Laura Santangelo

The stable repression of mesenchymal program is required for hepatocyte identity: A novel role for hepatocyte nuclear factor 4α

The concept that cellular terminal differentiation is stably maintained once development is complete has been questioned by numerous observations showing that differentiated epithelium may undergo an epithelial‐to‐mesenchymal transition (EMT) program. EMT and the reverse process, mesenchymal‐to‐epithelial transition (MET), are typical events of development, tissue repair, and tumor progression. In this study, we aimed to clarify the molecular mechanisms underlying these phenotypic conversions in hepatocytes. Hepatocyte nuclear factor 4α (HNF4α) was overexpressed in different hepatocyte cell lines and the resulting gene expression profile was determined by real‐time quantitative polymerase chain reaction. HNF4α recruitment on promoters of both mesenchymal and EMT regulator genes was determined by way of electrophoretic mobility shift assay and chromatin immunoprecipitation. The effect of HNF4α depletion was assessed in silenced cells and in the context of the whole liver of HNF4 knockout animals. Our results identified key EMT regulators and mesenchymal genes as new targets of HNF4α. HNF4α, in cooperation with its target HNF1α, directly inhibits transcription of the EMT master regulatory genes Snail, Slug, and HMGA2 and of several mesenchymal markers. HNF4α‐mediated repression of EMT genes induces MET in hepatomas, and its silencing triggers the mesenchymal program in differentiated hepatocytes both in cell culture and in the whole liver. Conclusion: The pivotal role of HNF4α in the induction and maintenance of hepatocyte differentiation should also be ascribed to its capacity to continuously repress the mesenchymal program; thus, both HNF4α activator and repressor functions are necessary for the identity of hepatocytes. (HEPATOLOGY 2011;)

Hepatitis C virus production requires apolipoprotein A-I and affects its association with nascent low-density lipoproteins

Background/aims The life cycle of hepatitis C virus (HCV) is intimately linked to the lipid metabolism of the host. In particular, HCV exploits the metabolic machinery of the lipoproteins in several steps of its life cycle such as circulation in the bloodstream, cell attachment and entry, assembly and release of viral particles. However, the details of how HCV interacts with and influences the metabolism of the host lipoproteins are not well understood. A study was undertaken to investigate whether HCV directly affects the protein composition of host circulating lipoproteins. Methods A proteomic analysis of circulating very low-, low- and high-density lipoproteins (VLDL, LDL and HDL), isolated from either in-treatment naïve HCV-infected patients or healthy donors (HD), was performed using two-dimensional gel electrophoresis and tandem mass spectrometry (MALDI-TOF/TOF). The results obtained were further investigated using in vitro models of HCV infection and replication. Results A decreased level of apolipoprotein A-I (apoA-I) was found in the LDL fractions of HCV-infected patients. This result was confirmed by western blot and ELISA analysis. HCV cellular models (JFH1 HCV cell culture system (HCVcc) and HCV subgenomic replicons) showed that the decreased apoA-I/LDL association originates from hepatic biogenesis rather than lipoprotein catabolism occurring in the circulation, and is not due to a downregulation of the apoA-I protein concentration. The sole non-structural viral proteins were sufficient to impair the apoA-I/LDL association. Functional evidence was obtained for involvement of apoA-I in the viral life cycle such as RNA replication and virion production. The specific siRNA-mediated downregulation of apoA-I led to a reduction in both HCV RNA and viral particle levels in culture. Conclusions This study shows that HCV induces lipoprotein structural modification and that its replication and production are linked to the host lipoprotein metabolism, suggesting apoA-I as a new possible target for antiviral therapy.

The Snail repressor recruits EZH2 to specific genomic sites through the enrollment of the lncRNA HOTAIR in epithelial-to-mesenchymal transition

The transcription factor Snail is a master regulator of cellular identity and epithelial-to-mesenchymal transition (EMT) directly repressing a broad repertoire of epithelial genes. How chromatin modifiers instrumental to its activity are recruited to Snail-specific binding sites is unclear. Here we report that the long non-coding RNA (lncRNA) HOTAIR (for HOX Transcript Antisense Intergenic RNA) mediates a physical interaction between Snail and enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of the polycomb-repressive complex 2 and the main writer of chromatin-repressive marks. The Snail-repressive activity, here monitored on genes with a pivotal function in epithelial and hepatic morphogenesis, differentiation and cell-type identity, depends on the formation of a tripartite Snail/HOTAIR/EZH2 complex. These results demonstrate an lncRNA-mediated mechanism by which a transcriptional factor conveys a general chromatin modifier to specific genes, thereby allowing the execution of hepatocyte transdifferentiation; moreover, they highlight HOTAIR as a crucial player in the Snail-mediated EMT.

An epistatic mini-circuitry between the transcription factors Snail and HNF4α controls liver stem cell and hepatocyte features exhorting opposite regulation on stemness-inhibiting microRNAs.

Preservation of the epithelial state involves the stable repression of epithelial-to-mesenchymal transition program, whereas maintenance of the stem compartment requires the inhibition of differentiation processes. A simple and direct molecular mini-circuitry between master elements of these biological processes might provide the best device to keep balanced such complex phenomena. In this work, we show that in hepatic stem cell Snail, a transcriptional repressor of the hepatocyte differentiation master gene HNF4α, directly represses the expression of the epithelial microRNAs (miRs)-200c and -34a, which in turn target several stem cell genes. Notably, in differentiated hepatocytes HNF4α, previously identified as a transcriptional repressor of Snail, induces the miRs-34a and -200a, b, c that, when silenced, causes epithelial dedifferentiation and reacquisition of stem traits. Altogether these data unveiled Snail, HNF4α and miRs-200a, b, c and -34a as epistatic elements controlling hepatic stem cell maintenance/differentiation.

TGFβ overrides HNF4α tumor suppressing activity through GSK3β inactivation: implication for hepatocellular carcinoma gene therapy

BACKGROUND & AIMS The tumor fate derives from cell autonomous properties and niche microenvironmental cues. The transforming growth factor β (TGFβ) is a major microenvironmental factor for hepatocellular carcinoma (HCC) influencing tumor dedifferentiation, induction of epithelial-to-mesenchymal transition (EMT) and acquisition of metastatic properties. The loss of the transcriptional factor HNF4α is a predominant mechanism through which HCCs progress to a more aggressive phenotype; its re-expression, reducing tumor formation and repressing EMT program, has been suggested as a therapeutic tool for HCC gene therapy. We investigated the influence of TGFβ on the anti-EMT and tumor suppressor HNF4α activity. METHODS Cell motility and invasion were analyzed by wound healing and invasion assays. EMT was evaluated by RT-qPCR and immunofluorescence. ChIP and EMSA assays were utilized for investigation of the HNF4α DNA binding activity. HNF4α post-translational modifications (PTMs) were assessed by 2-DE analysis. GSK3β activity was modulated by chemical inhibition and constitutive active mutant expression. RESULTS We demonstrated that the presence of TGFβ impairs the efficiency of HNF4α as tumor suppressor. We found that TGFβ induces HNF4α PTMs that correlate with the early loss of HNF4α DNA binding activity on target gene promoters. Furthermore, we identified the GSK3β kinase as one of the TGFβ targets mediating HNF4α functional inactivation: GSK3β chemical inhibition results in HNF4α DNA binding impairment while a constitutively active GSK3β mutant impairs the TGFβ-induced inhibitory effect on HNF4α tumor suppressor activity. CONCLUSIONS Our data identify in the dominance of TGFβ a limit for the HNF4α-mediated gene therapy of HCC.

Ferritin heavy chain is the host factor responsible for HCV-induced inhibition of apoB-100 production and is required for efficient viral infection

Hepatic fat export occurs by apolipoprotein B-100-containing lipoprotein production, whereas impaired production leads to liver steatosis. Hepatitis C virus (HCV) infection is associated to dysregulation of apoB-100 secretion and steatosis; however, the molecular mechanism by which HCV affects the apoB-100 secretion is not understood. Here, combining quantitative proteomics and computational biology, we propose ferritin heavy chain (Fth) as being the cellular determinant of apoB-100 production inhibition. By means of molecular analyses, we found that HCV nonstructural proteins and NS5A appear to be sufficient for inducing Fth up-regulation. Fth in turn was found to inhibit apoB-100 secretion leading to increased intracellular degradation via proteasome. Notably, intracellular Fth down-regulation by siRNA restores apoB-100 secretion. The inverse correlation between ferritin and plasma apoB-100 concentrations was also found in JFH-1 HCV cell culture systems (HCVcc) and HCV-infected patients. Finally, Fth expression was found to be required for robust HCV infection. These observations provide a further molecular explanation for the onset of liver steatosis and allow for hypothesizing on new therapeutic and antiviral strategies.

Functional Roles and Therapeutic Applications of Exosomes in Hepatocellular Carcinoma

Exosomes are important in intercellular communication. They assure the horizontal transfer of specific functional contents (i.e., proteins, lipids, RNA molecules, and circulating DNA) from donor to recipient cells. Notably, tumor-derived exosomes (TDEs) appear to be an important vehicle of specific signals in cancer, impacting on tumor growth and metastasis. Recent researches point to the characterization of exosomes in Hepatocellular Carcinoma (HCC), the major adult liver malignancy. In this review, we summarize current findings on HCC exosomes, focusing on the identification of noncoding RNAs as exosome-enriched functional regulators and new potential biomarkers. The great potential of exosomes in future HCC diagnostic and therapeutic approaches is underlined.

Hepatitis C virus direct-acting antivirals therapy impacts on extracellular vesicles microRNAs content and on their immunomodulating properties

Hepatitis C virus (HCV) infection is known to cause major alterations in the cross‐talk between hepatic and immune cells thus contributing to the liver disease pathogenesis. Extracellular vesicles have been proved to act as major players in cell‐cell communication, and their cargo changes in relation to pathophysiological states. The aim of this study was to evaluate the effects of chronic HCV infection and direct‐acting antivirals (DAA) on exosome‐delivered microRNAs and on their ability to modulate the innate immune response.

The lncRNA HOTAIR transcription is controlled by HNF4α-induced chromatin topology modulation

The expression of the long noncoding RNA HOTAIR (HOX Transcript Antisense Intergenic RNA) is largely deregulated in epithelial cancers and positively correlates with poor prognosis and progression of hepatocellular carcinoma and gastrointestinal cancers. Furthermore, functional studies revealed a pivotal role for HOTAIR in the epithelial-to-mesenchymal transition, as this RNA is causal for the repressive activity of the master factor SNAIL on epithelial genes. Despite the proven oncogenic role of HOTAIR, its transcriptional regulation is still poorly understood. Here hepatocyte nuclear factor 4-α (HNF4α), as inducer of epithelial differentiation, was demonstrated to directly repress HOTAIR transcription in the mesenchymal-to epithelial transition. Mechanistically, HNF4α was found to cause the release of a chromatin loop on HOTAIR regulatory elements thus exerting an enhancer-blocking activity.