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.

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.Tumor recurrence and metastasis represent the two major obstacles in the successful treatment of cancer. Increasing evidence suggests that the aggressive phenotype of this disease is associated with the activation of an embryonic program termed epithelial-mesenchymal transition (EMT), a process in which epithelial cells lose apical-basal cell polarity and change to a mesenchymal phenotype. [1][2][3] In order to initiate and complete an EMT, several distinct molecular programsKey words: cancer stem cells, epithelial-mesenchymal transition (EMT), metastasis, drug resistance, differential splicing Abbreviations: bHLH: basic helix-loop-helix; CD44s: cluster of differentiation 44, standard isoform; CD44v: cluster of differentiation 44, variant isoforms; ChIP: chromatin immunoprecipitation; CSC: cancer stem cell; Dox: doxycycline; EGF: epidermal growth factor; EMT: epithelial-mesenchymal transition; ESRP1: epithelial splicing regulatory protein 1; FGF: fibroblast growth factor; HGF/SF: hepatic growth factor/scatter factor; hnRNPM: heterogeneous nuclear ribonucleoprotein M; PDAC: pancreas ductal adenocarcinoma; shRNA: small hairpin ribonucleic acid; siRNA: small interference ribonucleic acid; TGFb: transforming growth factor b; ZEB: zinc-finger and E-box binding; ZFH: zinc-finger homeodomain

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.