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.

ZEB1 turns into a transcriptional activator by interacting with YAP1 in aggressive cancer types.

Early dissemination, metastasis and therapy resistance are central hallmarks of aggressive cancer types and the leading cause of cancer-associated deaths. The EMT-inducing transcriptional repressor ZEB1 is a crucial stimulator of these processes, particularly by coupling the activation of cellular motility with stemness and survival properties. ZEB1 expression is associated with aggressive behaviour in many tumour types, but the potent effects cannot be solely explained by its proven function as a transcriptional repressor of epithelial genes. Here we describe a direct interaction of ZEB1 with the Hippo pathway effector YAP, but notably not with its paralogue TAZ. In consequence, ZEB1 switches its function to a transcriptional co-activator of a 'common ZEB1/YAP target gene set', thereby linking two pathways with similar cancer promoting effects. This gene set is a predictor of poor survival, therapy resistance and increased metastatic risk in breast cancer, indicating the clinical relevance of our findings.

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.

The tyrosine phosphatase PTPN14 is a negative regulator of YAP activity.

The Hippo (Hpo) pathway is a novel signaling pathway that controls organ size in Drosophila and mammals and is deregulated in a variety of human cancers. It consists of a set of kinases that, through a number of phosphorylation events, inactivate YAP, a transcriptional co-activator that controls cellular proliferation and apoptosis. We have identified PTPN14 as a YAP-binding protein that negatively regulates YAP activity by controlling its localization. Mechanistically, we find that the interaction of ectopic YAP with PTPN14 can be mediated by the respective WW and PPxY motifs. However, the PTPN14 PPxY motif and phosphatase activity appear to be dispensable for the negative regulation of endogenous YAP, likely suggesting more complex mechanisms of interaction and modulation. Finally, we demonstrate that PTPN14 downregulation can phenocopy YAP activation in mammary epithelial cells and synergize with YAP to induce oncogenic transformation.

The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins.

The mitochondrial outer membrane contains translocase complexes for the import of precursor proteins. The translocase of the outer membrane complex functions as a general preprotein entry gate, whereas the sorting and assembly machinery complex mediates membrane insertion of ß-barrel proteins of the outer membrane. Several α-helical outer membrane proteins are known to carry multiple transmembrane segments; however, only limited information is available on the biogenesis of these proteins. We report that mitochondria lacking the mitochondrial import protein 1 (Mim1) are impaired in the biogenesis of multispanning outer membrane proteins, whereas overexpression of Mim1 stimulates their import. The Mim1 complex cooperates with the receptor Tom70 in binding of precursor proteins and promotes their insertion and assembly into the outer membrane. We conclude that the Mim1 complex plays a central role in the import of α-helical outer membrane proteins with multiple transmembrane segments.