Integrated stress response in hepatitis C promotes Nrf2-related chaperone-mediated autophagy: a novel mechanism for host-microbe survival and HCC development in liver cirrhosis

Abstract

The molecular mechanism(s) how liver damage during the chronic hepatitis C virus (HCV) infection evolve into cirrhosis and hepatocellular carcinoma (HCC) is unclear. HCV infects hepatocyte, the major cell types in the liver. During infection, large amounts of viral proteins and RNA replication intermediates accumulate in the endoplasmic reticulum (ER) of the infected hepatocyte, which creates a substantial amount of stress response. Infected hepatocyte activates a different type of stress adaptive mechanisms such as unfolded protein response (UPR), antioxidant response (AR), and the integrated stress response (ISR) to promote virus-host cell survival. The hepatic stress is also amplified by another layer of innate and inflammatory response associated with cellular sensing of virus infection through the production of interferon (IFN) and inflammatory cytokines. The interplay between various types of cellular stress signal leads to different forms of cell death such as apoptosis, necrosis, and autophagy depending on the intensity of the stress and nature of the adaptive cellular response. How do the adaptive cellular responses decode such death programs that promote host-microbe survival leading to the establishment of chronic liver disease? In this review, we discuss how the adaptive cellular response through the Nrf2 pathway that promotes virus and cell survival. Furthermore, we provide a glimpse of novel stress-induced Nrf2 mediated compensatory autophagy mechanisms in virus-cell survival that degrade tumor suppressor gene and activation of oncogenic signaling during HCV infection. Based on these facts, we hypothesize that the balance between hepatic stress, inflammation and different types of cell death determines liver disease progression outcomes. We propose that a more nuanced understanding of virus-host interactions under excessive cellular stress may provide an answer to the fundamental questions why some individuals with chronic HCV infection remain at risk of developing cirrhosis, cancer and some do not.

1. Introduction

Hepatitis C is a blood-borne viral pathogen that infects the liver [1,2] explicitly. Most of the individuals infected with HCV fail to clear the infection naturally, leading to a stage of chronic infection. The long-lasting liver inflammation due to chronic HCV infection causes the onset of advanced liver diseases, such as liver fibrosis, cirrhosis, and HCC resulting in death [3]. HCV infection is one of the leading causes of liver transplantation in many parts of the world. The cloning and sequencing of the HCV genome in 1989, has enabled swift progress in many areas of research that lead to a viral cure (Figure 1). The recent development of a combination of direct-acting antivirals (DAAs) targeting the NS3/4A protease, NS5B polymerase, and NS5A has rapidly changed the therapeutic landscape for curing HCV infection [4,5]. Treatments for chronic HCV infection incorporating an improved version of DAAs with lower costs are expected to be available in the future in many developing countries with high infection rates, providing hope for the global eradication of HCV infection. This progress offers encouragement for HCV infected patients for the potential of being cured with antiviral therapy. However, HCV eradication will require that all infected patients receive early diagnosis and access to antiviral treatments.

Figure 1:

Overview of the rapid progress made in HCV research that leads to significant breakthroughs in antiviral development and viral cure. HCV was cloned in 1989 by a group of researchers at Chiron Corporation, CA. The cloning and sequencing of the HCV genome have facilitated basic research on this virus, which allows a detailed understanding of its genome organization, development of infectious cDNA clones, in vitro cell culture and direct acting antivirals (DAAs) and viral cure.

In the future, HCV cure by DAA therapy is expected to reduce the incidence of chronic liver disease, liver cirrhosis, HCC and therefore will decrease liver-related mortality [6-8]. This conclusion is based on the results of many recent clinical studies indicating that the majority of patients after HCV cure by DAA therapy showed reduced liver inflammation and reverse liver fibrosis [9-14]. However, only a minority of patients is unable to experience the benefit of a viral cure on the reversal of cirrhosis, and they do not show a reduction in their liver inflammation after HCV cure [15-19]. The liver cirrhotic condition in these patients remains stable or become worse because these patients show persistent liver enzyme elevation even after curing HCV infection. Some of those patients with persistent liver enzyme elevation after HCV cure have underlying liver damage related to alcoholic and non-alcoholic fatty liver etiologies [20-25]. Based on these results, and some emerging data, it is recognized that HCV cure may not eliminate the risk of HCC development among patients who have liver cirrhosis before initiation of the antiviral therapy [26-32]. Although, the exact reason why some patients with advanced liver disease or cirrhosis develop HCC after viral clearance is not unknown. A better understanding of the mechanism(s) by which residual viral induced cellular stress factors contribute to the progression of non-viral liver cirrhosis should allow novel therapeutic interventions for the prevention of cirrhosis and HCC. Furthermore, this knowledge will be of great interest to hepatologists since non-infectious metabolic conditions such as alcoholism and non-alcoholic steatohepatitis (NASH) also expose humans to advanced liver disease, including cirrhosis and HCC [33-36].

We have been studying how chronic HCV infection induces ER stress and autophagy process for cell survival and liver disease progression [37-48]. The ER in the single large organelle plays a crucial role in maintaining hepatocyte function, including protein, lipids, and carbohydrate metabolisms [49-52]. HCV is a positive-stranded RNA virus that replicates in the cytoplasm. The viral protein accumulation and replication of HCV cause massive rearrangement of ER membranes [53]. For these reasons, the expression of ER stress chaperones and UPR gene increased during chronic HCV infection. Accumulating evidence indicates that the increased cellular stress response is associated with the development of many other human diseases such as neurodegeneration, type II diabetes, and cancer [54]. The purpose of this review is to compile our recent works as well as the work of other researchers supporting how the stress response is associated with HCV infection promoted cell survival program, and HCC progression. We begin with an introduction to the endoplasmic reticulum (ER), the single most crucial endomembrane system that handles different types of hepatic stress response during chronic HCV infection.

2. The importance of endoplasmic reticulum (ER) function in liver homeostasis:

The liver is one of the most important organs that perform many vital metabolic, synthetic, and secretory functions as well as filter and detoxify the blood in the human body [55]. For example, hepatocytes synthesize the majority of plasma proteins, including albumin, hormones, apolipoproteins, as well as clotting factors needed for blood coagulation. The hepatocytes in the liver play a central role in carbohydrate and lipid metabolism. The translation of most of the secreted proteins starts in the cytosolic ribosomes attached to the ER membrane. The ER is an essential organelle that plays a significant role in the production, processing, and transport of proteins and lipids. The hepatocytes are specialized cells in the liver with an elaborate ER network that covers the entire cytoplasm. The ER transports many essential proteins and fats for many other cell organelles such as lysosomes, mitochondria, secretory vesicles, the Golgi apparatus, endosome, and the cell membrane, therefore, plays an indispensable role in maintaining liver homeostasis. The progress made during the last decade, determining the super-resolution structure of this organelle has radically changed our knowledge on how ER coordinates multi-organelle functions for cell survival [56]. The ER is comprised of two distinct structures: the nuclear envelope and the peripheral ER. The ER-derived nuclear envelop protects the cell nucleus. The outer ER branches out with a series of cisternal sheets around the core nucleus, which then extends throughout the cytoplasm with a tubular structure. The cisternal leaves with ribosomes called the rough ER. The rough ER is arranged like a parking garage around the nucleus allowing an efficient packing of a maximum number of membrane sheets with more ribosomes for dynamic protein translation, translocation, and post-translational modification. In contrast, the tubular structures that are devoid of ribosomes are called smooth ER. The ER tubules have high-curvature due to the presence of specific sets of proteins called reticulons [57,58]. The highly curved ER tubules spread throughout the cytoplasm like a subway reaching various organelles through multiple contact sites [59]. The rough and smooth ER are interconnected so that the proteins made by the rough ER can move into the smooth ER then transferred to other locations. A wealth of literature implicates many positive-stranded RNA viruses, including HCV, uses rough ER as their favorite site for viral RNA replication.

The proteins synthesized in the ER must be correctly folded and undergo post-translational modifications such as glycosylation, disulfide bridge formation, and oligomerization. All of these processes take place in the ER. The increase in cellular energy demand due to persistent virus infection imposes all types of caloric restriction, leading to depletion in ATP, sugar, and fatty acid levels that triggers cellular autophagy. Viral replication decreases oxygen supply (hypoxia) and creates oxidative stress that generates reactive oxygen species (ROS), which can cause endoplasmic reticulum stress. Persistent virus replication increases cellular DNA damage response, a frequency of DNA repair, transcriptional fidelity, and genomic instability, which create additional cellular stress [60]. Besides, the hepatic innate immune system detects the infection using a microbial sensing mechanism called the pattern recognition receptors (PRRs) [61]. These receptors recognize conserved microbial structures, which are distinct from the host nucleic acids due to long and short double-stranded RNA and RNAs containing 5′-triphosphate. Detection of viral pathogens by the innate immune system has two significant consequences: first, it leads to the induction of the natural antiviral mechanisms through the production of type-I interferons (IFNs). Second, it leads to the activation of the adaptive immune response, which provides a more directed, antigen-specific, and long-lasting antiviral immunity. These activities contribute to the cellular stress response called the innate and inflammatory stress. Infected hepatocytes accumulate a multi-faceted stress response in a coordinated fashion in addition to virus-associated ER stress. Infected hepatocytes can manage the stress response through the induction of integrated stress response (ISR) that promotes the transcription of numerous genes for cell survival. Otherwise, unresolved stress could lead to cell death (Figure 2). The stress signal also changes the expression of cell surface proteins; their composition also secretes vesicles outside to support cell survival. When cell signaling cascade reprograms switches cell death to cell survival, it will abet the emergence of malignancies like liver cancer. Overwhelming the natural defense of the liver through various gene regulation compromise its function, leading to severe liver disease such as fibrosis, cirrhosis, and eventually to HCC. In the following sections, we will review various types of cellular stress generated during chronic HCV infection and how the infected hepatocytes promote their cell survival programs under the ISR.

Figure 2:

Hepatitis C virus infection of hepatocyte leads to the activation of diverse stress response in the endoplasmic reticulum of infected hepatocytes. Hepatocyte activates an adaptive response to deal with the stress response, called the integrative stress response (ISR) to improve cell survival. The cell survival program activated during ISR plays an essential role in chronic HCV infection and liver disease outcome.

3. Hepatic ER stress/ UPR response in chronic HCV infection:

The replication cycle of HCV spans through six different stages with the assistance from the ER: entry, translation of viral proteins, replication of virus genome, assembly, packaging of virus particles with the ER membrane and exocytosis. Some of these processes may need help from other organelles, but ER is involved in all stages of the virus replication cycle. The continuous cycle of intracellular virus replication, virus release, and re-infection is central to mechanisms of HCV persistence and pathogenesis. After initial infection, HCV replicates in hepatocytes, the predominant cell type in the liver, producing millions of new virus particles. In infected cells, the cellular translation machinery is used extensively to produce a large amount of viral structural and non-structural proteins [62-64]. The newly synthesized viral proteins assemble in the ER membrane vesicles to reproduce HCV genomic RNA. Multiple rounds of viral replication lead to increased accumulation of positive-strand HCV RNA, replicative intermediates (negative-strand RNA) and many viral proteins in the ER. The accumulation of viral products results in the proliferation and remodeling of the ER membranes. When the viral protein load in the lumen exceeds the capacity of the ER, this leads to a stress response called ER-stress. Mammalian cells generate a cytoprotective gene transcription cascade in the nucleus called the unfolded protein response (UPR) to handle the severe stress response [65-67]. There are three classes of cytoprotective ER-stress signaling: PERK, ATF6, and IRE1 (Figure 3). Mechanistically, the adaptive UPR signaling restores ER homeostasis at multiple levels: (i). increased expression of ER-resident chaperones; (ii). increased production of an enzyme involved in protein folding; (iii). promote the ER-associated degradation (ERAD) of the misfolded proteins and mRNAs; (iv). inhibits protein synthesis to decrease the protein load in the ER; (v). expand the size of ER membranes to accommodate more proteins. In normal conditions, the ER luminal-glucose related proteins (GRP78 is also known as BiP) minimize the ER signaling by binding and inactivating three branches of ER-stress transducers (PERK; IRE1 and ATF6) [68-70]. When sufficient viral proteins accumulate in the ER lumen, GRP78 is titrated away by binding to the misfolded viral proteins triggering the activation of PERK, IRE1, and ATF6 signaling. The first ER stress sensor, PERK, phosphorylates eukaryotic initiation factor-2alpha (eIF2α) at Ser51, preventing the exchange of GDP and GTP, effectively blocking the initiation of mRNA translation by preventing 80S ribosome assembly from recovering from ER stress. The activation of PERK favors increased translation of ATF4 under ER stress [71]. The ATF4 plays a crucial role in the cellular adaptation to the stress response by inducing transcription of C/EBP-homologous protein (CHOP), and GADD34 to trigger apoptosis upon prolonged activation of PERK [72-74]. The activation of the second sensor, ATF6 leads to the transcription of numerous genes involved in protein folding and protein degradation to reduce the stress response [75,76]. In mammals, the ATF6 present in two isoforms, ATF6α (670 amino acids) and ATF6β (703 amino acids). ATF6α is the predominant isoform that regulates expression of most of the ER stress-responsive genes. In normal conditions, ATF6 retained within the ER as disulfide-linked oligomers attached to the ER chaperone, BiP (77). During ER stress, the disulfide bonds reduced to a monomeric form that allows ATF6 trafficking to the Golgi and proteolytic processing by site-1 and site-2 proteases (S1P and S2P). Both the isoforms undergo processing in the Golgi. Protein disulfide isomerase (PDI) is implicated in the reduction of the ATF6 disulfide bond and generation of monomeric ATF6. The C-terminal of ATF6 isoforms protrudes into the ER lumen, whereas the N-terminal face the cytosol. The proteolytic cleavage releases the cytosolic, N-terminal ATF6 transcription factor domain that localizes to the nucleus, homo- or heterodimerizes, and promotes ATF6-regulated transcriptional activity. Both ATF6α and ATF6β are cleaved by the site-1 and site-2 protease, liberating the 50kDa amino-terminal cytoplasmic fragment (ATF6f) [78,79]. The two ATF6 isoforms have significant sequence homology, ATF6α is a potent transcriptional inducer compared to ATF6β [77]. The functional implications of ATF6β activity remain poorly defined. The ATF6β isoform acts as an endogenous repressor that fine-tunes the strength and duration of ATF6 signaling during ER stress. This ATF6f transcription factor enters the nucleus and binds to ER stress-response elements [80]. The cleaved version of ATF6f homodimerizes and binds to promoter regions of target genes containing canonical ATF6 binding motifs, such as the ER stress element (ERSE). The ATF6f induces expression of chaperone genes GRP78 (BiP), GRP94, GRP170, GRP75, and other heat-shock protein families (e.g., Hsc70) and X-box binding protein 1 (XBP1) [81]. ATF6 signaling integrates with additional stress signaling to adapt cellular ER stress. The ATF6f heterodimerizes with XBP1s to induce expression of genes involved in ER-associated degradation (ERAD). The third sensor, IRE1, is the most conserved ER stress sensor that has both serine/threonine kinase and endoribonuclease activities [82]. In response to ER, activated IRE1 by autophosphorylation, dimerization, and oligomerization leads unconventional processing of the mRNA encoding the transcriptional factor XBP1 [83-85]. A 26-nucleotide intron of the XBP1 mRNA is spliced out by IRE1a, leading to a shift in the codon reading frame of the mRNAs that generate new protein (XBPs) with potent transactivation domain. This transcription factor enters the nucleus and induces transcription of UPR-related genes involved in protein folding, protein entry to the ER, and ER-associated degradation (ERAD) [82,86]. In the liver, stress-induced XBPs indirectly regulates the biosynthesis of the ER and Golgi by enhancing the production enzymes involved in phospholipid synthesis [87,88]. The activated IRE1a targets the degradation of a subset of cellular mRNAs via a process called regulated IRE1-dependent decay (RIDD) [89,90]. For example, when IRE1a exists as dimer/tetramer, its RNase activity is largely restricted to XBP splicing, but when the stress level is high the oligomerization of IRE1 can expand its RNase activities to many localized mRNAs, ribosomal RNA, and microRNAs having a consensus sequence, CUGCAG with a stem-loop structure [91]. The activation of IRE1 RNase activity causes resistance to apoptosis during HCV infection, a progression of fibrosis through the degradation of selected microRNAs and promotes carcinogenesis through the production of pro-tumorigenic growth factors [92-94]. The expression of ER stress/UPR response has been confirmed in human liver tissue samples from infected patients, suggesting that stress response persists during chronic HCV infection and plays an important role in liver disease progression.

Figure 3:

Schematic representation of unfolded protein response (UPR) associated with ER stress in HCV infection. The three ER stress sensors, protein kinase RNA (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring protein-1 (IRE1), enable gene expression that is involved in reducing ER-stress through a wide variety of mechanisms. PERK phosphorylates eukaryotic translation initiation factor 2A (eIF2α) to decrease translation while it activates activating transcription factor 4 (ATF4). ATF4 translocates into the nucleus to initiate transcription of genes involved in cellular autophagy. ATF6 monomers traffic to the Golgi where they are cleaved by S1P and S2P. The active form of N-terminal ATF6 translocates to the nucleus to activate genes involved in protein degradation and protein folding. The IRE1 decreases protein loads in the ER by enhancing mRNA degradation. The IRE1 axis also generates spliced versions of XBP1, which translocate to the nucleus to induce genes involved for protein degradation, lipogenesis, and inflammation.

4. Chronic HCV infection induces oxidative stress response:

Chronic HCV infection creates an imbalance in the cellular detoxification mechanism of reactive oxygen species (ROS) leading to increased oxidation of essential biological molecules such as proteins, lipids, and nucleic acids, leading to a stress response called oxidative stress (OS) [95-97]. Increased oxidative stress means an accumulation of more ROS such as hydrogen peroxide (H2O2), superoxide anion (O2-), and hydroxyl radicals (HO) in the infected cells. Hepatocytes in the liver contain the highest density of peroxisomes to regulate lipid metabolism, and detoxification as well as synthesis of phospholipids, bile acids, and cholesterol [98]. This organelle contains the highest level of peroxide-consuming enzyme (peroxidase) activity; therefore, it is called peroxisome. Peroxisomes are involved in the metabolisms of long-chain fatty acids and branched fatty acids that cannot be broken down in the mitochondria. Several peroxisomal enzymes produce reactive oxygen or nitrogen as a byproduct of metabolism including acyl-CoA oxidase, L-pipecolic acid oxidase, L-a-hydroxy acid oxidase, and xanthine oxidase, all of which generate H202 in oxidizing their substrates [99]. The ER connects to peroxisomes and mitochondria through a tubular network by multiple contact sites (MCS) [59]. During HCV infection, many different enzymes present in the mitochondria, ER, and peroxisomes generate ROS. For example, ROS may be produced from mitochondria by oxidative phosphorylation or the activation of NADPH oxidase, xanthine oxidase, cyclooxygenase, and lipoxygenase [100]. Several groups have shown that HCV replication in the ER membrane of hepatocytes triggers protein oxidation that results in the accumulation of ROS, leading to oxidative stress using HCV infected liver culture [101-112]. Other researchers have confirmed these findings and showed that markers of oxidative stress were increased in liver tissue of patients chronically infected with HCV, indicating that ROS levels increased during chronic HCV infection [110-112]. Studies have shown that almost all HCV proteins can trigger oxidative stress, but HCV core found to be a potent inducer of oxidative stress in infected liver cell culture. One recent study showed that HCV RNA sensing by inflammatory cells could trigger activation of inflammasome and production of inflammatory cytokine, IL-1b secretion [106]. Some studies showed that core and NS5A expressing cells generate ROS and reactive nitrogen species (RNS) such as NO and peroxynitrite free radicals [113]. Like ROS, RNS production can have detrimental effects on cell survival. A few recent studies have demonstrated that alteration at the mitochondria-associated ER membranes causes a release of Ca2+ from the ER during HCV infection. The sustained calcium release ensures progressive mitochondrial dysfunction leading to the generation of reactive oxygen and nitrogen species and a progressive metabolic adaptive response consisting of decreased oxidative phosphorylation and enhanced aerobic glycolysis and lipogenesis [114]. The adaptive cellular response to oxidative stress results in the expression of antioxidant enzymes that eliminate ROS and free radicals. The Nrf2 (nuclear factor erythroid two related factors 2) is the major antioxidant transcription factor implicated in the transcription of numerous genes involved in the antioxidative response. We found that oxidative stress was significantly high in HCV infected culture compared to uninfected Huh-7.5 cells leading to the activation of Nrf2 signaling [47,48].

5. Hepatic stress response due to innate sensing of the HCV:

The liver constituted with many cell types including hepatocytes, bile duct epithelial cells, Kupffer cells (hepatic macrophages), perisinusoidal cells consists of stellate cells and pit cells. The liver resident immune cells (Kupffer cells/ macrophages) play an essential role in the induction of innate and adaptive immune response against varieties of dietary and microbial products from the gut. The liver frequently exposed to a wide range of bacterial products, environmental toxins, and food antigens. Hepatocytes are the dominant cell type that provides protective immunity in the liver, which produces 80-90% of the innate immunity proteins in the body [115]. Hepatocytes recognize cellular and microbial products through two classes of molecules: pathogen-associated molecular patterns (PAMP), which are associated with microbial pathogens and damage-associated molecular patterns (DAMP), which are components of host cells released during cell damage or death. These molecules are called primitive pattern recognition receptors (PRR) because they evolved before the adaptive immunity. They play an essential role in the event of an early innate immune response against microbial pathogens. Monocytes, macrophages and non-immune cells such as hepatocytes usually sense RNA virus through RIG-I-like receptors (RLR) or Toll-like receptors (TLR) 3, whereas TLR 7 is the primary RNA sensor in plasmacytoid dendritic cells (PDC) [116]. The innate immune response in the infected hepatocyte gets activated as a result of direct sensing of HCV through RLR and TLR3 pathways, whereas the natural immune response in dendritic cells gets activated through TLR7. Both hepatic and non-hepatic innate immune recognition events lead to the production IFN and proinflammatory cytokines [117]. During HCV infection, dsRNAs and 5'-triphosphate containing single-stranded HCV RNAs are recognized by MDA5 and RIG-1, respectively [118]. These two DEAD-box helicases signal through the adaptor protein mitochondrial antiviral signaling protein (MAVS) to promote the transcription of antiviral and pro-inflammatory cytokines [119-121]. Activated MAVS, in turn, recruits tumor necrosis factor receptor-associated factor 2 (TRAF2), TRAF5 or TRAF6, which are ubiquitin ligases that signal to inhibitor of nuclear factor-κB (NF-κB) kinase (IKK) complex proteins and to TANK-binding kinase 1 (TBK1); this ultimately results in the activation of the transcription factors NF-κB and IFN-regulatory factor 3 (IRF3), respectively. Activated NF-κB and IRF3 translocate to the nucleus, where they activate the transcription of genes that encode type I IFNs and other cytokines that orchestrate the antiviral immune response. The same RNA ligands signaling can activate PKR in the infected cells simultaneously. Indeed, transfection of cells with polyinosinic:polycytidylic acid (poly(I:C)) or infection with mutant viruses that are unable to suppress dsRNA recognition demonstrates that elF2α phosphorylation, stress granule formation, nuclear translocation of IRF3 and the transcription of type I IFNs occur in parallel [122]. Most notably, PKR, RIG-I, MDA5, TRIM25, and TRAF2 are recruited to stress granules in response to eIF2α phosphorylation [123-125]. One of the antiviral responses triggered by PKR activation is the inhibition of cellular translation through eIF2α phosphorylation. The untranslated mRNAs packaged into RNA cytoplasmic granules called processing bodies (P-bodies). These RNA granules are present in a low number in normal growth conditions, but they increase in size and number in response to virus infection. In addition to mRNAs, P-bodies are enriched in mRNA decay machinery that results in mRNA degradation during high stress [126]. The mechanism detailing how P-bodies formation leads to translation attenuation and mRNA degradation occurs during chronic HCV infection has been described nicely in several review articles [126,127]. The number of stress granules is increased during HCV infection, suggesting that they are as a part of the cellular response to stress generated during the virus infection [128].

6. Hepatic stress response due to inflammation:

Inflammasome activation has been proposed as a significant contributor to immune cell activation and amplification of inflammatory reaction [129]. The inflammasome activation occurs due to a combination of microbial (PAMP) and non-microbial signals released by dead cells (DAMP). The DAMP can be intracellular proteins (heat-shock protein or chromatin-associated high-mobility group box 1, HMGB1) and non-protein substances (ATP, uric acid, and cellular DNA). Inflammasomes are multiprotein complexes that sense danger signals via nucleotide-binding oligomerization domain receptors (NOD-like receptors, NLR) leading to the secretion of cytokines such as IL-1b and IL-18 through activation of the serine protease caspase-1 (CASP-1) [130]. HCV can activate the inflammasome complex in monocytes and macrophages and induce expression of pro-inflammatory cytokine production that contributes to the progression of HCV-induced liver fibrosis [131-133]. Direct activation of inflammasome can also occur after HCV infection using Huh-7.5 liver cells suggesting that multiple cell types present in the liver microenvironment could activate inflammasome and contribute to inflammation during chronic HCV infection [134]. Different branches of the UPR, particularly IRE1a and XBP1, could cross talk with microbe sensing pathways RLRs and PAMP, which could amplify inflammatory cytokines production and IFN response [135-138]. There is not much work that has been done showing the synergistic interaction of UPR and PPR pathways in the HCV infection model. Based on these publications, we can speculate that such synergistic interaction is possible between innate sensing of HCV infection and cellular UPR activation.

7. Integrative stress response (ISR) promotes cell survival by promoting cap-independent translation:

Numerous abiotic agents such as excess fat, alcohol, drugs perturb ER homeostasis during chronic HCV infection. Hepatocytes activate an elaborative cell-signaling pathway in response to the combination of viral (biotic) and non-viral (abiotic) stress called the integrative stress response (ISR) to promote cell survival [139]. The ISR measured by the level of phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 alpha subunit on amino acid serine 52. We published data showing that increased expression of stress kinases, chaperones (Hsc70, GRP78) and increased expression of the phosphorylated eIF2alpha subunit in the chronically infected liver and liver cirrhosis tissue samples derived from patients with HCV [42,45]. The expressions of stress chaperones are high in the cirrhotic liver tissues as compared to normal uninfected liver tissues (Figure 4). There are four members of the family of eIF2 alpha kinases sensing different cellular stress: PKR-like ER kinase (PERK), double-stranded RNA-dependent protein kinase (PKR), heme-regulated eIF2a kinase (HRI) and general control non-derepressible 2 (GCN2). All these four kinases phosphorylate eIF2alpha in response to wide varieties of cellular stress (ATP depletion, glucose deprivation, amino acid deprivation, ribosomal stress, growth factor deprivation, stress granules accumulation, oxidative stress, nitric oxide, hypoxia, heat shocks, and osmotic stress) [139-147]. For example, the activation of PERK occurs in response to glucose deprivation and ATP depletion during virus infection. The mammalian PKR becomes activated by double-stranded RNA (dsRNA), oxidative stress, growth factor deprivation, ribosomal stress, and stress granules accumulation [146]. The heme-regulated eIF2α kinase (HRI) becomes activated due to iron deficiency, oxidative stress, heat shock, osmotic stress, and nitric oxide accumulation. GCN2 becomes activated in response to the depletion of certain amino acids [142]. One of the mechanisms of cell survival under ISR is to reduce stress through inhibition in global protein translation (Figure 5). During this process, the dephosphorylation of the eIF2α signal is central to ISR termination to restore protein synthesis [148,149]. The protein phosphatase 1 (PP1), which is a complex of two regulatory subunits (PPP1R15A also called growth arrest and DNA-damage-inducible protein, GADD34, and PPP1R15B, a constitutive repressor of eIF2α phosphorylation called CRP). During the ISR, phosphorylation of eIF2α subunit of eIF2 complex inhibits eIF2B-mediated exchange of eIF2-GDP to eIF2-GTP and therefore reduces the formation eIF2-GTP-methionyl-initiator tRNA ternary complex formation. The inhibition of ternary complex (eIF2-GTP-tRNAMet) formation results in polysome disassembly and inhibition of cap-dependent mRNA translation [150]. However, certain mRNAs such as ATF4, ATF5, CHOP, and GADD34 that contain a short upstream open reading frame (uORF) in their 5' untranslated region (5'UTR) preferentially translated in the presence of ISR [139]. Most of the ISR-induced proteins are translated from mRNAs and harbor a series of uORFs in the 5' untranslated region that limits the ribosome access to the main coding sequence (CDS) [151-153]. Under normal conditions, ATF4 protein is produced a low level due to eIF2α-mediated translation control. For example, the high concentration of ternary complex available in normal cells, initiate ribosome scanning at the upstream open reading frame uORF1 of ATF4 transcript and quickly re-initiate at uORF2. The uORF2 overlaps with out-of-frame with ATF4 main coding sequences (CDS) therefore prevent translation of functional ATF4 protein. During stressed conditions, low availability of ternary complex leads to a longer ribosomal scanning along with the ATF4 mRNA enabling re-initiation of ATF4 CDS (Figure 5). A recent study by Stark et al. shows that ISR protects BiP (Grp78) and Hsc70 mRNAs from translational shutdown upon eIF2α phosphorylation [154]. Another recent study by Zhou et al. showed that RNA methylation such as N6-methyladenosine (M6) could promote translation independent of cap-dependent translation through leaky ribosome scanning [155]. The translation of c-Myc is increased in cancer cells due to extreme ER stress through cap-independent IRES-mediated translation [156,157]. These data indicate that eIF2α phosphorylation is required for the expression of selected mRNAs needed for cell survival. How translation of certain mRNA occurs under severe stress is unknown, remains an active area of research. A recent review claims that cells employ a variety of mechanisms to achieve selective translation under stress utilizing cis-acting sequence elements on the mRNAs and trans-regulatory factors recognizing the mRNA features [158]. The cis-elements present in the untranslated region of the mRNAs include the internal ribosome entry sites (IRES), upstream open reading frames (uORFs), motifs with special sequences, secondary structures, and microRNA binding sites. One such example is the translation of ATF4, Hsc70, and BiP (GRP78) under stress. These results suggest that their many additional mechanisms are utilized to translate proteins needed for cell survival under stress. A better understanding of the translational program under stress may allow the development of novel therapies for human disease related to increased stress.

Figure 4:

Shows the expression of ER-stress chaperones (GRP78, BiP), Hsc70 proteins in HCV infected cirrhotic liver as compared to uninfected liver determined by immunostaining. Both proteins are known to be translated through cap-independent translation under ISR.

Figure 5:

Different stress signals during HCV infection activates PKR, GCN2, PERK and HRI kinases that stimulate phosphorylation of eIF2α the core element of the stress response that inhibits cellular translation. Under normal conditions with low levels of eIF2α promotes mobile cap-dependent translation. During the ISR cellular translation is attenuated due to increased eIF2α phosphorylation, which supports the translation of specific genes needed to cell survival. Bottom panel shows an example of ATF4 mRNA translation by upstream open reading frame (uORF1 and uORF2). In normal condition, ATF4 translation is inhibited by uORF2-mediated frameshift mechanism. Under stress condition, frameshift mechanism is inhibited leading to ATF4 translation at the open reading frame.

8. Integrative stress response promotes cellular autophagy to improve cell survival:

Autophagy is a catabolic process that generates energy through the lysosomal degradation of cytoplasmic organelles in the autolysosomes [159]. If the nutrient supply is low only for a short period, then the UPR activates ubiquitin and proteasome-mediated degradation to generate energy, which is called ER-assisted degradation (ERAD). If the nutrient supply is low for an extended period during chronic viral infection, the UPR induces cellular autophagy process to generate energy through lysosomal degradation [160]. A multi-layered control system regulates cellular autophagy. There are three types of autophagy described in mammalian cells (Figure 6). 1. Chaperone-mediated autophagy (CMA): It causes the silent degradation of cytosolic proteins with a consensus pentapeptide motif (KFERQ) in the lysosome. Cytosolic proteins with this motif are selectively binds to Hsc70 chaperone and delivered proteins across the lysosome membrane for degradation. This type of autophagy is highly specific because the target protein first forms a complex with Hsc70 and then binds to the LAMP2A receptor expressed in the lysosome surface for the transport of a protein across the lysosomal membrane and subsequent degradation. 2. Microautophagy: It causes direct engulfment of nearby cytosolic materials or protein cargoes into endosome or lysosome via direct inward membrane rearrangement. 3. Macroautophagy referred to as autophagy. It covers a significant portion of cellular protection under normal conditions. During the process of macroautophagy, a part of the cytosol sequestered in a double-membrane structure called the autophagosome, which fuses with the endosome and lysosome for degradation. The initiation and termination of autophagy linked to cellular nutrient sensing mechanisms [161]. The molecule AMP-kinase (AMPK) senses mobile energy requirements through AMP to ATP ratios in the cell cytoplasm. High AMP levels reflect low energy states in the cell, and under these conditions, AMPK can initiate autophagy through inactivation of mTOR1 (a mechanistic target of rapamycin complex 1) or by phosphorylation of ULK1/2 protein. Autophagy activation follows several steps as initiation, nucleation, elongation, maturation, and degradation (Figure 7).

Figure 6:

Schematic illustrations of different types of autophagy. (A). In macroautophagy the cargo targeted for degradation is enclosed in a double membrane vesicle called autophagosome that delivers the contents to the interior of lysosome through membrane fusion. The metabolites: amino acids, sugars, and lipids are then released into the cytoplasm for the synthesis of new macromolecules or as a source of energy. (B). In microautophagy, the cargo directly enters the lysosome by membrane invagination that leads to its degradation. (C). In CMA, proteins with KFERQ-like sequences are recognized by the Hsc70 chaperone. This complex then binds to LAMP2A on the lysosome membrane for subsequent internalization and degradation.

Figure 7:

Molecular interactions involved in macroautophagy. Activation of UPR due to HCV initiates autophagy. Autophagy responses are also launched due to low energy conditions by AMP-activated kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTOR1). The phosphorylation of the ULK complex can be initiated by AMPK activation due to low ATP or mTOR inhibition due to low nutrients sensing. Activation of the ULK complex begins a series of reactions that lead to the engulfment of the cellular constituents in a double-membrane structure called autophagosome. The autophagosome then fuses with a lysosome to form autolysosome in which lysosomal enzymes digest the contents into essential nutrients (sugars, lipids, amino acids, and nucleosides), which are released into the cytoplasm for subsequent use.

The first step of autophagy initiation occurs due to a decrease in mTOR activity that allows phosphorylation, translocation and subsequent interaction of the four ULK protein complex (ULK1–ATG13–FIP200–ATG101) with ER-resident proteins [162]. The second step, nucleation, occurs when the ULK complex becomes large due to the additional interactions with the class III phosphatidylinositol 3-kinase (PI3K) compounds consisting of either Beclin 1-ATG14L-PI3KCIII-p150-Ambra1 or Beclin 1-UVRAG-PI3KCIII-p150-Bif1 [163,164] with ER-resident proteins (DFCPI and WIPI). This process, also called phagophore formation, usually starts with a curved double-membrane structure derived from the ER. The third step is called elongation in which the two edges of the phagophore membrane are extended by ATG7-dependent ubiquitin-like protein conjugation systems that catalyze the covalent linkage of the ATG12-ATG5 and ATG16 L1 for membrane expansion [165]. The ubiquitination-like modification is also required for LC3 conjugation to phosphatidylethanolamine (PE), thus converting LC3B-1 to LC3-II [166]. The formation of LC3-II is increased in cells carrying out autophagy. Therefore, LC3-I/LC3-II ratio measurement is used to detect increased autophagy. The LC3 family is required for phagophore expansion, closure, and cargo recruitment. During elongation, the LC3 protein on the autophagosome can interact with misfolded and polyubiquitinated proteins for degradation through autophagy such as p62 and NBR1 [167,168]. The fourth step, maturation, is related to autophagosome completion. The autophagosome undergoes two maturation steps: (i). The autophagosome fuses with multivesicular endosomes to form an amphisome, where proton pumps acquired for acidification; (ii). The amphisome combines with a lysosome to become an autolysosome. The final step involves the degradation of materials present inside the autolysosome by the action of different lysosomal enzymes into amino acids, lipids, and sugars. The degradation products, such as amino acids, fats, and sugars, are released from the autolysosome via lysosome efflux transporters for reuse. The release of nutrients from the autolysosome reactivates mTOR, which triggers autophagy termination and the formation of nascent lysosomes. This process is called autolysosome reformation (ALR). Under conditions of low nutrition, the cycle is repeated. Other researchers and we have shown that HCV induces autophagy through ER stress and mTOR pathway [169-171].

If the autophagy process is not controlled or remains activated for a prolonged period, this can lead to the accumulation of large scale autophagic vacuoles in the cytoplasm leading to cell death. Autophagic cell death occurs during adenovirus infection [172]. Many researchers have shown that tumor cells infected with oncolytic adenoviruses activate expression of the autophagy-related proteins, microtubule-associated protein one light chain 3, and p62 degradation [173]. The ER membranes are exhausted during high levels of autophagy induction as a source of autophagosomes during virus replication. Autophagic cell death can selectively release DAMP such as ATP, HMGB1, and uric acid, leading to increased antitumor immunity [174,175]. Autophagy levels increased during chronic HCV infection, and the accumulation of autophagic vesicles has been demonstrated using quantitative electron microscopy [176,177]. In contrast, autophagy dysfunction associated with the development of HCC related chronic viral infection and non-viral etiology under high stress [44,45]. In the following section, we review our recent observation on how cell survival program and autophagy processes modulate under severe stress response during chronic HCV infection that may imply understanding the mechanisms of HCC development.

9. Integrative stress response activates CMA through Nrf2 for cell survival:

We are interested in understanding the molecular switch from autophagic pro-death to pro-survival signaling when the ER-stress becomes severe or prolonged in HCV infected culture model. We examined the mRNA and protein expression levels of three UPR genes in Huh-7.5 liver cells and primary human hepatocyte after HCV infection in a kinetic study over several months. We found that the level of PERK mRNA expression (>40 fold) was the highest compared to ATF6 and IRE1, suggesting that the PERK axis is the dominant player in the cell survival mechanism during chronic HCV infection. The PERK activates two significant cell survival programming under stress by promoting eIF2α and Nrf2 phosphorylation (Figure 8). We examined the involvement of the cell survival switch during persistent HCV infection. Interestingly, we found that expression of PERK activated ATF4 and CHOP were decreased significantly during the late stage of persistent HCV infection of Huh-7.5 liver cells (unpublished data). These results suggest that persistent HCV infection inhibits the major cell death pathway activated by the PERK axis of UPR. Previous studies by Diehl laboratory showed that the Nrf2 transcription factor activated by the PERK axis of UPR independent of eIF2α phosphorylation [178]. These results lead us to examine whether the cell survival switch during persistent HCV infection involves the activation of Nrf2, the major transcription factor that mediates cellular protection during oxidative stress, and ER stress [179,180]. We demonstrated in our publication that the Nrf2 activation and nuclear translocation occurs in the majority of Huh-7.5 cells persistently infected with HCV suggesting that PERK-Nrf2 activation is the primary cell survival mechanism under prolonged stress during chronic HCV infection [47,48]. We showed that the mRNA levels of Nrf2 are increased during HCV replication, suggesting that the PERK axis probably controls the transcription of the antioxidant gene. Based on the published reports, we conclude that Nrf2 activation by the PERK pathway is another mechanism in addition to the canonical (ROS-dependent, KEAP dependent), non-canonical (p62-dependent) mechanisms of Nrf2 activation [181-185]. CMA is responsible for the degradation of cytosolic proteins that contain a consensus pentapeptide motif (KFERQ). Hsc70 and LAMP2A are vital to the uptake of cellular substrates required for the lysosomal degradation of cytosolic proteins during CMA. The Nrf2 is a transcription factor induces Hsc70 and LAMP2A by directly binding to their promoter regions. We demonstrated multiple ARE (TGAnnnnGC) and ARE-like (TGAnnnGC or TGAnnnnnGC) binding sites that were present in LAMP2A and Hsc70 promoter [48]. We showed Nrf2 activation increased mRNA and protein levels of LAMP2A and Hsc70 in a persistently infected HCV cell culture model. Furthermore, we verified that Nrf2 silencing decreased LAMP2A and Hsc70 expression in HCV cell culture. To test whether Nrf2-induced CMA activation as a mechanism of cell survival related to ER stress, the viability of HCV infected cells measured after silencing LAMP2A and Nrf2. These results indicate that silencing LAMP2A decreased the viability of HCV infected culture more than 90% and silencing Nrf2 reduced cell viability by 70%. The amalgamation of these data suggests that Nrf2-induced CMA activation is required to improve cell survival during chronic HCV infection [186,187]. There is evidence that autophagy processes compensate for each other under extreme stressful environments for promoting cell survival [188,189]. Our data show that high-level cellular stress associated with persistent HCV infection activates other forms of autophagy to enhance cell survival. In the following sections, we review some of our published work that explains how the activation of Nrf2 signaling activates cell survival program through the degradation of tumor suppressors and activation of oncogenic signaling.

Figure 8:

Working model was illustrating how integrated stress response prefers PERK-dependent Nrf2 signaling that leads to activation of stress chaperones, CMA and degradation of tumor suppressors: p53, Beclin 1, and pRb1 for cell survival.

10. Integrative stress response degrades the major tumor suppressors in chronic HCV infection:

Viruses intrinsically manipulate host cell surveillance during infection for the establishment of chronic infection. Particularly, the host cell uses two different surveillance mechanisms to block virus replication and spread. One mechanism involves the production of interferon, which directly inhibits virus replication [190]. The other mechanism involves blocking the spread of infection by inducing p53-mediated cellular apoptosis [191,192]. In this section, we present supporting data from our publication showing how HCV induced stress response degrades the major tumor suppressors: (i). Persistent HCV infection degrades p53: Many viruses, as well as bacteria, regulate p53 signaling in favor of their continued survival. A review article by Rusyn and Lemon claimed that the majority of previous publications demonstrated HCV replication represses p53 functions [193]. We now have new evidence suggesting that HCV-infected PHH or Huh-7.5 cells adapt to the stress response through the rapid degradation of p53 to improve cellular surveillance during persistent HCV infection [46]. We showed that p53 is a target of CMA because it harbors two pentapeptide motifs (200 NLRVE204 and 341FRELN345) that are similar to the Hsc70 recognition sequence. Due to the presence of such pentapeptide motifs, this tumor suppressor degraded under excessive stress. We showed that stress response degrades both wild-type p53 PHHs and mutant p53 using a persistent HCV infection model. Using co-immunoprecipitation followed by Western blot analysis, we verified that HCV infection or serum starvation of Huh-7.5 cells promotes interaction between Hsc70, LAMP2A and p53 [46]. In contrast, PHHs cultured under similar conditions over 15 days showed stable expression of p53, suggesting that the degradation of p53 is not related to stress associated with culturing PHH in Petri dishes. We also demonstrated that the tumor suppressor p14ARF is also a CMA target protein, which is why the expression of p14ARF decreased in HCV infection under stress [46]. The treatment with ER stress inducer, thapsigargin (TG), induced p53 degradation more in HCV infected culture. The p53 expression in HCV culture was restored by ER stress inhibitor (tauroursodeoxycholic acid [TUDCA]) and a lysosome inhibitor (hydroxychloroquine [HCQ]). Mdm2-mediated degradation of p53 is dependent on the direct interaction, so we examined whether disruption of the interaction between p53 and Mdm2 could result in p53 stabilization in HCV cell culture. We showed that treatment with either Nutlin-3 or MG132 alone or in combination did not stabilize p53 expression levels in HCV infected culture, indicating that p53 degradation is independent of Mdm2-mediated degradation. Restoration of p53 by HCQ and LAMP2A silencing suggests that the degradation of p53 is through the lysosomal pathway. We found that p53 protein expression is significantly low in HCV infected explant livers. The expression of the ER stress chaperone (BiP), CMA related proteins (Hsc70, LAMP2A), and Mdm2 increased in HCV positive liver cirrhosis compared with HCV negative cirrhotic samples. (ii). Persistent HCV infection degrades pRb tumor suppressor: The loss of tumor suppressor genes such as retinoblastoma, p53 gene, and activation of the Mdm2 pathway are involved in HCC development related to HCV infection in humans [194]. We showed that persistent HCV replication induces chronic ER stress with a significant accumulation of reactive oxygen species (ROS), which resulted in the nuclear translocation of Nrf2 in 100% of infected cells. The nuclear translocation of Nrf2 prevented by a PERK inhibitor or TUDCA and silencing the PERK also inhibits Nrf2 activation in infected culture, suggesting that the activation of the PERK pathway due to oxidative stress is responsible for sustained nuclear translocation of Nrf2 [47]. To understand the significance of Nrf2 activation on HCV related HCC mechanism, we examined the Nrf2-mediated induction of Mdm2 and the degradation of Rb tumor suppressor. In this study, we showed that persistent HCV infection activated Nrf2-mediated Mdm2 transcription. Increase Mdm2 expression degraded Rb tumor suppressor through activation of Nrf2 signaling. The silencing the Nrf2 pathway, treatment with the PERK inhibitor and ER stress inhibitor (TUDCA) decreased Nrf2 nuclear translocation, inhibited Mdm2 levels, and restored Rb degradation. The silencing of Mdm2 by siRNA or inhibition of Mdm2 restored Rb degradation in HCV culture, providing direct evidence of how ER stress controls Rb and p53 degradation. Previous studies by Stanley Lemon's group have shown that Rb degradation in HCV infected culture is mediated by direct binding of Rb with viral RNA-dependent RNA polymerase (NS5B) [195]. They also showed that NS5B-dependent ubiquitination of pRb and subsequent degradation mediated by ubiquitin ligase activity of E6-associated protein (E6AP) [196]. The loss of Rb function can also occur through the mutation in the Rb gene itself, hypermethylation of Rb promoter, bindings of viral proteins such as NS5B protein of HCV, E7 of the papillomavirus or E1A protein of adenovirus or post-transcriptional modification with tumor-associated kinase activity [197]. Phosphorylation is also another well-characterized mechanism involved in Rb inhibition during cell cycle control, suggesting that Rb function inhibited by a wide variety of mechanisms [198,199]. Our results show another potential novel ER stress related mechanism controlling Rb degradation through Mdm2 during HCV infection, which may have implications for understanding HCV-related HCC development. (iii). Persistent HCV infection promotes Beclin 1 degradation. HCC development in human and mouse models is associated with an impaired autophagy response. The mechanism of autophagy regulation in the setting of a highly stressed ER during chronic HCV infection is unknown. Therefore, we are interested in understanding the mechanism through which the hepatic autophagy process functions as a pro-survival defense mechanism under high ER stress and sets the course of HCC development in the cirrhotic liver. In this study, we examined how macroautophagy and CMA compensate for each other to promote hepatocyte survival using a persistently infected HCV culture model. We demonstrate that persistent HCV infection resulted in the prolonged activation of the PERK axis of the UPR, leading to the activation of Nrf2 signaling and its nuclear translocation. Nrf2 nuclear translocation induced Hsc70 and LAMP2A expression and subsequently activated CMA. Beclin 1 is a key molecule involved in autophagy initiation and autophagosome-lysosome fusion. Beclin 1 interacts with class III type P13CIII/Vps34 during the formation of Beclin 1-Vps34-Vps15 core complex [163,164]. CMA activation leads to Beclin 1 degradation and inhibition of autophagy. We found that there are four KFERQ-like motifs in human Beclin 1 protein, which is why this protein preferentially selected for degradation under stress. The decreased Beclin 1 protein expression in persistently infected HCV culture was not due to mRNA transcription or degradation. In this study, we showed that silencing LAMP2A and HCQ treatment restored Beclin 1 expression in infected Huh-7.5 cells. We demonstrated a functional interaction between Beclin 1, and CMA machinery was examined using co-immunoprecipitation with antibodies against Beclin 1, LAMP2A, and Hsc70 in HCV infected cell culture. We showed that cells treated with a known Nrf2 activator, sulforaphane, induced Nrf2 nuclear stabilization and increased Beclin 1 degradation along with LAMP2A induction. Many previous publications, including those from our laboratory, have shown that p62 is an autophagy flux protein and that its expression increased in HCC due to autophagy inhibition. Because decreased autophagy flux observed in HCC due to p62 accumulation, we examined whether Beclin 1 degradation during chronic HCV infection alters p62 levels. The accumulation of autophagy flux protein, p62, and HCV core protein expressions was measured in infected cells with HCV over 30 days by flow cytometric analysis. We found a high p62 expression in uninfected Huh-7.5 cells, whereas HCV infection resulted in a time-dependent decrease in p62 expression. On day 12, the number of cells expressing p62 had decreased by 75% compared with the uninfected control. After day 12 onwards the p62 expression was increased and the level reached to a comparison with the uninfected culture. The percentage of infected cells increased rapidly from day 3 to day 12 (10% to 83%) and remained constant afterward. The percentage of HCV-infected cells decreased slightly (86% to 79%) between days 23 and 30 due to impaired autophagy. Bafilomycin A1 treatment increased LC3B-I/ II and p62 levels in infected Huh-7.5 cells. In contrast, no further enhancement of LC3-II or p62 levels was observed in the HCV-infected culture, thereby confirming that autophagy flux blocked in persistently infected Huh-7.5 cells. To verify that the loss of Beclin 1 is the reason for the impaired autophagy flux in late-infected culture, levels of LC3B-I/II, and p62 examined after silencing or overexpression of Beclin 1. Silencing Beclin 1 accumulated p62 and LC3B-I/ II, whereas the overexpression of Beclin 1 decreased expression of p62 and LC3B-I/ II in a dose-dependent manner. This treatment did not alter HCV core expression. A low-level Beclin 1 expression has been implicated in several disease-associated processes, such as neurodegeneration and HCC tumorigenesis. A previous report claims that monoallelic deletion of Beclin 1 results in an increased incidence of spontaneous malignancies, including lung cancer, lymphoma, and HCC. Moreover, loss of Beclin 1 has been correlated with poor prognosis in various cancers, including HCC, in clinical studies [200-203]. Similarly, many earlier studies reported spontaneous HCC development in mice as a result of a mosaic deletion of an ATG5 or hepatic disruption of ATG7, supporting our observations that impairment in autophagy could promote HCC development [204].

11. Integrative stress response activates oncogenic signaling:

The simultaneous loss of tumor suppressor and activation of oncogenic signaling could promote cell cycle progression and malignant transformation [205]. Our published data show that activation of CMA leads to degradation of the dominant tumor suppressor and impaired autophagic degradation through Beclin 1 degradation. Beclin 1 is involved in the initiation of the canonical form of autophagy and autophagosome-endosome fusion [200]. We examined whether Beclin 1 loss through CMA inhibited autophagy initiation and autophagosome formation using a GFP tagged-ATG5 (K130R) mutant plasmid construct that is incapable of conjugating to ATG12. The number of membrane-associated puncta is significantly lower in late-infected culture than in uninfected culture, suggesting that Beclin 1 loss also reduced autophagy initiation. The expression levels of ATG16L1 and ATG7 were comparable in uninfected, and HCV infected cultures, implying that Beclin 1 did not affect the elongation step of autophagy machinery or autophagosome formation. Autophagy proceeds through several stages, beginning with the creation of a phagophore complex, which then elongates to form a complete double-membrane vesicle called an autophagosome. The fusion of an autophagosome with a lysosome creates an autolysosome where contents degraded. Beclin 1 is a member of the phosphatidylinositol 3-kinase complex, which is involved in vesicle trafficking during autophagy and other cellular processes. We examined whether Beclin 1 loss blocks the fusion of autophagosomes with endosomes and lysosomes. Interactions among Beclin 1, ATG14L, and UV radiation resistance-associated gene protein (UVRAG) are needed for autophagosome-endosome fusion, whereas Rab7, various tethering proteins, and soluble N-ethylmaleimide-sensitive factor activating protein receptor protein required for autophagosome-lysosome fusion. The expressions of Rab5 and Rab7 decreased during persistent HCV infection without altering the appearance of VPS34. Immunostaining showed increased accumulation of the EGFR on the surface of infected Huh-7.5 cells 21 days after infection compared with uninfected Huh-7.5 cells. Receptor-mediated endocytosis and lysosomal degradation of EGFR are thought to be necessary for the attenuation of ligand-induced EGFR signaling. We showed that persistent HCV infection activated EGFR signaling through impaired degradation at the level of endocytosis by specifically blocking autophagosomes and endocytic vesicle fusion. Impaired autophagic degradation could activate many cancer pathways [206]. EGFR is a transmembrane receptor tyrosine kinase that is overexpressed in 68% of human HCCs and correlates with aggressive tumors and patient survival [207-210]. Receptor-mediated endocytosis and lysosomal degradation is a significant negative feedback loop for EGFR signaling [211]. We hypothesize that HCV induced impaired autophagy response will inhibit the degradation of EGFR at the level of autophagosome-lysosome fusion leading to the activation of downstream RAS/RAF/MEK/ERK signaling. Autophagy processes also negatively regulate Wnt/β-catenin signaling by promoting the degradation of Dishevelled (Dvl) and β-catenin in the cytoplasm [212]. We hypothesize that stress-induced impaired autophagic degradation of Dvl and β-catenin will activate the Wnt/ β-catenin pathway implicated in HCC and cancer stemness.

12. The interplay among stress, cell survival, autophagy switching and HCC risk in cirrhosis:

Mechanisms of acute and chronic liver injury in humans have been linked mainly to infection by hepatitis viruses, alcohol abuse, and excess fat accumulation. In some individuals, combinations of multiple insults may contribute to liver disease progression. One of the host-related factors responsible for the progression of liver disease is the degree of hepatocellular injury. Aminotransferases are a group of enzymes that synthesize and break down amino acids and to convert energy storage molecules. Increased level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood is a direct indication of a liver injury, which has been widely used by physicians as one of the surrogate markers to assess the extent of hepatic injury in patients with chronic liver diseases of both viral and non-viral etiologies. As discussed in the earlier sections, cellular stress signaling can promote three types of cell death (apoptosis, necrosis, and autophagy) with distinct morphological features [213-215]. Apoptosis is a caspase-mediated cell death that is characterized by chromosome condensation, nuclear fragmentation, and membrane blessing. In contrast, necrosis is an accidental cell death caused by cellular stress that is characterized by the expansion of cell organelles, plasma membrane rupture, and subsequent release of intracellular content that leads to an inflammatory response. Autophagy is a cellular degradation mechanism that improves cell survival in a stressed environment. However, excessive stress such as hypoxia and infection with lytic viruses such as oncolytic adenovirus can lead to autophagic cell death. The autophagic cell death induced by cellular stress characterized by the formation of excessive autophagic vesicles containing the damaged organelles, including the destruction of the endoplasmic reticulum [216-218]. Among the three types of cell death occurring in the HCV infected liver, autophagy provides the highest cell survival superiority, as autophagy flux p62 level remains undetectable in liver cirrhosis (Figure 9). All three types of cell death pathways are linked to UPR signaling through multiple cascades [217]. Chronic HCV infection of hepatocytes causes different levels of cellular stress throughout the disease process. Activation of various cell death cascades (apoptotic, necrotic, or autophagic) demonstrated in HCV-induced chronic hepatitis. Necrotic cell death also contributes to the chronic inflammation and other pathological changes associated with chronic HCV infection. The release of DAMP from lysed necrotic cells can produce reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are known to cause lipid peroxidation [219]. This process can activate the UPR and NF-kB pathway, which is the hallmark of an inflammatory response implicated in the development of liver fibrosis and HCC during chronic HCV infection [220]. There are compelling pieces of evidence that the perpetuation of inflammation is associated with the development of liver cirrhosis and HCC [221-224]. Apoptotic cell death is considered a weak inducer of inflammation as it causes the limited release of molecules and cellular components called damage-associated molecular patterns (DAMP). Histologically, apoptosis of hepatocytes is characteristically present in chronic HCV infection throughout the disease process. The engulfment of apoptotic hepatocytes has been shown to lead to the production of TGF-β, which is known to activate hepatic stellate cells and promote liver fibrosis [225-227]. A direct correlation between the apoptotic index, histological grading, and the activation of caspase-3 and caspase-7 has been seen in liver biopsies of chronic HCV patients [228,229]. Hepatocellular apoptosis after HCV infection has been detected in liver cells of SCID/ALB-UPA humanized mice [230]. Four separate publications document the presence of HCV induced UPR gene expression using human liver tissues from chronic HCV patients [231]. Shuda and coworkers [232] examined the expression of GRP78, ATF6, and XBP levels using surgically resected HCC and surrounding non-cancerous liver tissues from 13 patients infected with HBV or HCV. They found a high expression of GRP78 in the cytoplasm, nuclear translocation of activated ATF6, and spliced version of XBP mRNA, indicating that the presence of chronic ER-stress response in advanced liver disease. Asselah and coworkers [233] investigated ER-stress and UPR expression in liver samples from 28 untreated chronic HCV patients, 13 with mild fibrosis, and 15 with advanced fibrosis. They showed the activation of UPR (ATF6, IRE1, and PERK) pathways in mild and advanced liver fibrosis, which is indicative of the presence of chronic ER-stress in advanced liver disease. The authors also confirmed altered hepatocyte ER-morphology in livers from patients with hepatitis C when compared to normal livers. Studies from our lab have shown increased UPR gene expression in advanced chronic HCV patients and liver cirrhosis patients with viral and non-viral etiologies [42]. Our data also demonstrate increased expression of UPR genes in chronic HCV infection as well as in cirrhotic livers in comparison to the normal uninfected liver. Therefore, the presence of integrative stress response may represent a key mechanism for viral persistence and viral carcinogenesis. Notably, the ER stress response and UPR gene expression seen in liver diseases are associated with hepatitis B [234] and hepatitis C virus infection [235], alcoholic liver diseases [236], and nonalcoholic steatohepatitis (NASH) [237,238]. In most of the patients, HCC develops on the background of chronic liver diseases and is closely associated with liver cirrhosis. However, the exact mechanism for HCC development in cirrhotic microenvironment remains unknown. Recent studies from our group show that HCC grown in the highly stressed cirrhotic liver undergo autophagy switching from a protective state characterized by high macroautophagy and low chaperone-mediated autophagy (CMA) to an HCC-promoting state characterized by low macroautophagy and high CMA [48]. The expression of p62 and glypican-3 increased in HCC areas as compared to the adjacent non-tumorous cirrhotic liver (Figure 10). In three separate publications, we have shown that virus-associated stress response degrades the tumor suppressors and activates oncogenic EGFR signaling [46-48]. These results are consistent with the hypothesis that loss of tumor suppressor with activation of oncogenic signaling promotes cell survival and the onset of cell cycle progression leading to liver tumor development. Our results support the hypothesis that autophagy may play as a pro-death (tumor suppressor) during chronic infection but pro-survival (oncogenic) during HCC development in the cirrhotic liver. Our findings support previous studies from other investigators on how ER stress activates adaptive signaling to restore hepatic homeostasis and drives human fatty liver disease progression, cirrhosis, and HCC [49]. The mechanisms for how the integrative stress response is associated with the evolution of the chronic liver disease, cirrhosis, and HCC through the autophagy pathway remain incompletely understood. Our results suggest that the interplay between hepatic stress response, autophagy, and cell survival pathways could determine the risk of advanced liver disease, and HCC induced by chronic HCV infection. Given that HCV is a curable disease, this information can be used to help understand the mechanism of liver disease progression related to non-viral etiologies.

Figure 9:

Relationship of ISR and cell survival superiority during chronic HCV infection. ISR associated with virus infection induces multiple cell death events: necrosis, apoptosis, autophagic cell death or cell survival through activation of cellular autophagy processes.

Figure 10:

Shows the expression of p62 and glypican-3 in HCC and surrounding non-tumor areas of the cirrhotic livers. The appearance of p62 and glypican-3 was significantly higher in HCC as compared to the adjacent non-tumorous cirrhotic liver, suggesting HCC have impaired autophagy response. Autophagy is impaired in HCC related to viral (HBV and HCV) as well as non-viral etiologies.

13. Summary and conclusions:

Hepatitis C virus utilizes ER extensively during all stages of liver disease process leading to the creation of cellular stress and UPR response. The UPR response is an integral part of liver tissue homeostasis that leads to different types of cell death pathways to fuel into a common tumor suppressor mechanism. The selection of cellular cell death or cell survival program ultimately determined by the magnitude of the stress response during infection. Our data show that aberrant stress response can promote cell survival through autophagy switching. This review summarizes the molecular and cellular basis of autophagy switching in which tumor suppressor autophagy switches to tumor-promoting form. The mechanism of autophagy switching during the excessive UPR response described here opens new perspectives for the development of targeted molecular treatment approaches for HCC. HCC initiation is a multi-step process with many oncogenes genes coordinately regulate cell division, proliferation, and tumor development. Since ER acts as an essential regulator of hepatocyte function, this review provides updated information on how excessive ER stress response generated during chronic HCV infection promotes cell survival pathway that links to HCC development.