pool of strongly differentially regulated miRNAs. In a combination of screens, we tested their functionality during EMT and mesenchymal tumor cell migration and identified miR-1199-5p as a novel EMT-regulatory miRNA. MiR-1199-5p is transcriptionally downregulated during EMT, and forced expression of miR-1199-5p prevented TGFβ-induced EMT and decreased mesenchymal mammary tumor cell migration and invasion. Furthermore, we report a new double-negative feedback regulation between miR-1199-5p and the EMT transcription factor Zeb1, exemplifying the close interconnections of transcriptional and post-transcriptional networks facilitating epithelial plasticity. In summary, both studies provided new insights into the molecular mechanisms orchestrating EMT and its functional consequences.