Margherita Melegari

Risk factors for hepatocellular carcinoma in italy. Male sex, hepatitis b virus, non-a non-B infection, and alcohol

To investigate risk factors for hepatocellular carcinoma (HCC) in Italy—a country with medium (south: 5% to 10%) to low (north: 1% to 2%) incidence of hepatitis B virus (HBV) infection—we studied 646 consecutive patients: 58 chronic active hepatitis (CAH), 428 cirrhosis, and 160 HCC, 49% from Southern and 51% from Northern Italy. Hepatitis B surface antigen (HBsAg) was positive in 41.4% of the CAH, in 23.1% of cirrhotic patients, and in 26.2% of HCC. In the latter, HBV DNA assay increased the number of subjects with active HBV infection by about 12%. Alcohol abuse was evenly distributed in all three categories of HBV markers. Males were preferentially affected. The HCC was superimposed on cirrhosis in more than 90% of patients. Our data suggest that, in our epidemiologic setting, different factors (HBV, non‐A, non‐B agents, alcohol) may cooperate in the development of HCC, mainly through their potential for causing cirrhosis.

Hepatitis B Virus DNA Replication Is Coordinated by Core Protein Serine Phosphorylation and HBx Expression

ABSTRACT The hepatitis B virus (HBV) core protein forms the capsid of viral particles and is essential for viral genome DNA replication and maturation. The C terminus of core protein contains three serines at positions 155, 162, and 170, phosphorylation of which is important for viral DNA replication. We demonstrate that the phosphorylation of these serines is stimulated by the viral HBx protein, a regulatory protein that activates signal transduction pathways and viral replication. HBx is therefore shown to stimulate HBV replication by increasing core serine phosphorylation. Mutational, biochemical, and mixing studies of C-terminal core serine mutants demonstrate that multiple serine phosphorylations occur on the same core protein. Mutation of individual core protein serines is shown to inhibit HBV replication at distinct stages corresponding to encapsidation of viral pregenomic RNA, reverse transcription, and restriction to synthesis of specific DNA replicative intermediates. We therefore demonstrate that a primary target of HBV replication that is regulated by HBx protein corresponds to increased phosphorylation of the viral core protein. We also demonstrate that core phosphorylation mediated by HBx promotes sequential progression of viral replication through the assembly of capsids primed for different stages of DNA synthesis.

Use of dominant negative mutants of the hepadnaviral core protein as antiviral agents

Chronic hepatitis B virus (HBV) infection is a major cause of acute and chronic liver diseases. We have recently described HBV and woodchuck hepatitis virus (WHV) dominant negative (DN) core mutants that were capable of inhibiting wild‐type viral replication by 95%. These mutants may represent a potent class of antiviral agents that act as “intracellular immunogens.” To facilitate their potential use in animal model systems, we now have studied the duck HBV (DHBV) and placed the DN mutant constructs in recombinant retroviral and adenoviral expression vectors. Transient expression of the DHBV molecular equivalent of the WHV and HBV DN constructs inhibited wild‐type DHBV replication by 98%. Recombinant retroviral and adenoviral vectors containing the HBV and DHBV DN complementary DNAs (cDNAs) were used to transiently and stably transduce hepatoma‐derived cell lines constitutively expressing replicating wild‐type virus. These investigations show that the DN core mutants were powerful inhibitors of HBV and DHBV replication when delivered intracellularly and appear as promising antiviral agents for gene therapy of persistent viral infection of the liver.

HCV RNA in serum of asymptomatic blood donors involved in post-transfusion hepatitis (PTH)

A group of blood donors involved in post-transfusion hepatitis was investigated for the presence of the anti-HCV antibody and of HCV RNA as a more direct infection marker. RNA was extracted from serum, reverse transcribed and amplified using primers which belonged to the non structural region. The amplified product of the PCR reaction was 582 base pairs. Seven (25.9%) of the 27 blood donors examined were found anti-HCV-positive by ELISA; five (71.4%) of these were HCV RNA positive. Among the 20 anti-HCV-negative blood donors, four (20.0%) were HCV RNA positive. ALT levels were below 45 UI/l in 18 donors, while the other nine had ALTs over the limit accepted for transfusion. The anti-HCV-negative HCV RNA-positive blood donors had normal ALTs. Our study offers a direct explanation for the substantial proportion of residual cases of anti-HCV-positive post-transfusion hepatitis and suggests the necessity of creating a register of blood donors who have at some time presented blood enzyme abnormalities and for whom second level investigations such as HCV RNA should be used.

Recent advances in the molecular biology of hepatitis B virus.

Hepatitis B virus (HBV) is an enveloped hepatotropic DNA virus. Acute and chronic HBV infection causes significant liver diseases such as acute hepatis, fulminant hepatitis and chronic active hepatitis that may lead to liver cirrhosis and the development of hepatocellular carcinoma. The use of molecular biological techniques has substantially improved our understanding of the HBV life cycle. In this review, we discuss recent advances that have contributed to a better understanding of HBV biology. Recent studies in the understanding of the life cycle of HBV such as viral entry, replication, transcriptional regulation, viral regulatory proteins, viral assembly and secretion, and nucleic acid based approaches to antiviral therapy will be emphasized. These advances in molecular biology and relationship to clinical disease will be instrumental in developing effective therapeutic approaches for the estimated 300 million individuals worldwide chronically infected with HBV.

Nucleic acid-based antiviral and gene therapy of chronic hepatitis B infection

Persistent hepatitis B virus (HBV) infection often leads to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. There is a need to develop new antiviral approaches for the treatment of this disease. We have explored various nucleic acid‐based strategies designed to inhibit HBV replication including: the use of antisense RNA and DNA constructs, DNA‐based immunization techniques to stimulate broad‐based cellular immune responses with particular emphasis on the generation of cytotoxic lymphocyte (CTL) activity to viral structural proteins, hammerhead ribozymes to cleave HBV pregenomic RNA in vitro and dominant negative HBV core mutant proteins as inhibitors of nucleocapsid formation within cells. In order to optimize these antiviral effects, various novel expression vectors have been developed to deliver such DNA constructs to cells. For example, adenoviral vectors carrying genes that encode for dominant negative proteins have been employed to transfect hepatocytes in vitro and in vivo. In addition, plasmid vectors have been produced to promote expression of HBV structural genes following injection into muscle cells as a means to stimulate the hosts cellular and humoral immune response in the context of histocompatibility antigen (HLA) class I and II antigen presentation. These experimental approaches may have important implications for the generation of efficient antiviral effects during chronic HBV infection.

Conserved core protein sequences in hepatitis B virus infected patients without anti-HBc

The absence of detectable anti-HBc antibodies in some hepatitis B virus (HBV) infected patients may be due to altered core-protein (HBc) sequences. To investigate this possibility we sequenced the pre-C/C-region of HBV isolated from 12 juvenile cancer patients who incurred a nosocomial infection of HBV during chemotherapy but did not develop anti-HBc antibodies or acute cytolytic episodes. The sequences demonstrated the highest sequence homology to the pre-C/C region of a previously cloned HBV genome (subtype ayw) and no deletions or striking mutations were detected. Up to 7 years after infection almost all the survivors developed low titers of anti-HBc antibodies but no clinical signs of hepatic damage. These results suggest that chemotherapy may induce a tolerance status to HBcAg, the most immunogenic HBV protein.

Focal adhesion kinase regulates the DNA damage response and its inhibition radiosensitizes mutant KRAS lung cancer

Purpose: Non–small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide due to the limited availability of effective therapeutic options. For instance, there are no effective strategies for NSCLCs that harbor mutant KRAS, the most commonly mutated oncogene in NSCLC. Thus, our purpose was to make progress toward the generation of a novel therapeutic strategy for NSCLC. Experimental Design: We characterized the effects of suppressing focal adhesion kinase (FAK) by RNA interference (RNAi), CRISPR/CAS9 gene editing or pharmacologic approaches in NSCLC cells and in tumor xenografts. In addition, we tested the effects of suppressing FAK in association with ionizing radiation (IR), a standard-of-care treatment modality. Results: FAK is a critical requirement of mutant KRAS NSCLC cells. With functional experiments, we also found that, in mutant KRAS NSCLC cells, FAK inhibition resulted in persistent DNA damage and susceptibility to exposure to IR. Accordingly, administration of IR to FAK-null tumor xenografts causes a profound antitumor effect in vivo. Conclusions: FAK is a novel regulator of DNA damage repair in mutant KRAS NSCLC and its pharmacologic inhibition leads to radiosensitizing effects that could be beneficial in cancer therapy. Our results provide a framework for the rationale clinical testing of FAK inhibitors in NSCLC patients. Clin Cancer Res; 22(23); 5851–63. ©2016 AACR.

PIAS1-FAK Interaction Promotes the Survival and Progression of Non-Small Cell Lung Cancer

The sequence of genomic alterations acquired by cancer cells during tumor progression and metastasis is poorly understood. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that integrates cytoskeleton remodeling, mitogenic signaling and cell survival. FAK has previously been reported to undergo nuclear localization during cell migration, cell differentiation and apoptosis. However, the mechanism behind FAK nuclear accumulation and its contribution to tumor progression has remained elusive. We report that amplification of FAK and the SUMO E3 ligase PIAS1 gene loci frequently co-occur in non-small cell lung cancer (NSCLC) cells, and that both gene products are enriched in a subset of primary NSCLCs. We demonstrate that endogenous FAK and PIAS1 proteins interact in the cytoplasm and the cell nucleus of NSCLC cells. Ectopic expression of PIAS1 promotes proteolytic cleavage of the FAK C-terminus, focal adhesion maturation and FAK nuclear localization. Silencing of PIAS1 deregulates focal adhesion turnover, increases susceptibility to apoptosis in vitro and impairs tumor xenograft formation in vivo. Nuclear FAK in turn stimulates gene transcription favoring DNA repair, cell metabolism and cytoskeleton regulation. Consistently, ablation of FAK by CRISPR/Cas9 editing, results in basal DNA damage, susceptibility to ionizing radiation and impaired oxidative phosphorylation. Our findings provide insight into a mechanism regulating FAK cytoplasm-nuclear distribution and demonstrate that FAK activity in the nucleus promotes NSCLC survival and progression by increasing cell-ECM interaction and DNA repair regulation.

PIAS1 Promotes Lymphomagenesis through MYC Upregulation

The MYC proto-oncogene is a transcription factor implicated in a broad range of cancers. MYC is regulated by several post-translational modifications including SUMOylation, but the functional impact of this post-translational modification is still unclear. Here, we report that the SUMO E3 ligase PIAS1 SUMOylates MYC. We demonstrate that PIAS1 promotes, in a SUMOylation-dependent manner, MYC phosphorylation at serine 62 and dephosphorylation at threonine 58. These events reduce the MYC turnover, leading to increased transcriptional activity. Furthermore, we find that MYC is SUMOylated in primary B cell lymphomas and that PIAS1 is required for the viability of MYC-dependent B cell lymphoma cells as well as several cancer cell lines of epithelial origin. Finally, Pias1-null mice display endothelial defects reminiscent of Myc-null mice. Taken together, these results indicate that PIAS1 is a positive regulator of MYC.