The miR-200-Zeb1 axis regulates key aspects of ß-cell function and survival in vivo.

OBJECTIVE: The miR-200-Zeb1 axis regulates the epithelial-to-mesenchymal transition (EMT), differentiation, and resistance to apoptosis. A better understanding of these processes in diabetes is highly relevant, as ß-cell dedifferentiation and apoptosis contribute to the loss of functional ß-cell mass and diabetes progression. Furthermore, EMT promotes the loss of ß-cell identity in the in vitro expansion of human islets. Though the miR-200 family has previously been identified as a regulator of ß-cell apoptosis in vivo, studies focusing on Zeb1 are lacking. The aim of this study was thus to investigate the role of Zeb1 in ß-cell function and survival in vivo. METHODS: miR-200 and Zeb1 are involved in a double-negative feedback loop. We characterized a mouse model in which miR-200 binding sites in the Zeb1 3'UTR are mutated (Zeb1200), leading to a physiologically relevant upregulation of Zeb1 mRNA expression. The role of Zeb1 was investigated in this model via metabolic tests and analysis of isolated islets. Further insights into the distinct contributions of the miR-200 and Zeb1 branches of the feedback loop were obtained by crossing the Zeb1200 allele into a background of miR-141-200c overexpression. RESULTS: Mild Zeb1 derepression in vivo led to broad transcriptional changes in islets affecting ß-cell identity, EMT, insulin secretion, cell-cell junctions, the unfolded protein response (UPR), and the response to ER stress. The aggregation and insulin secretion of dissociated islets of mice homozygous for the Zeb1200 mutation (Zeb1200M) were impaired, and Zeb1200M islets were resistant to thapsigargin-induced ER stress ex vivo. Zeb1200M mice had increased circulating proinsulin levels but no overt metabolic phenotype, reflecting the strong compensatory ability of islets to maintain glucose homeostasis. CONCLUSIONS: This study signifies the importance of the miR-200-Zeb1 axis in regulating key aspects of ß-cell function and survival. A better understanding of this axis is highly relevant in developing therapeutic strategies for inducing ß-cell redifferentiation and maintaining ß-cell identity in in vitro islet expansion.

Stabilized epithelial phenotype of cancer cells in primary tumors leads to increased colonization of liver metastasis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is therapeutically recalcitrant and metastatic. Partial epithelial to mesenchymal transition (EMT) is associated with metastasis; however, a causal connection needs further unraveling. Here, we use single-cell RNA sequencing and genetic mouse models to identify the functional roles of partial EMT and epithelial stabilization in PDAC growth and metastasis. A global EMT expression signature identifies ∼50 cancer cell clusters spanning the epithelial-mesenchymal continuum in both human and murine PDACs. The combined genetic suppression of Snail and Twist results in PDAC epithelial stabilization and increased liver metastasis. Genetic deletion of Zeb1 in PDAC cells also leads to liver metastasis associated with cancer cell epithelial stabilization. We demonstrate that epithelial stabilization leads to the enhanced collective migration of cancer cells and modulation of the immune microenvironment, which likely contribute to efficient liver colonization. Our study provides insights into the diverse mechanisms of metastasis in pancreatic cancer and potential therapeutic targets.

Inactivation of endothelial ZEB1 impedes tumor progression and sensitizes tumors to conventional therapies.

Current antiangiogenic therapy is limited by its cytostatic property, scarce drug delivery to the tumor, and side toxicity. To address these limitations, we unveiled the role of ZEB1, a tumor endothelium-enriched zinc-finger transcription factor, during tumor progression. We discovered that the patients who had lung adenocarcinomas with high ZEB1 expression in tumor endothelium had increased prevalence of metastases and markedly reduced overall survival after the diagnosis of lung cancer. Endothelial ZEB1 deletion in tumor-bearing mice diminished tumor angiogenesis while eliciting persistent tumor vascular normalization by epigenetically repressing TGF-ß signaling. This consequently led to improved blood and oxygen perfusion, enhanced chemotherapy delivery and immune effector cell infiltration, and reduced tumor growth and metastasis. Moreover, targeting vascular ZEB1 remarkably potentiated the anticancer activity of nontoxic low-dose cisplatin. Treatment with low-dose anti-programmed cell death protein 1 (anti-PD-1) antibody elicited tumor regression and markedly extended survival in ZEB1-deleted mice, conferring long-term protective anticancer immunity. Collectively, we demonstrated that inactivation of endothelial ZEB1 may offer alternative opportunities for cancer therapy with minimal side effects. Targeting endothelium-derived ZEB1 in combination with conventional chemotherapy or immune checkpoint blockade therapy may yield a potent and superior anticancer effect.

Inactivation of Zeb1 in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure.

Cleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like (Grhl1-3) genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, Grhl2, is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. Grhl2-null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of Grhl2-null embryos, dysregulation of transcription factors that drive epithelial-mesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors Ovol1 and Ovol2, which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of Zeb1 expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in Zeb1-/- mice is evident, with rescue of both palatal and facial fusion seen in Grhl2-/-;Zeb1-/- embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients.

ZEB1 protects skeletal muscle from damage and is required for its regeneration.

The mechanisms linking muscle injury and regeneration are not fully understood. Here we report an unexpected role for ZEB1 regulating inflammatory and repair responses in dystrophic and acutely injured muscles. ZEB1 is upregulated in the undamaged and regenerating myofibers of injured muscles. Compared to wild-type counterparts, Zeb1-deficient injured muscles exhibit enhanced damage that corresponds with a retarded p38-MAPK-dependent transition of their macrophages towards an anti-inflammatory phenotype. Zeb1-deficient injured muscles also display a delayed and poorer regeneration that is accounted by the retarded anti-inflammatory macrophage transition and their intrinsically deficient muscle satellite cells (MuSCs). Macrophages in Zeb1-deficient injured muscles show lower phosphorylation of p38 and its forced activation reverts the enhanced muscle damage and poorer regeneration. MuSCs require ZEB1 to maintain their quiescence, prevent their premature activation following injury, and drive efficient regeneration in dystrophic muscles. These data indicate that ZEB1 protects muscle from damage and is required for its regeneration.

Loss of the candidate tumor suppressor ZEB1 (TCF8, ZFHX1A) in Sézary syndrome.

Cutaneous T-cell lymphoma is a group of incurable extranodal non-Hodgkin lymphomas that develop from the skin-homing CD4+ T cell. Mycosis fungoides and Sézary syndrome are the most common histological subtypes. Although next-generation sequencing data provided significant advances in the comprehension of the genetic basis of this lymphoma, there is not uniform consensus on the identity and prevalence of putative driver genes for this heterogeneous group of tumors. Additional studies may increase the knowledge about the complex genetic etiology characterizing this lymphoma. We used SNP6 arrays and GISTIC algorithm to prioritize a list of focal somatic copy-number alterations in a dataset of multiple sequential samples from 21 Sézary syndrome patients. Our results confirmed a prevalence of significant focal deletions over amplifications: single well-known tumor suppressors, such as TP53, PTEN, and RB1, are targeted by these aberrations. In our cohort, ZEB1 (TCF8, ZFHX1A) spans a deletion having the highest level of significance. In a larger group of 43 patients, we found that ZEB1 is affected by deletions and somatic inactivating mutations in 46.5% of cases; also, we found potentially relevant ZEB1 germline variants. The survival analysis shows a worse clinical course for patients with ZEB1 biallelic inactivation. Multiple abnormal expression signatures were found associated with ZEB1 depletion in Sézary patients we verified that ZEB1 exerts a role in oxidative response of Sézary cells. Our data confirm the importance of deletions in the pathogenesis of cutaneous T-cell lymphoma. The characterization of ZEB1 abnormalities in Sézary syndrome fulfils the criteria of a canonical tumor suppressor gene. Although additional confirmations are needed, our findings suggest, for the first time, that ZEB1 germline variants might contribute to the risk of developing this disease. Also, we provide evidence that ZEB1 activity in Sézary cells, influencing the reactive oxygen species production, affects cell viability and apoptosis.

Zinc finger E-box-binding homeobox 1 (ZEB1) is required for neural differentiation of human embryonic stem cells.

Human pluripotent stem cells hold great promise for improving regenerative medicine. However, a risk for tumor formation and difficulties in generating large amounts of subtype derivatives remain the major obstacles for clinical applications of stem cells. Here, we discovered that zinc finger E-box-binding homeobox 1 (ZEB1) is highly expressed upon differentiation of human embryonic stem cells (hESCs) into neuronal precursors. CRISPR/Cas9-mediated ZEB1 depletion did not impede neural fate commitment, but prevented hESC-derived neural precursors from differentiating into neurons, indicating that ZEB1 is required for neuronal differentiation. ZEB1 overexpression not only expedited neural differentiation and neuronal maturation, which ensured safer neural cell transplantation, but also facilitated the generation of excitatory cortical neurons, which were valuable for managing certain neurological disorders, such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Our study provides useful information on how human neural cells are generated, which may help in forming strategies for developing and improving replacement therapies for treating patients with neurological diseases.

Co-optation of Tandem DNA Repeats for the Maintenance of Mesenchymal Identity.

Tandem repeats (TRs) are generated by DNA replication errors and retain a high level of instability, which in principle would make them unsuitable for integration into gene regulatory networks. However, the appearance of DNA sequence motifs recognized by transcription factors may turn TRs into functional cis-regulatory elements, thus favoring their stabilization in genomes. Here, we show that, in human cells, the transcriptional repressor ZEB1, which promotes the maintenance of mesenchymal features largely by suppressing epithelial genes and microRNAs, occupies TRs harboring dozens of copies of its DNA-binding motif within genomic loci relevant for maintenance of epithelial identity. The deletion of one such TR caused quasi-mesenchymal cancer cells to reacquire epithelial features, partially recapitulating the effects of ZEB1 gene deletion. These data demonstrate that the high density of identical motifs in TRs can make them suitable platforms for recruitment of transcriptional repressors, thus promoting their exaptation into pre-existing cis-regulatory networks.

Knocking down TCF8 inhibits high glucose- and angiotensin II-induced epithelial to mesenchymal transition in podocytes.

Epithelial to mesenchymal transition (EMT) is a physiological phenomenon in mammalian embryogenesis by which epithelial cells become mesenchymal stem cells. Studies have indicated that an inappropriate EMT plays a key role in a variety of pathogenic processes such as embryonic development and tumor metastasis. Moreover, recent studies have indicated EMT also plays an important role in renal fibrosis. In the current study, glucose and angiotensin II promoted EMT in podocytes as well as changes in the cellular morphology of podocytes. A high concentration of glucose and angiotensin II also promoted podocyte movement and migration. Moreover, a high concentration of glucose and angiotensin II promoted TCF8 expression. Inhibiting TCF8 expression with siRNA reversed EMT in podocytes in the presence of a high concentration of glucose and angiotensin. Inhibiting TCF8 expression also reversed changes in cellular morphology and podocyte movement and migration. Therefore, glucose and angiotensin II may promote EMT in podocytes via TCF8.