Reciprocal regulatory circuits coordinating EMT plasticity

Epithelial to mesenchymal transition (EMT) as well as its reversal process, mesenchymal to epithelial transition (MET), are essential and well-controlled cellular processes during embryonic development. Tightly controlled regulatory mechanisms guide an EMT/MET plasticity and enable cells to switch forth and back between different cell morphologies and functional capabilities to endow the necessity of tissue plasticity. However, aberrant and uncontrolled activation of these processes during malignant tumor progression promotes primary tumor cell invasion, cancer cell dissemination and metastatic outgrowth. In a recent study (Nat Commun; doi: 10.1038/s41467-017-01197-w), we have reported on the post-transcriptional control of normal and cancer-associated EMT by miRNAs and identified a novel, critical double-negative feedback regulation of the thus far unknown miRNA miR1199 and the key EMT transcription factor Zeb1

Live slow-frozen human tumor tissues viable for 2D, 3D, ex vivo cultures and single-cell RNAseq

Biobanking of surplus human healthy and disease-derived tissues is essential for diagnostics and translational research. An enormous amount of formalin-fixed and paraffin-embedded (FFPE), Tissue-Tek OCT embedded or snap-frozen tissues are preserved in many biobanks worldwide and have been the basis of translational studies. However, their usage is limited to assays that do not require viable cells. The access to intact and viable human material is a prerequisite for translational validation of basic research, for novel therapeutic target discovery, and functional testing. Here we show that surplus tissues from multiple solid human cancers directly slow-frozen after resection can subsequently be used for different types of methods including the establishment of 2D, 3D, and ex vivo cultures as well as single-cell RNA sequencing with similar results when compared to freshly analyzed material

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

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

Tead2 expression levels control the subcellular distribution of Yap and Taz, zyxin expression and epithelial-mesenchymal transition

The cellular changes during an epithelial-mesenchymal transition (EMT) largely rely on global changes in gene expression orchestrated by transcription factors. Tead transcription factors and their transcriptional co-activators Yap and Taz have been previously implicated in promoting an EMT; however, their direct transcriptional target genes and their functional role during EMT have remained elusive. We have uncovered a previously unanticipated role of the transcription factor Tead2 during EMT. During EMT in mammary gland epithelial cells and breast cancer cells, levels of Tead2 increase in the nucleus of cells, thereby directing a predominant nuclear localization of its co-factors Yap and Taz via the formation of Tead2-Yap-Taz complexes. Genome-wide chromatin immunoprecipitation and next generation sequencing in combination with gene expression profiling revealed the transcriptional targets of Tead2 during EMT. Among these, zyxin contributes to the migratory and invasive phenotype evoked by Tead2. The results demonstrate that Tead transcription factors are crucial regulators of the cellular distribution of Yap and Taz, and together they control the expression of genes critical for EMT and metastasis

A dual role of Irf1 in maintaining epithelial identity but also enabling EMT and metastasis formation of breast cancer cells.

An epithelial to mesenchymal transition (EMT) is an embryonic dedifferentiation program which is aberrantly activated in cancer cells to acquire cellular plasticity. This plasticity increases the ability of breast cancer cells to invade into surrounding tissue, to seed metastasis at distant sites and to resist to chemotherapy. In this study, we have observed a higher expression of interferon-related factors in basal-like and claudin-low subtypes of breast cancer in patients, known to be associated with EMT. Notably, Irf1 exerts essential functions during the EMT process, yet it is also required for the maintenance of an epithelial differentiation status of mammary gland epithelial cells: RNAi-mediated ablation of Irf1 in mammary epithelial cells results in the expression of mesenchymal factors and Smad transcriptional activity. Conversely, ablation of Irf1 during TGFβ-induced EMT prevents a mesenchymal transition and stabilizes the expression of E-cadherin. In the basal-like murine breast cancer cell line 4T1, RNAi-mediated ablation of Irf1 reduces colony formation and cell migration in vitro and shedding of circulating tumor cells and metastasis formation in vivo. This context-dependent dual role of Irf1 in the regulation of epithelial-mesenchymal plasticity provides important new insights into the functional contribution and therapeutic potential of interferon-regulated factors in breast cancer

Early morphological changes and junction disassembly can be attributed to non-canonical TGFβ signaling pathways.

<p>(<b>A</b>) Smad-mediated canonical TGFβ signaling is dispensable for early changes in morphology and junction disruption. Cells were transfected with a pool of siRNAs against Smad4 or a non-targeting pool and were then treated or not with TGFβ for 1 day as indicated. Fixed cells were stained for the adherens junction components E-cadherin and β-catenin, or for E-cadherin and the tight junction component ZO-1. Note the relocalization of β-catenin from adherens junctions to the cytoplasm upon TGFβ-treatment. Scale bars, 50 µm. (<b>B</b>) Immunoblot analysis of lysates from the experiment described in (A) to control for Smad4 knockdown efficiency. (<b>C</b>) Requirement of non-canonical TGFβ signaling pathways on early morphological changes and junction disassembly. Cells were pre-treated for 4 hours with chemical inhibitors of the kinases indicated, and were then treated with TGFβ for 1 day and analyzed as described in (A). Scale bars, 50 µm. (<b>D</b>) RhoA expression levels during the early stages of EMT. Cells were treated or not with TGFβ for 1 day and RhoA expression levels were analysed by immunoblotting. (<b>E</b>) Importance of RhoA levels for tight- and adherens junction integrity. Epithelial Py2T cells were separately transfected with two different siRNAs targeting RhoA to achieve expression levels comparable to those observed in Py2T cells treated with TGFβ (see D). Cells were stained for the adherens junction components E-cadherin and β-catenin, or for E-cadherin and the tight junction component ZO-1. (<b>F</b>) Immunoblotting analysis to determine the RhoA knockdown efficiency in the experiment described in (E).</p

Establishment of a murine breast cancer cell line undergoing TGFβ-induced EMT.

<p>(<b>A</b>) Primary tumor cells were isolated from an advanced breast tumor of a MMTV-PyMT transgenic female mouse and were cultured for at least 2 months prior to further experimentation, resulting in a novel cell line termed Py2T. (<b>B</b>) Py2T cells maintain the MMTV-PyMT transgene. The MMTV-PyMT transgene was detected by PCR and agarose gel electrophoresis. DNA from an MMTV-PyMT tumor and from normal murine mammary gland (NMuMG) cells served as positive and negative controls, respectively. (<b>C</b>) Py2T cells lost the expression of the MMTV-PyMT transgene. Immunoblotting for the PyMT protein was performed on lysates of Py2T cells untreated or treated with 0.1 µM Dexamethasone for up to 72 h to induce the MMTV promoter. Lysates of an MMTV-PyMT tumor and NMuMG cells served as positive and negative controls, respectively. (<b>D</b>) Treatment of Py2T cells with known EMT inducers. Cells were continuously treated with the indicated growth factors and cytokines for 10 days (2 ng/mL TGFβ1; 50 ng/mL EGF; 10 ng/mL IGF-I; 50 ng/mL HGF; 20 ng/mL FGF-2; 20 ng/mL PDGF-BB; 50 ng/mL IL-6). Potential morphological changes were analyzed by phase-contrast microscopy. (<b>E</b>) Expression of epithelial (E-cadherin) and mesenchymal (N-cadherin, fibronectin) markers were analyzed by immunoblotting of the lysates of cells treated in (D). (<b>F</b>) Immunoblotting analysis of EMT marker expression in Py2T and Py2T LT cells. The mesenchymal subline Py2T LT (long-term) was generated by TGFβ-treatment of Py2T cells for at least 20 days, and was subsequently maintained in TGFβ containing growth medium. (<b>G</b>) Analysis of markers for EMT and breast cell type before and after TGFβ-induced EMT. Immunofluorescence staining was performed with antibodies against E-Cadherin (epithelial marker), vimentin (mesenchymal marker), estrogen receptor alpha (ERα), cytokeratin 8/18 (luminal markers) and cytokeratin 14 (basal marker). Scale bar, 20 µm.</p

Kinetics and reversibility of TGFβ-induced EMT in Py2T cells.

<p>(<b>A</b>) Morphological changes of Py2T cells during a time-course of TGFβ-treatment. Cells were cultured in growth medium containing TGFβ (2 ng/ml) and phase-contrast microscopy pictures were taken at the indicated times. (<b>B</b>) Immunoblotting analysis of lysates prepared from Py2T cells treated as in (A). The expression of epithelial (E-cadherin), mesenchymal (N-cadherin, fibronectin), luminal (CK8/18) and basal (CK14) markers was analyzed. (<b>C</b>) Changes in the expression of EMT markers during TGFβ-induced EMT of Py2T cells. Py2T cells were treated for 10 days with TGFβ as described in (A). RNA was extracted at the indicated time points of TGFβ-treatment and quantitative RT-PCR was performed with primers specific for the EMT markers indicated. Expression levels are shown as mean fold difference of untreated cells (0d) ± S.E.M of 5 independent experiments. (<b>D–E</b>) Reversibility of TGFβ-induced EMT. Py2T cells were treated with TGFβ for 30 days to induce EMT and were then further cultured without TGFβ for additional 30 days. Phase-contrast microscopy images were taken at the indicated time points (E). E-cadherin expression levels were analyzed throughout the experiment by immunoblotting (F).</p

Tumors of TGFβ-resistant Py2T cells contain areas with a more epithelial phenotype.

<p>(<b>A</b>) Morphology of tumors generated from Py2T cells stably overexpressing a dominant-negative TGFβRII (Py2T TBRDN) or empty vector control cells (Py2T). 1×10⁶ cells were injected into fat pads of nude mice and tumors were grown for 24 days. Paraffin sections were stained with H&E. Note the appearance of more differentiated epithelial areas in Py2T TBRDN tumors. <i>Top</i>: Epithelial (E) and mesenchymal (M) regions are separated by the dashed line (Scale bar, 200 µm). Bottom panels show larger magnification (Scale bar, 50 µm). (<b>B</b>) Expression of EMT and lineage markers in Py2T tumors and in the more epithelial areas of Py2T TBRDN tumors. Immunohistochemical staining of paraffin sections was performed using the specified antibodies. White squares show higher magnification. Scale bar, 100 µm. (<b>C</b>) Immunofluorescence staining of frozen sections of GFP-labeled Py2T and Py2T TBRDN tumors as described in (A) with antibodies against E-cadherin <i>(red)</i> and Py2T tumor cells <i>(green)</i>. Scale bar, 20 µm. (<b>D</b>) Immunoblotting analysis of epithelial and cytokeratin lineage markers in a series of Py2T and Py2T TBRDN tumors as indicated. (<b>E</b>) Immunofluorescence staining of frozen sections of GFP-labeled Py2T and Py2T TBRDN tumors as described in (A) with antibodies against vimentin <i>(red)</i> and Py2T tumor cells <i>(green)</i>. Scale bar, 20 µm.</p

Orthotopic transplantation of Py2T cells into syngeneic mice results in the formation of invasive tumors.

<p>(<b>A</b>) H&E staining of histological sections from tumors of MMTV-PyMT transgenic mice and from transplanted Py2T tumors. 1×10⁶ Py2T cells were transplanted into the fat pad of 8 weeks old female FVB/N mice and allowed to grow tumors for 27 days. Late-stage MMTV-PyMT tumors were from 12 weeks old female mice. <i>Bottom panels</i>: enlarged regions indicated by the white squares in the top panels. Note the typical pushing borders in MMTV-PyMT tumors in contrast to stream-like invasion of fat tissue in Py2T tumors. Scale bars, 200 µm. (<b>B</b>) Polyoma-middle-T (PyMT) expression in MMTV-PyMT and Py2T tumors. Paraffin sections were stained with an antibody against PyMT. Immunohistochemical staining in the absence of primary antibody (1°) was used as negative control. Scale bar, 100 µm. (<b>C</b>) Immunoblotting analysis for EMT markers in tumor lysates of MMTV-PyMT and Py2T tumors. Lysate from cultured Py2T cells is included as a control. Note the loss of E-cadherin expression and upregulation of mesenchymal markers (N-cadherin, fibronectin) in Py2T tumors.</p