Carracedo, Arkaitz

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease associated with autoimmune phenomena targeting intrahepatic bile duct cells (cholangiocytes). Although its etiopathogenesis remains obscure, development of antimitochondrial autoantibodies against pyruvate dehydrogenase complex E2 is a common feature. MicroRNA (miR) dysregulation occurs in liver and immune cells of PBC patients, but its functional relevance is largely unknown. We previously reported that miR‐506 is overexpressed in PBC cholangiocytes and directly targets both Cl – / anion exchanger 2 and type III inositol 1,4,5‐trisphosphate receptor, leading to cholestasis. Here, the regulation of miR‐506 gene expression and its role in cholangiocyte pathophysiology and immune activation was studied. Several proinflammatory cytokines overexpressed in PBC livers (such as interleukin‐8 [IL8], IL12, IL17, IL18, and tumor necrosis factor alpha) stimulated miR‐506 promoter activity in human cholangiocytes, as revealed by luciferase reporter assays. Experimental overexpression of miR‐506 in cholangiocytes dysregulated the cell proteomic profile (by mass spectrometry), affecting proteins involved in different biological processes including mitochondrial metabolism. In cholangiocytes, miR‐506 (1) induced dedifferentiation with down‐regulation of biliary and epithelial markers together with up‐regulation of mesenchymal, proinflammatory, and profibrotic markers; (2) impaired cell proliferation and adhesion; (3) increased oxidative and endoplasmic reticulum stress; (4) caused DNA damage; and (5) sensitized to caspase‐3‐dependent apoptosis induced by cytotoxic bile acids. These events were also associated with impaired energy metabolism in mitochondria (proton leak and less adenosine triphosphate production) and pyruvate dehydrogenase complex E2 overexpression. Coculture of miR‐506 overexpressing cholangiocytes with PBC immunocytes induced activation and proliferation of PBC immunocytes. Conclusion : Different proinflammatory cytokines enhance the expression of miR‐506 in biliary epithelial cells; miR‐506 induces PBC‐like features in cholangiocytes and promotes immune activation, representing a potential therapeutic target for PBC patients. (H epatology 2018;67:1420‐1440)

Abstract Purpose: The limited supply of oxygen and nutrients is thought to result in rigorous selection of cells that will eventually form the tumor. Experimental Design: Nupr1 expression pattern was analyzed in human tissue microarray (TMA) and correlated with survival time of the patient. Microarray analysis was conducted on MiaPaCa2 cells subjected to metabolic stress in Nupr1-silenced conditions. DNA repair and cell cycle–associated gene expression was confirmed by real-time quantitative PCR (qRT-PCR). Nupr1 and AURKA protective role were analyzed using RNA interference (RNAi) silencing or overexpression. DNA damage and autophagy were analyzed by Western blot analysis and immunofluorescence. Results: We showed that both Nupr1 and HIF1α are coexpressed in human pancreatic ductal adenocarcinoma (PDAC) samples and negatively correlate with survival time. PDAC-derived cells submitted to hypoxia and/or glucose starvation induce DNA damage–dependent cell death concomitantly to the overexpression of stress protein Nupr1. Affymetrix-based transcriptoma analysis reveals that Nupr1 knockdown enhances DNA damage and alters the expression of several genes involved in DNA repair and cell-cycle progression. Expression of some of these genes is common to hypoxia and glucose starvation, such as Aurka gene, suggesting that Nupr1 overexpression counteracts the transcriptional changes occurring under metabolic stress. The molecular mechanism by which hypoxia and glucose starvation induce cell death involves autophagy-associated, but not caspase-dependent, cell death. Finally, we have found that AURKA expression is partially regulated by Nupr1 and plays a major role in this response. Conclusions: Our data reveal that Nupr1 is involved in a defense mechanism that promotes pancreatic cancer cell survival when exposed to metabolic stress. Clin Cancer Res; 18(19); 5234–46. ©2012 AACR.