Simone Brabletz

E-cadherin, β-catenin, and ZEB1 in malignant progression of cancer

The embryonic program ‘epithelial-mesenchymal transition’ (EMT) is activated during tumor invasion in disseminating cancer cells. Characteristic to these cells is a loss of E-cadherin expression, which can be mediated by EMT-inducing transcriptional repressors, e.g. ZEB1. Consequences of a loss of E-cadherin are an impairment of cell-cell adhesion, which allows detachment of cells, and nuclear localization of β-catenin. In addition to an accumulation of cancer stem cells, nuclear β-catenin induces a gene expression pattern favoring tumor invasion, and mounting evidence indicates multiple reciprocal interactions of E-cadherin and β-catenin with EMT-inducing transcriptional repressors to stabilize an invasive mesenchymal phenotype of epithelial tumor cells.

The ZEB/miR-200 feedback loop—a motor of cellular plasticity in development and cancer?

Epithelial‐to‐mesenchymal transition (EMT) is a fundamental process in development and disease. Zinc‐finger enhancer binding (ZEB) transcription factors (ZEB1 and ZEB2) are crucial EMT activators, whereas members of the miR‐200 family induce epithelial differentiation. They are reciprocally linked in a feedback loop, each strictly controlling the expression of the other. Now data show that EMT not only confers cellular motility, but also induces stem‐cell properties and prevents apoptosis and senescence. Thus the balanced expression of ZEB factors and miR‐200 controls all these processes. We therefore propose that the ZEB/miR‐200 feedback loop is the molecular motor of cellular plasticity in development and disease, and in particular is a driving force for cancer progression towards metastasis by controlling the state of cancer stem cells.

The ZEB1/miR‐200 feedback loop controls Notch signalling in cancer cells

Notch signalling is important for development and tissue homeostasis and activated in many human cancers. Nevertheless, mutations in Notch pathway components are rare in solid tumours. ZEB1 is an activator of an epithelial–mesenchymal transition (EMT) and has crucial roles in tumour progression towards metastasis. ZEB1 and miR‐200 family members repress expression of each other in a reciprocal feedback loop. Since miR‐200 members target stem cell factors, ZEB1 indirectly induces stemness maintenance and associated drug resistance. Here, we link ZEB1 and its cancer promoting properties to Notch activation. We show that miR‐200 members target Notch pathway components, such as Jagged1 (Jag1) and the mastermind‐like coactivators Maml2 and Maml3, thereby mediating enhanced Notch activation by ZEB1. We further detected a coordinated upregulation of Jag1 and ZEB1, associated with reduced miR‐200 expression in two aggressive types of human cancer, pancreatic adenocarcinoma and basal type of breast cancer. These findings explain increased Notch signalling in some types of cancers, where mutations in Notch pathway genes are rare. Moreover, they indicate an additional way how ZEB1 exerts its tumour progressing functions.

The ZEB1 pathway links glioblastoma initiation, invasion and chemoresistance

Glioblastoma remains one of the most lethal types of cancer, and is the most common brain tumour in adults. In particular, tumour recurrence after surgical resection and radiation invariably occurs regardless of aggressive chemotherapy. Here, we provide evidence that the transcription factor ZEB1 (zinc finger E‐box binding homeobox 1) exerts simultaneous influence over invasion, chemoresistance and tumourigenesis in glioblastoma. ZEB1 is preferentially expressed in invasive glioblastoma cells, where the ZEB1‐miR‐200 feedback loop interconnects these processes through the downstream effectors ROBO1, c‐MYB and MGMT. Moreover, ZEB1 expression in glioblastoma patients is predictive of shorter survival and poor Temozolomide response. Our findings indicate that this regulator of epithelial‐mesenchymal transition orchestrates key features of cancer stem cells in malignant glioma and identify ROBO1, OLIG2, CD133 and MGMT as novel targets of the ZEB1 pathway. Thus, ZEB1 is an important candidate molecule for glioblastoma recurrence, a marker of invasive tumour cells and a potential therapeutic target, along with its downstream effectors.

The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer

Metastasis is the major cause of cancer-associated death. Partial activation of the epithelial-to-mesenchymal transition program (partial EMT) was considered a major driver of tumour progression from initiation to metastasis. However, the role of EMT in promoting metastasis has recently been challenged, in particular concerning effects of the Snail and Twist EMT transcription factors (EMT-TFs) in pancreatic cancer. In contrast, we show here that in the same pancreatic cancer model, driven by Pdx1-cre-mediated activation of mutant Kras and p53 (KPC model), the EMT-TF Zeb1 is a key factor for the formation of precursor lesions, invasion and notably metastasis. Depletion of Zeb1 suppresses stemness, colonization capacity and in particular phenotypic/metabolic plasticity of tumour cells, probably causing the observed in vivo effects. Accordingly, we conclude that different EMT-TFs have complementary subfunctions in driving pancreatic tumour metastasis. Therapeutic strategies should consider these potential specificities of EMT-TFs to target these factors simultaneously.

ZEB1-associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat

Therapy resistance is a major clinical problem in cancer medicine and crucial for disease relapse and progression. Therefore, the clinical need to overcome it, particularly for aggressive tumors such as pancreatic cancer, is very high. Aberrant activation of an epithelial–mesenchymal transition (EMT) and an associated cancer stem cell phenotype are considered a major cause of therapy resistance. Particularly, the EMT‐activator ZEB1 was shown to confer stemness and resistance. We applied a systematic, stepwise strategy to interfere with ZEB1 function, aiming to overcome drug resistance. This led to the identification of both its target gene miR‐203 as a major drug sensitizer and subsequently the class I HDAC inhibitor mocetinostat as epigenetic drug to interfere with ZEB1 function, restore miR‐203 expression, repress stemness properties, and induce sensitivity against chemotherapy. Thereby, mocetinostat turned out to be more effective than other HDAC inhibitors, such as SAHA, indicating the relevance of the screening strategy. Our data encourage the application of mechanism‐based combinations of selected epigenetic drugs with standard chemotherapy for the rational treatment of aggressive solid tumors, such as pancreatic cancer.

A self-enforcing CD44s/ZEB1 feedback loop maintains EMT and stemness properties in cancer cells

Invasion and metastasis of carcinomas are often activated by induction of aberrant epithelial–mesenchymal transition (EMT). This is mainly driven by the transcription factor ZEB1, promoting tumor‐initiating capacity correlated with increased expression of the putative stem cell marker CD44. However, the direct link between ZEB1, CD44 and tumourigenesis is still enigmatic. Remarkably, EMT‐induced repression of ESRP1 controls alternative splicing of CD44, causing a shift in the expression from the variant CD44v to the standard CD44s isoform. We analyzed whether CD44 and ZEB1 regulate each other and show that ZEB1 controls CD44s splicing by repression of ESRP1 in breast and pancreatic cancer. Intriguingly, CD44s itself activates the expression of ZEB1, resulting in a self‐sustaining ZEB1 and CD44s expression. Activation of this novel CD44s‐ZEB1 regulatory loop has functional impact on tumor cells, as evident by increased tumor‐sphere initiation capacity, drug‐resistance and tumor recurrence. In summary, we identified a self‐enforcing feedback loop that employs CD44s to activate ZEB1 expression. This renders tumor cell stemness independent of external stimuli, as ZEB1 downregulates ESRP1, further promoting CD44s isoform synthesis.

MicroRNAs coordinately regulate protein complexes

BackgroundIn animals, microRNAs (miRNAs) regulate the protein synthesis of their target messenger RNAs (mRNAs) by either translational repression or deadenylation. miRNAs are frequently found to be co-expressed in different tissues and cell types, while some form polycistronic clusters on genomes. Interactions between targets of co-expressed miRNAs (including miRNA clusters) have not yet been systematically investigated.ResultsHere we integrated information from predicted and experimentally verified miRNA targets to characterize protein complex networks regulated by human miRNAs. We found striking evidence that individual miRNAs or co-expressed miRNAs frequently target several components of protein complexes. We experimentally verified that the miR-141-200c cluster targets different components of the CtBP/ZEB complex, suggesting a potential orchestrated regulation in epithelial to mesenchymal transition.ConclusionsOur findings indicate a coordinate posttranscriptional regulation of protein complexes by miRNAs. These provide a sound basis for designing experiments to study miRNA function at a systems level.

The ZEB1/miR-200c feedback loop regulates invasion via actin interacting proteins MYLK and TKS5.

Epithelial to mesenchymal transition (EMT) is a developmental process which is aberrantly activated during cancer invasion and metastasis. Elevated expression of EMT-inducers like ZEB1 enables tumor cells to detach from the primary tumor and invade into the surrounding tissue. The main antagonist of ZEB1 in controlling EMT is the microRNA-200 family that is reciprocally linked to ZEB1 in a double negative feedback loop. Here, we further elucidate how the ZEB1/miR-200 feedback loop controls invasion of tumor cells. The process of EMT is attended by major changes in the actin cytoskeleton. Via in silico screening of genes encoding for actin interacting proteins, we identified two novel targets of miR-200c - TKS5 and MYLK (MLCK). Co-expression of both genes with ZEB1 was observed in several cancer cell lines as well as in breast cancer patients and correlated with low miR-200c levels. Depletion of TKS5 or MYLK in breast cancer cells reduced their invasive potential and their ability to form invadopodia. Whereas TKS5 is known to be a major component, we could identify MYLK as a novel player in invadopodia formation. In summary, TKS5 and MYLK represent two mediators of invasive behavior of cancer cells that are regulated by the ZEB1/miR-200 feedback loop.

A novel ZEB1/HAS2 positive feedback loop promotes EMT in breast cancer

Cancer metastasis is the main reason for poor patient survival. Tumor cells delaminate from the primary tumor by induction of epithelial-mesenchymal transition (EMT). EMT is mediated by key transcription factors, including ZEB1, activated by tumor cell interactions with stromal cells and the extracellular matrix (ECM). ZEB1-mediated EMT and motility is accompanied by substantial cell reprogramming and the acquisition of a stemness phenotype. However, understanding of the underlying mechanism is still incomplete. We identified hyaluronic acid (HA), one major ECM proteoglycan and enriched in mammary tumors, to support EMT and enhance ZEB1 expression in cooperation with CD44s. In breast cancer cell lines HA is synthesized mainly by HAS2, which was already shown to be implicated in cancer progression. ZEB1 and HAS2 expression strongly correlates in various cancer entities and high HAS2 levels associate with an early relapse. We identified HAS2, tumor cell-derived HA and ZEB1 to form a positive feedback loop as ZEB1, elevated by HA, directly activates HAS2 expression. In an in vitro differentiation model HA-conditioned medium of breast cancer cells is enhancing osteoclast formation, an indicator of tumor cell-induced osteolysis that facilitates formation of bone metastasis. In combination with the previously identified ZEB1/ESRP1/CD44s feedback loop, we found a novel autocrine mechanism how ZEB1 is accelerating EMT.