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A dual role for hypoxia inducible factor-1α in the hepatitis C virus lifecycle and hepatoma migration

 

Garrick K. Wilson, Claire L. Brimacombe, Ian A. Rowe, Gary M. Reynolds, Nicola F. Fletcher, Zania Stamataki, Ricky H. Bhogal, Maria Simöes, Margaret Ashcroft, Simon C. Afford, Ragai R, Mitry, Anil Dhawan, Christopher J Mee, Stefan G. Hübscher, Peter Balfe and Jane A. McKeating.

Journal of Hepatology 56:803-809, 2012.

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Synopsis:

Recent advances allowing the assembly of infectious HCV particles in vitro have enabled the complete virus lifecycle to be studied. HCV encodes two glycoproteins E1 and E2 that mediate virus attachment to the host cell receptors tetraspanin CD81 and scavenger receptor class BI (SR-BI). More recently, the tight junction proteins Claudin-1 and Occludin have also been implicated in HCV entry (reviewed here). We previously reported that HCV infection reduced hepatoma polarity reminiscent of epithelial to mesenchymal transition (EMT) (Mee, 2010). Given the many reports detailing aberrant tight junction protein expression and EMT in malignant neoplasms, including HCC, and the knowledge that viruses frequently down regulate expression of their cellular receptors we investigated the effect(s) of HCV infection on hepatoma migration and invasion.

In this study we demonstrate that HCV glycoproteins and virus infection reduce tight junction integrity and E-Cadherin expression, promote the expression of the EMT markers Snail and Twist and enhance hepatoma migration via stabilizing hypoxia inducible factor 1a (HIF-1α), a transcriptional regulator that activates vascular endothelial growth factor (VEGF) and transforming growth factor (TGFβ) expression. We found a role for VEGF and TGFβ in de-regulating hepatoma polarity and promoting the migration of infected cells. Inhibition of HIF-1α reversed the viral effects on hepatoma migration and significantly reduced HCV replication, suggesting a dual role for HIF-1α in deregulating cellular processes associated with tumor growth and in the viral life cycle.

HIF-1α is expressed in many human cancers and is considered a therapeutic target for treating malignant disease of diverse aetiologies. These data provide new insights into a role for HCV in HCC pathogenesis and suggest new approaches for treatment.


Abstract:

Background & Aims: Hepatitis C virus (HCV) causes progressive liver disease and is a major risk factor for the development of hepatocellular carcinoma (HCC). However, the role of infection in HCC pathogenesis is poorly understood. We investigated the effect(s) of HCV infection and viral glycoprotein expression on hepatoma biology to gain insights into the development of HCV associated HCC.

Methods: We assessed the effect(s) of HCV and viral glycoprotein expression on hepatoma polarity, migration and invasion.

Results: HCV glycoproteins perturb tight and adherens junction protein expression, increase hepatoma migration and expression of epithelial to mesenchymal transition markers Snail and Twist via stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α regulates many genes involved in tumor growth and metastasis, including vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β). Neutralization of growth factors show different roles for VEGF and TGF-β in regulating hepatoma polarity and migration, respectively. Importantly, we confirmed these observations in virus infected hepatoma and primary human hepatocytes. Inhibition of HIF-1α reversed the effect(s) of infection and glycoprotein expression on hepatoma permeability and migration and significantly reduced HCV replication, demonstrating a dual role for HIF-1α in the cellular processes that are deregulated in many human cancers and in the viral life cycle.

Conclusions: These data provide new insights into the cancer-promoting effects of HCV infection on HCC migration and offer new approaches for treatment.


figure 1

Figure 1. HCV glycoproteins perturb tight junction protein localization.
Polarized HepG2 cells expressing glycoproteins from HCV strains H77 and JFH-1 or control vesicular stomatitis virus glycoprotein (VSV-G) were stained for tight junction protein localization. Green shows Occludin and Claudin-1 distribution in parental and glycoprotein expressing cells, where cell nuclei are counterstained with DAPI (blue); scale bar represents 20μm.


figure 2

Figure 2. Occludin localization in normal and diseased liver tissue.
Representative immunohistochemical stains of Occludin in normal, early and late stage HCV infected livers (x200), where the arrow shows Occludin at bile canalicular (BC) or basolateral (BM) hepatocyte membranes. The basolateral distribution in all inflamed tissue was significantly higher compared to normal tissue (P < 0.05 Dunn's test).


figure 3

Figure 3. HCV glycoproteins perturb hepatoma polarity, tight junction integrity and stabilize HIF-1α.
(A) Cells were grown for 1, 3 and 5 days and their polarity assessed by measuring MRP-2 (multi-drug resistant protein-2) positive bile canalicular structures/100 nuclei (left) . Cells were allowed to polarize for 5 days and tight junction integrity quantified by enumerating the number of bile canaliculi retaining the fluorescent dye CMFDA (right). (*** = P < 0.001, t test).
(B) HCV glycoproteins and VSV-G stabilize HIF-1α: nuclear HIF-1α (green) was visualized by confocal microscopy; scale bar represents 10μm.


figure 4

Figure 4. Mechanism(s) of HCV glycoproteins modulation of HepG2 polarity.
(A) HCV glycoprotein expressing cells demonstrate increased migration. HIF-1α inhibitor (NSC) reduced migration and restored tight junction integrity indicating an HIF-1α dependent mechanism.
(B) Representative image of HIF-1α distribution in a HCV cirrhotic nodule, arrow heads indicate the nuclear localization of HIF-1α. Box represents a magnified area from the image



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