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. © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

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

Genome-wide expression profiling of lymphoblastoid cell lines distinguishes different forms of autism and reveals shared pathways.

Autism is a heterogeneous condition that is likely to result from the combined effects of multiple genetic factors interacting with environmental factors. Given its complexity, the study of autism associated with Mendelian single gene disorders or known chromosomal etiologies provides an important perspective. We used microarray analysis to compare the mRNA expression profile in lymphoblastoid cells from males with autism due to a fragile X mutation (FMR1-FM), or a 15q11-q13 duplication (dup(15q)), and non-autistic controls. Gene expression profiles clearly distinguished autism from controls and separated individuals with autism based on their genetic etiology. We identified 68 genes that were dysregulated in common between autism with FMR1-FM and dup(15q). We also identified a potential molecular link between FMR1-FM and dup(15q), the cytoplasmic FMR1 interacting protein 1 (CYFIP1), which was up-regulated in dup(15q) patients. We were able to confirm this link in vitro by showing common regulation of two other dysregulated genes, JAKMIP1 and GPR155, downstream of FMR1 or CYFIP1. We also confirmed the reduction of the Jakmip1 protein in Fmr1 knock-out mice, demonstrating in vivo relevance. Finally, we showed independent confirmation of roles for JAKMIP1 and GPR155 in autism spectrum disorders (ASDs) by showing their differential expression in male sib pairs discordant for idiopathic ASD. These results provide evidence that blood derived lymphoblastoid cells gene expression is likely to be useful for identifying etiological subsets of autism and exploring its pathophysiology