Our studies show that HCV infection and the viral encoded glycoproteins reduce hepatoma polarity and increase cell migration by stabilizing HIF-1α expression and upregulating downstream effectors, VEGF and TGFβ. Neutralization of both growth factors, or inhibition of HIF-1α, restored the polarity and migratory capacity of infected cells. Furthermore, inhibiting HIF-1α significantly reduced HCV replication in hepatoma cell lines and primary hepatocytes, highlighting a dual role for this transcription factor in the viral life cycle and hepatoma migration.
HIF-1α expression has been reported to associate with EMT, a reversible developmental process where epithelial cells reduce intercellular adhesion and acquire fibroblastoid properties that promote an invasive and metastatic phenotype [12,26]
. EMT plays a major role in the invasive and metastatic potential of human cancers [27,28]
. EMT transcription factors Snail and Twist are expressed in 40–70% of HCCs and associate with adherens junction disruption and poor prognosis [29,30]
. Similarly, ectopic expression of Snail or Twist in hepatoma cell lines enhances their motility and invasiveness [30,31]
. Yang and co-authors reported that increased Twist expression was more frequently observed in HCC associated with HCV infection than with other liver diseases 
. The poor prognosis of HCC is largely due to the invasive nature of the tumor, with frequent intrahepatic and extrahepatic metastases 
. Therapeutic options for patients with HCC are limited. Curative approaches, including surgical resection and liver transplantation are attempted in approximately 30% of patients, however, cancer recurrence following surgery is in the order of 60–70% within 5 years 
. This study provides a potential explanation for the clinical observation that HCV associated HCC is frequently more aggressive 
and highlights a mechanism for HCV to accelerate the malignant process.
Recent reports demonstrate that HCV promotes TGFβ expression and activates p38MAPK, JNK, ERK and NFκB pathways [23,24]
. TGFβ promotes EMT and plays a major role in the dissemination of malignant hepatocytes during HCC progression [33,34]
. Our data showing a role for TGFβ in the increased migratory capacity of HCV infected hepatoma cells, supports a role for HCV in promoting tumor spread rather than a direct role in the oncogenic process per se.
We failed to observe any morphological fibroblast features of infected or HCV glycoprotein expressing hepatoma cells, suggesting a partial de-differentiation process in vitro.
Mazzocca et al.
recently reported that inhibition of TGFβ receptor I kinase blocked HCC growth, supporting a rationale for therapeutic targeting of TGFβ signaling in HCV associated HCC 
Our observation that VEGF and TGFβ perturb tight junction protein localization and function supports our in vivo
observations demonstrating a widespread reorganization of Occludin in the diseased human liver. Benedicto and colleagues reported that HCV glycoproteins associate with Occludin and alter protein trafficking in Huh-7 cells 
. In contrast, we failed to immunoprecipitate Occludin and HCV glycoproteins in a variety of cell lines transduced to express HCV E1E2 or in HCV infected cells (data not shown). Furthermore, VEGF or TGFβ had no detectable effect on the expression or localization of Occludin or other tight junction proteins in Huh-7 cells, suggesting that these cells are refractory to these cytokines 
HCV infection is one of the leading indications for liver transplantation and the number of patients requiring transplantation for chronic hepatitis C is increasing. HCV infects the newly transplanted liver in all cases, leading to a more rapidly progressive disease and frequent graft loss 
. Recurrent HCV is recognized as one of the major challenges facing liver transplantation in the next decade 
. Currently available antiviral treatments are poorly tolerated and have limited efficacy in patients after transplant and new therapies are urgently required. Injury to the liver at the time of transplantation (i.e. ischaemia reperfusion injury, IRI) has been associated with more aggressive recurrent HCV disease 
, however, the factors governing viral replication rate(s) in the newly transplanted liver are poorly understood. Our demonstration that hypoxia, a key event during hepatic IRI, increases virus replication provides a potential explanation for these clinical observations and highlights the potential value of short term anti-oxidant or HIF-1a inhibitor treatment at the time of transplantation to limit HCV replication.
In summary, we have shown a central role for HIF-1α in stimulating VEGF and TGFβ that alters hepatocyte behavior and promotes malignancy in the HCV infected microenvironment. We have also demonstrated a role for HIF-1α in HCV infection. These findings highlight a potential role for HIF-1α inhibitors as therapeutics in patients with both HCC and HCV infection.