Hepatitis C virus suppresses Hepatocyte Nuclear Factor 4 alpha, a key regulator of hepatocellular carcinoma

Highlights

• HCV suppresses HNF4α expression in both RNA and protein levels.

• HCV viral proteins reduce HNF4α expression.

• HCV viral proteins downregulate HNF4α promoter activity.

• HCV perturbs HNF4α-related microRNA (miR-24, miR-629 and miR-124) expression.

Abstract

Hepatitis C Virus (HCV) infection presents with a disturbed lipid profile and can evolve to hepatic steatosis and hepatocellular carcinoma (HCC). Hepatocyte Nuclear Factor 4 alpha (HNF4α) is the most abundant transcription factor in the liver, a key regulator of hepatic lipid metabolism and a critical determinant of Epithelial to Mesenchymal Transition and hepatic development. We have previously shown that transient inhibition of HNF4α initiates transformation of immortalized hepatocytes through a feedback loop consisting of miR-24, IL6 receptor (IL6R), STAT3, miR-124 and miR-629, suggesting a central role of HNF4α in HCC. However, the role of HNF4α in Hepatitis C Virus (HCV)-related hepatocarcinoma has not been evaluated and remains controversial. In this study, we provide strong evidence suggesting that HCV downregulates HNF4α expression at both transcriptional and translational levels. The observed decrease of HNF4α expression correlated with the downregulation of its downstream targets, HNF1α and MTP. Ectopic overexpression of HCV proteins also exhibited an inhibitory effect on HNF4α levels. The inhibition of HNF4α expression by HCV appeared to be mediated at transcriptional level as HCV proteins suppressed HNF4α gene promoter activity. HCV also up-regulated IL6R, activated STAT3 protein phosphorylation and altered the expression of acute phase genes. Furthermore, as HCV triggered the loss of HNF4α a consequent change of miR-24, miR-629 or miR-124 was observed. Our findings demonstrated that HCV-related HCC could be mediated through HNF4α-microRNA deregulation implying a possible role of HNF4α in HCV hepatocarcinogenesis. HCV inhibition of HNF4α could be sustained to promote HCC.

Introduction

Hepatitis C Virus (HCV) infection is one of the most common causes of liver disease. HCV patients often develop chronic infection, which is associated with liver inflammation, fibrosis, steatosis, cirrhosis and hepatocellular carcinoma (HCC) (Adinolfi et al., 2005, Negro and Sanyal, 2009). HCV infection presents with a disturbed lipid profile as HCV uses cellular host lipids for entry, replication, virion assembly, secretion, trafficking and escape from host immune surveillance (Popescu et al., 2014, Syed et al., 2010).

HCV possesses a single-stranded, positive sense RNA genome with a nucleotide length of 9.6 kb that encodes a polyprotein which is processed by cellular and viral proteases to yield 10 structural (core, E1, E2 and p7) and non-structural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) (Bartenschlager et al., 2013, Moradpour et al., 2007). An additional HCV protein that is synthesised by a coding sequence that overlaps HCV core protein coding sequence in the +1 reading frame, core +1, has been recently identified (Kotta-Loizou et al., 2015, Vassilaki and Mavromara, 2009). The main role for core protein in the viral life cycle is to form a nucleocapsid to protect the viral genome. Non-structural proteins are believed to comprise the viral replicase complex and mediate all the functions necessary for the replication of HCV viral genome (Lohmann et al., 1999). A considerable number of in vitro studies have suggested that expression of HCV proteins may lead to alterations of lipid metabolism and transport, cell cycle dyregulation and cellular transformation (Hoshida et al., 2014, Popescu et al., 2014). There is strong evidence that HCV proteins, including core and NS5A, are implicated in several oncogenic pathways and contribute to carcinogenesis by modulating metabolic and intracellular signalling processes (Banerjee et al., 2010). In particular, liver expression of HCV proteins in transgenic mice is linked to the development of progressive hepatic steatosis (Lerat et al., 2002, Moriya et al., 1997, Perlemuter et al., 2002) and HCC (Lerat et al., 2002, Moriya et al., 1998). Furthermore, HCV proteins directly and indirectly interact with numerous transcription factors (Banerjee et al., 2010, Tsutsumi et al., 2002) and regulate genes involved in immune phase response (Heim and Thimme, 2014, Lin et al., 2006, Polyak et al., 2001). There is sufficient evidence that HCV infection affects nuclear receptor mediated pathways and leads to problems in hepatic metabolism (Dharancy et al., 2005, Raglow et al., 2011, Wu et al., 2011). Nuclear receptors are major regulators of lipid homeostasis and inflammation; processes intricately involved in HCV infection and progression, and regulate processes essential to the progression of chronic hepatitis C.

Hepatocyte Nuclear Factor 4 alpha (HNF4α) is a member of nuclear receptor superfamily of transcription factors that is indispensable for liver development and hepatocyte differentiation and function. HNF4α is the most abundant transcription factor in the liver and a central regulator of genes involved in hepatic lipid metabolism (Hayhurst et al., 2001). HNF4α functions as master regulator of liver transcriptome by binding directly to promoters of actively transcribed genes (Odom et al., 2004). In addition to its metabolic role, HNF4α is associated with HCC. We have previously shown that HNF4α exerts a key role in hepatocellular carcinogenesis, as transient inhibition of HNF4α in immortalized hepatocytes leads to hepatocellular transformation (Hatziapostolou et al., 2011). We have also established a dynamic relationship between HNF4α and microRNAs, miR-24 and miR-629, that are direct regulators of HNF4α. Moreover, HNF4α regulates the expression of miR-124 that directly targets interleukin 6 receptor (IL6R), thus implicating HNF4α in liver inflammation and liver cancer development. Alterations in HNF4α functionality may increase the risk of development of HCC. Indeed, several mouse and human studies have shown that HNF4α expression is diminished in HCC and its loss may directly contribute to HCC development (Kalkuhl et al., 1996, Lazarevich et al., 2004, Lazarevich and Fleishman, 2008, Ning et al., 2010, Tanaka et al., 2006, Yin et al., 2008). Additionally, it was demonstrated that overexpression of HNF4α in HCC models blocks carcinogenesis and metastasis, thus indicating a potential role for HNF4α as a tumor suppressor (Ning et al., 2010, Yin et al., 2008).

In this study, we provide strong evidence that HCV remarkably decreases HNF4α expression in both protein and RNA levels. HCV proteins, core, NS5A and NS proteins, significantly suppress HNF4α expression and downregulate HNF4α promoter activity indicating regulation of HNF4α at transcriptional level. In addition, HCV leads to overexpression of miR-24 and miR-629 that we have previously shown to downregulate HNF4α expression. HCV also leads to induction of miR-124 expression and activation of IL6R-STAT3 axis. Overall, our data suggest that HCV perturbs HNF4α-microRNA pathway implying a pivotal role of HNF4α in HCV related HCC.