Activation of PERK-Nrf2 oncogenic signaling promotes Mdm2-mediated Rb degradation in persistently infected HCV culture

www.nature.com/scientificreports OPEN Received: 24 April 2017 Accepted: 26 July 2017 Published: xx xx xxxx Activation of PERK-Nrf2 oncogenic signaling promotes Mdm2mediated Rb degradation in persistently infected HCV culture Yucel Aydin2, Milad Chedid1, Srinivas Chava1, Donkita Danielle Williams1, Shuanghu Liu4, Curt H. Hagedorn5, Suchitra Sumitran-Holgersson6, Krzysztof Reiss7, Krzysztof Moroz1, Hua Lu3, Luis A. Balart2 & Srikanta Dash1,2 The mechanism of how chronic hepatitis C virus (HCV) infection leads to such a high rate of hepatocellular carcinoma (HCC) is unknown. We found that the PERK axis of endoplasmic reticulum (ER) stress elicited prominent nuclear translocation of Nrf2 in 100% of HCV infected hepatocytes. The sustained nuclear translocation of Nrf2 in chronically infected culture induces Mdm2-mediated retinoblastoma protein (Rb) degradation. Silencing PERK and Nrf2 restored Mdm2-mediated Rb degradation, suggesting that sustained activation of PERK/Nrf2 axis creates oncogenic stress in chronically infected HCV culture model. The activation of Nrf2 and its nuclear translocation were prevented by ER-stress and PERK inhibitors, suggesting that PERK axis is involved in the sustained activation of Nrf2 signaling during chronic HCV infection. Furthermore, we show that HCV clearance induced by interferon-α based antiviral normalized the ER-stress response and prevented nuclear translocation of Nrf2, whereas HCV clearance by DAAs combination does neither. In conclusion, we report here a novel mechanism for how sustained activation of PERK axis of ER-stress during chronic HCV infection activates oncogenic Nrf2 signaling that promotes hepatocyte survival and oncogenesis by inducing Mdm2-mediated Rb degradation. Approximately 3% of the world population is infected with hepatitis C virus (HCV)1. The majority of people infected with HCV develop chronic liver disease that often progresses to liver cirrhosis and hepatocellular carcinoma (HCC)2–6. Patients with cirrhosis have an increased risk to develop HCC3, 4. The highly effective DAA based antiviral therapy results in a high cure rate of chronic HCV infection7, 8. Additional versions of highly effective DAA combination therapies are expected to be available in the future, which provides hope that HCV infection can be globally eliminated8. This will require that all infected patients receive early diagnosis and access to antiviral treatment. However, chronically infected individuals who do not receive appropriate care have the highest risk of developing liver cirrhosis and HCC9. Some recently reported clinical studies show that HCV cure using DAA based antiviral therapy decreases HCC risk significantly among patients with advanced liver cirrhosis but does not eliminate the risk completely10–12. The risk of HCC persists for several years after viral cure among patients with advanced liver disease9. The mechanism of how chronic HCV infection causes hepatocellular carcinoma is unknown13, 14. HCV is a positive-stranded RNA virus belonging to the flaviviridae family. The HCV genome is a single-stranded RNA molecule of 9600nts in length. The viral genome is translated in the endoplasmic reticulum into a large polyprotein, which is post-translationally processed by cellular and viral proteases into structural (core, E1 and E2) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B)15, 16. The viral mRNA 1 Department of Pathology and Laboratory Medicine, New Orleans, Louisiana, USA. 2Department of Medicine, Division of Gastroenterology and Hepatology, New Orleans, Louisiana, USA. 3Department of Biochemistry, Tulane University Health Sciences Center, New Orleans, Louisiana, USA. 4Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT, USA. 5Department of Medicine and Genetics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA. 6Laboratory of Transplantation Surgery and Regenerative Medicine, University of Gothenburg, Gothenburg, Sweden. 7School of Medicine, LSU Health Sciences Center, New Orleans, Louisiana, USA. Correspondence and requests for materials should be addressed to S.D. (email: ) SCieNTifiC REPOrtS | 7: 9223 | DOI:10.1038/s41598-017-10087-6 1 www.nature.com/scientificreports/ translation and replication processes occur within the endoplasmic reticulum (ER), a membrane-enclosed organelle specific to eukaryotic cells. Sustained RNA translation and replication in the hepatocytes results in an accumulation of large amounts of viral proteins in the ER, which generates a substantial amount of stress response called ER-stress17, 18. The low level accumulation of misfolded or unassembled proteins in the ER is cleared by ubiquitination and proteosomal degradation pathway called ER-associated degradation (type I). When this mode of protein degradation is not sufficient, the ER initiates a second line of protein degradation mechanism through induction of UPR-mediated autophagy (type II). Chronic ER-stress activates several well-orchestrated cellular transcription programs, called unfolded protein response (UPR), in order to restore cellular homeostasis and prevent cell death19. The UPR is orchestrated by three different cellular transcription factors: protein kinase-like endoplasmic reticulum kinase (PERK), activation of transcription factor 6 (ATF6), and inositol requiring enzyme 1 (IRE1), to maintain ER homeostasis18. Long-term ER-stress induces macroautophagy, and the UPR can regulate expression of autophagy genes and autophagosome formation. We and many other researchers have shown that acute HCV infection induces UPR and autophagy response to promote cell survival20–35. To understand the significance of ER-stress in chronic liver disease and liver cirrhosis, the expression levels of UPR genes were examined using liver biopsies from chronically infected patients plus explant cirrhotic livers with HCC33–35. These findings show that ER-stress persists during chronic liver disease, liver cirrhosis and HCC, suggesting that chronic ER-stress plays a major role in HCC development. The detailed mechanism of how chronic ER-stress induces liver injury and HCC is not fully understood. The hepatic UPR activation during HCV infection is associated with the increased production of reactive oxygen intermediates (ROI) from the mitochondria due to calcium release from the ER22. A number of studies have shown that HCV infection induces oxidative stress through Ca++signaling in the ER23–25. Several HCV proteins including core, E1, E2, NS3, NS4A and NS5A induce oxidative stress and induce cytoprotective genes harboring a short cis-acting sequence, the antioxidant response elements (ARE) in their promoters36. The antioxidant response is mainly mediated by NF-E2 related factor 2 (Nrf2) activation and binding to the ARE elements in the nucleus. The activation of Nrf2 through phosphorylation is mediated by a number of kinases including protein kinase C (PKC), phosph (...truncated)