Dottorato in BIOLOGIA UMANA E GENETICA MEDICA

Titolo della tesi: Role of the transcription factor ZNF281 in intestinal fibrogenesis

Intestinal fibrosis is a serious complication of inflammatory bowel disease (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC). A comprehensive understanding of the pathogenesis of intestinal fibrosis in IBD remains uncertain hampering the development of effective therapeutic strategies. The currently accepted hypothesis indicates that a chronic inflammatory state stimulates the trans-differentiation of mesenchymal cells into fibroblasts and myofibroblasts, the major effector cells involved in intestinal fibrosis. Myofibroblasts also originate from local epithelial cells via epithelial-to-mesenchymal transition (EMT). ZNF281 is a zinc-finger transcriptional regulator that has been characterized as an EMT-inducing transcription factor and recently shown to be actively involved in intestinal inflammation. Further, ZNF281 is suggested to play a pro-tumoral role in several human malignancies, including breast, colorectal and pancreatic carcinomas. The aims of this study are 1) to set up patient-derived organoid cultures (PDOs) from IBD and control (non-IBD) pediatric patients as a new model of inflammatory-driven intestinal fibrosis 2) to investigate in vivo and in vitro the role of ZNF281 in intestinal fibrogenesis, 3) to assess the expression of ZNF281 in cholangiocarcinoma (CCA), a deadly cancer of the hepatic biliary tree. The results of the first and the third point will be presented and discussed in the thesis. The results and the conclusion of the second point will be presented attaching the published original paper to the thesis (ZNF281 Promotes Colon Fibroblast Activation in TGFβ1-Induced Gut Fibrosis). Briefly, PDOs were generated from intestinal stem cells contained in crypts isolated from the mucosal biopsies of IBD and non-IBD subjects. The inflammation-driven fibrosis was induced by exposing IBD- and non-IBD PDOs to pro-inflammatory (TNF-α) and pro-fibrotic (TGF-β) cytokines for 4 days. Treatments caused morphological changes towards a mesenchymal-like phenotype, paralleled by the upregulation of the inflammatory marker IL-1β and the mesenchymal marker SLUG. Interestingly, IBD-PDOs seem to be more responsive to inflammation-driven fibrosis. Indeed, in IBD-PDOs also the expression of fibrotic markers, such as ZNF281, PAI1 and COL4A1, is increased upon TNF-α and TGF-β treatment. C57BL/6J mice were treated with repeated cycles of dextran sulfate sodium (DSS) to induce inflammation-driven fibrosis. ZNF281 was significantly increased in colon tissue of treated mice as compared to controls. Accordingly, ZNF281 was also strongly upregulated in human colon fibroblasts (CCD-18Co) exposed to TGF-β. Transcriptomic analysis in CCD-18Co cells, in which ZNF281 was silenced, showed a downregulation of genes involved in fibroblast activation and differentiation pathways. Finally, ZNF281 expression levels were assessed in surgical specimens obtained from 44 patients with histologically confirmed intrahepatic CCA. Data showed that ZNF281 was overexpressed in the tumor as compared to non-neoplastic bile duct. In conclusion, this study demonstrates that PDOs are a reliable tool to study inflammation-driven intestinal fibrosis, opening new perspectives for deeply investigating molecular mechanisms underlying fibrosis and offering a new opportunity to develop alternative therapeutic strategies for its management. Moreover, ZNF281 is a central regulator of intestinal fibrosis with a role in colon fibroblast activation and myofibroblast differentiation. Hence, ZNF281 may represent a novel therapeutic target for the cure of fibrosis complications in IBD patients. Finally, preliminary results suggest that ZNF281 is implicated in the pathobiology of CCA.