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Metabolic dysfunction and cancer in HCV: Shared pathways and mutual interactions

  • Jack Leslie
    Affiliations
    Newcastle Fibrosis Research Group, Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
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  • Daniel Geh
    Affiliations
    Newcastle Fibrosis Research Group, Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
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  • Ahmed M. Elsharkawy
    Affiliations
    Liver Unit, University Hospitals Birmingham NHS Foundation Trust, Queen Elizabeth Hospital, Queen Elizabeth Medical Centre, Birmingham, B15 2TH UK

    National Institute for Health Research, Birmingham Biomedical Research Centre at University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK
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  • Derek A. Mann
    Correspondence
    Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, NE2 4HH, Newcastle upon Tyne, UK. Tel.: +44(0) 191 208 3851.
    Affiliations
    Newcastle Fibrosis Research Group, Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK

    Department of Gastroenterology and Hepatology, School of Medicine, Koç University, Istanbul, Turkey
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  • Michele Vacca
    Correspondence
    Corresponding authors. Address: Clinica Medica “Cesare Frugoni”, Interdisciplinary Department of Medicine, Università degli Studi di Bari “Aldo Moro”, Piazza Giulio Cesare 11, Bari, Italy. Tel.: +39 3792401521.
    Affiliations
    Interdisciplinary Department of Medicine, Università degli Studi di Bari “Aldo Moro”, Bari, Italy
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Open AccessPublished:February 11, 2022DOI:https://doi.org/10.1016/j.jhep.2022.01.029

      Summary

      HCV hijacks many host metabolic processes in an effort to aid viral replication. The resulting hepatic metabolic dysfunction underpins many of the hepatic and extrahepatic manifestations of chronic hepatitis C (CHC). However, the natural history of CHC is also substantially influenced by the host metabolic status: obesity, insulin resistance and hepatic steatosis are major determinants of CHC progression toward hepatocellular carcinoma (HCC). Direct-acting antivirals (DAAs) have transformed the treatment and natural history of CHC. While DAA therapy effectively eradicates the virus, the long-lasting overlapping metabolic disease can persist, especially in the presence of obesity, increasing the risk of liver disease progression. This review covers the mechanisms by which HCV tunes hepatic and systemic metabolism, highlighting how systemic metabolic disturbance, lipotoxicity and chronic inflammation favour disease progression and a precancerous niche. We also highlight the therapeutic implications of sustained metabolic dysfunction following sustained virologic response as well as considerations for patients who develop HCC on the background of metabolic dysfunction.

      Keywords

      Introduction

      HCV is a leading cause of end-stage liver disease and hepatocellular carcinoma (HCC). It is estimated that 71.1 million people worldwide have chronic HCV infection,
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      resulting in 400,000 predicted liver-related deaths per year. Whilst the hepatic manifestations are well known, chronic hepatitis C (CHC) is a multisystem disease with several extrahepatic manifestations. These include: mixed cryoglobulinaemic vasculitis, autoimmune disease, lymphoma, and cardiometabolic disease.
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      The biological pathways underlying this multisystem disease are not fully understood; however, host metabolic dysfunction overlapping with and/or in response to HCV infection is a unifying process for some of these manifestations. Specifically, CHC is often associated with obesity and/or an impairment of glucose and lipid metabolism, as well as with a higher prevalence of the metabolic syndrome (MetS) and its components/complications, such as insulin resistance and type 2 diabetes mellitus (T2DM), hepatic steatosis, dyslipidaemia, and cardiovascular disease (CVD).
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      The recent development of direct-acting antivirals (DAAs) has revolutionised the treatment of CHC, enabling sustained virological response (SVR) rates of well above 95-98% in the majority of patients. That being said, a high rate of early tumour recurrence was recently observed in patients with HCV-related HCC who had undergone DAA therapy.
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      While this was a relatively small cohort of patients, it does suggest that there are long-term carcinogenic consequences of HCV infection that are independent of active viral proteins. Metabolic impairment can persist after SVR, increasing the risk of liver disease progression, cancer development and cardiovascular disease, as seen in patients with non-alcoholic fatty liver disease (NAFLD), a form of fatty liver associated with MetS which many experts propose renaming as “metabolic dysfunction-associated fatty liver disease” (MAFLD).
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      Therefore, whilst eradication of HCV is an important goal, consideration and treatment of the metabolic consequences of HCV are equally important in order to improve patient outcomes. It is therefore crucial to be aware of the high prevalence of metabolic disturbances in patients with HCC and to treat them alongside HCV infection, in order to prevent progression of liver disease, and mortality from HCC or CVD. Herein, we explore how insulin resistance (IR) and MetS worsen fatty liver and promote cancer development in CHC, and we look at the implications for cancer treatment and prevention in patients with CHC.

      Viral influences on HCV-mediated steatosis development

      Accumulation of lipids in the liver, known as steatosis, results from imbalances in hepatic metabolic fluxes
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      An intriguing feature of HCV infection is its tight connection with hepatic and systemic metabolism. Viral HCV proteins, especially in genotype 3, have been proven to hijack the host's glucose and lipid metabolism at multiple levels (Fig. 1) since: i) HCV stimulates de novo lipogenesis (DNL) to favour HCV lipoviroparticle assembly via the activation of multiple lipogenic transcription factors
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      (and) iv) lipoviroparticle assembly interferes with VLDL assembly and export by hijacking the VLDL secretion pathway.
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      Moreover, other possible HCV-mediated effects involve the rewiring of nuclear receptor transcriptional machinery
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      Nuclear receptors control pro-viral and antiviral metabolic responses to hepatitis C virus infection.
      and the upregulation of the pentose phosphate pathway.
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      All together these effects have a major impact on hepatic fat content and liver function as described below.
      HCV rewires hepatic metabolism, driving liver steatosis and contributing to the development of metabolic dysfunction.
      Figure thumbnail gr1
      Fig. 1Mechanisms of HCV-induced steatosis and hepatic insulin resistance.
      HCV hijacks hepatocyte metabolism at multiple levels either directly, via a direct action exerted by viral proteins on intracellular signalling/metabolic pathways, or indirectly by promoting peripheral insulin resistance, hyperinsulinemia and hyperglycaemia. ChREBP, carbohydrate response element-binding protein; DNL, de novo lipogenesis; FA, fatty acid; FOXO1, forkhead box O1; GLUT, glucose transporter; INSR1/2, insulin receptor 1/2; LVP, lipoviroparticle; MTP, microsomal triglyceride transfer protein; PTEN, phosphatase and tensin homolog; ROS, reactive oxygen species; SREBPs, sterol regulatory element-binding proteins; TG, triglyceride.
      On top of direct viral protein effects on hepatic metabolism, patients with CHC frequently present with IR and T2DM; these metabolic features have been associated with an increased risk of progression toward fibrosis (Fig. 2). Intriguingly IR can develop independently from obesity and correlate with HCV replication rates.
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      Indeed, deep metabolic profiling (including clamps and indirect calorimetry) of lean patients with CHC at different stages of liver disease have suggested that IR primarily affects peripheral organs, while hepatic IR seems to depend on the stage of hepatic damage and/or HCV genotype.
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      Interestingly, especially in lean patients with CHC, HCV-mediated peripheral IR does not seem to be associated with the canonical effects of peripheral insulin on lipid metabolism in adipose tissue (AT; e.g. suppression of AT lipolysis), indicating substantial mechanistic differences from forms of IR that are AT dysfunction-led, and implicating a possible muscle involvement.
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      Since HCV does not infect extrahepatic (metabolically-relevant) tissues, it is conceivable that HCV infection might exert some indirect peripheral effects (e.g. by unbalancing the secretion of hepatokines involved in tuning peripheral insulin sensitivity and/or systemic inflammation) and that part of these effects can be reversed by viral eradication.
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      Sites and mechanisms of insulin resistance in nonobese, nondiabetic patients with chronic hepatitis C.
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      • Montet X.
      • Tappy L.
      • Clément S.
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      Treatment with direct-acting antivirals improves peripheral insulin sensitivity in non-diabetic, lean chronic hepatitis C patients.
      Figure thumbnail gr2
      Fig. 2HCV promotes systemic metabolic dysfunction.
      Schematic summarising the rewiring of metabolic pathways in metabolically active organs in subjects with insulin resistance (e.g. obesity, metabolic syndrome, and chronic hepatitis C); direct actions exerted by HCV proteins are also highlighted to suggest how HCV rewires systemic metabolism. DNL, de novo lipogenesis.
      However the following issues are still unresolved: i) what is the mechanism of peripheral IR in HCV infection? ii) is peripheral IR a direct consequence of HCV infection per se, or is it an outcome of chronic disease progression? 3) is this change in (AT-independent) IR pathophysiologically relevant for the progression of chronic liver disease? Future research should focus on identifying the mechanism responsible for the liver-to-periphery communication that drives IR in CHC. Moreover, it should be said that the direct association of HCV infection and IR/T2DM is still disputed and conflicting results have been reported even in consecutive studies on the same large cohorts (e.g. US NHANES studies have not continuously reported the association).
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      This raises i) concerns that the specific composition of the study populations might influence the results and interpretation; ii) the question of how best to characterise co-factors that could potentially impact IR (such as age, race, BMI, disease stage, or socio-economic status) in large cohort studies, to allow for a metabolically meaningful interpretation of the data and to definitively address this issue.
      In the following paragraphs we will discuss how the combination of HCV infection, obesity and its metabolic complications alter the natural history of CHC and its progression toward HCC, as well as looking at their impact on patient management.

      The role of host obesity and adipose tissue dysfunction in HCV-mediated steatosis

      It is estimated that up to 78% of patients with CHC are also overweight or obese
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      Abnormalities of lipid metabolism in hepatitis C virus infection.
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      Excess weight gain after cure of hepatitis C infection with direct-acting antivirals.
      : obesity is known to potentiate fatty liver development and boost CHC progression in those patients where both co-exist. Obesity is a global epidemic characterised by increased energy intake and/or reduced energy expenditure leading to adipocyte dysfunction and causing a cluster of cardiometabolic risk factors (IR and T2DM, atherogenic dyslipidaemia, hypertension) grouped under the umbrella definition of MetS.
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      Adipose tissue-liver cross talk in the control of whole-body metabolism: implications in nonalcoholic fatty liver disease.
      Obesity and MetS are associated with increased all-cause mortality and enhanced cancer risk.
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      Obesity is also the main driver of the increasing NAFLD burden and HCC rates in developed countries.
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      Metabolic dysfunction develops when AT reaches it expandability limit (e.g. in overweight/obesity or in lipodystrophy): AT expands under nutritional/metabolic pressure as a result of a fine balance of both adipocyte hyperplasia and hypertrophy, but has a finite storage capacity; if adipocyte hyperplasia (increased number of functional and insulin sensitive adipocytes) is “metabolically healthy”, AT hypertrophy (that prevails when adipocyte’s hyperplasia is impaired) results in adipocyte dysfunction and IR with subsequent increased AT lipolysis (leading to the spill over of fat to other organs including the liver and muscle).
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      Adipose tissue-liver cross talk in the control of whole-body metabolism: implications in nonalcoholic fatty liver disease.
      ,
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      Adipose tissue expandability, lipotoxicity and the Metabolic Syndrome--an allostatic perspective.
      Different clinical studies have confirmed that AT health and function and systemic/AT IR are much better predictors of systemic metabolic disturbances and hepatic steatosis than BMI or waist circumference (WC); moreover AT dysfunction and systemic/AT IR, prominent features of both non-alcoholic steatohepatitis (NASH) and CHC, have been associated with severe steatohepatitis and fibrosis.
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      • Carli F.
      • Rosso C.
      • Younes R.
      • D’Aurizio R.
      • Bugianesi E.
      • et al.
      Altered metabolic profile and adipocyte insulin resistance mark severe liver fibrosis in patients with chronic liver disease.
      ,
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      • Micu L.
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      Prevalence of steatosis and insulin resistance in patients with chronic hepatitis B compared with chronic hepatitis C and non-alcoholic fatty liver disease.
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      Additionally, ethnic-specific differences in body composition mandate the redefinition of cut-offs for overweight/obesity.
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      Adipose tissue-liver cross talk in the control of whole-body metabolism: implications in nonalcoholic fatty liver disease.
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      Nonalcoholic fatty liver disease in lean individuals in the United States.
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      Altogether a better understanding of these complex underlying mechanisms necessitate a critical re-evaluation of clinical studies where the BMI (or WC) has solely been used to define the role of metabolic dysfunction as a co-factor in HCV-related liver disease progression. Ideally, a better clinical assessment of AT dysfunction and IR would be preferable to anthropometrics, as it would also explain the apparent “lean fatty liver” paradox whereby individuals within “normal” BMI/WC ranges, who also exhibit IR and MetS, can still develop fatty liver and steatohepatitis.
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      NASH in lean individuals.
      This redefinition would also help to solve the puzzle of apparently conflicting results described in lean patients with CHC as discussed above.
      There is also increasing evidence that obesity, IR and fatty liver are associated with AT (especially visceral) inflammation, systemic low grade inflammation and fibrosis, which correlate with the degree of metabolic impairment and complications (e.g. NASH, atherosclerosis). Despite this, it is still debated if the activation of these processes in AT are cause or consequence of AT dysfunction/IR and metabolic disease (the results of clinical trials targeting these pathways have been disappointing from a metabolic perspective). AT (especially visceral) inflammation and systemic low grade inflammation might still be an extremely attractive therapeutic target to improve liver disease outcomes considering their strong association with the progression of steatohepatitis.
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      It is therefore our opinion that AT/systemic inflammation might be an important co-factor for the stratification of patients when assessing disease outcomes in clinical studies.
      In the interferon era of HCV management, it was recognised that obesity was associated with decreased rates of SVR
      • Poynard T.
      • Ratziu V.
      • McHutchison J.
      • Manns M.
      • Goodman Z.
      • Zeuzem S.
      • et al.
      Effect of treatment with peginterferon or interferon alfa-2b and ribavirin on steatosis in patients infected with hepatitis C.
      and as such, weight reduction used to play a significant role in HCV management. This is no longer the case in the DAA era where SVR rates do not appear to be affected by obesity.
      • Krassenburg L.A.P.
      • Maan R.
      • Ramji A.
      • Manns M.P.
      • Cornberg M.
      • Wedemeyer H.
      • et al.
      Clinical outcomes following DAA therapy in patients with HCV-related cirrhosis depend on disease severity.
      A recent large study examined the effect of DAA therapy on weight after 2 years of therapy.
      • Do A.
      • Esserman D.A.
      • Krishnan S.
      • Lim J.K.
      • Taddei T.H.
      • Hauser R.G.
      • et al.
      Excess weight gain after cure of hepatitis C infection with direct-acting antivirals.
      It is important to note that 78% of the 11,469 patients included were overweight or obese at treatment initiation (defined as a BMI ≥25) and that SVR rates were 97% in the cohort. Those who achieved SVR gained 0.56 ± 12.8 lbs vs. a − 3.43 ± 14.6 lbs weight loss in those who did not achieve SVR (p <0.0001). Furthermore, 22% of those who had a BMI ≤25 at treatment initiation became overweight during the follow-up period. Weight gain is common with some medications (e.g. anti-psychotics, anti-depressants, glucocorticoids and beta blockers) including antiviral medications (e.g. anti-HIV), but why DAA-treated patients gain weight is not fully understood; it is likely the result of a combination of factors including improved quality of life and increased nutritional intake following SVR.
      • Mukhtar N.A.
      • Fox R.K.
      Hepatitis C virus cure and obesity: watch the weight.
      The worsening of the systemic metabolic profile associated with body weight gain might thus explain, at least in part, the blunted clinical response (with regards to hepatic outcomes) associated with SVR in patients with advanced fibrosis, and/or the lack of protection from HCC in patients who achieved SVR; this is of particular concern in the presence of significant fibrosis and/or other comorbidities associated with liver injury. However, it should be mentioned that the effects of DAAs on systemic IR are debated since some studies have shown an improvement of extrahepatic insulin sensitivity after SVR.
      • Lim T.R.
      • Hazlehurst J.M.
      • Oprescu A.I.
      • Armstrong M.J.
      • Abdullah S.F.
      • Davies N.P.
      • et al.
      Hepatitis C virus infection is associated with hepatic and adipose tissue insulin resistance that improves after viral cure.
      ,
      • Gastaldi G.
      • Gomes D.
      • Schneiter P.
      • Montet X.
      • Tappy L.
      • Clément S.
      • et al.
      Treatment with direct-acting antivirals improves peripheral insulin sensitivity in non-diabetic, lean chronic hepatitis C patients.
      Also, it is important to note that alcohol intake should always be considered as a co-factor when assessing post-SVR data: heavy alcohol consumption promotes steatosis per se, and drives liver disease progression toward HCC thus making the picture even more complex.
      • Minami T.
      • Tateishi R.
      • Fujiwara N.
      • Nakagomi R.
      • Nakatsuka T.
      • Sato M.
      • et al.
      Impact of obesity and heavy alcohol consumption on hepatocellular carcinoma development after HCV eradication with antivirals.
      HCV infection and obesity promote lipotoxicity, senescence, chronic inflammation and fibrosis which drive malignant hepatocyte transformation and HCC development.
      In summary, these authors wonder if HCV-associated hepatic steatosis is best considered an “overlap syndrome” between HCV infection (and its metabolic/chronic liver disease implications) and NAFLD where, under the metabolic pressure of a positive energy balance and systemic/AT-IR, hepatic fat accumulation is boosted, leading to enhanced lipotoxicity-mediated wound healing with potential for a fast progression toward fibrosis and increased HCC risk. Moreover, it is our opinion that patients should be carefully metabolically re-phenotyped post-SVR: the persistence/worsening of metabolic disturbances and alcohol intake after treatment should be carefully characterised and therapeutically targeted to prevent the progression of liver disease and HCC once HCV has been eradicated.
      • Kanwal F.
      • Kramer J.
      • Asch S.M.
      • Chayanupatkul M.
      • Cao Y.
      • El-Serag H.B.
      Risk of hepatocellular cancer in HCV patients treated with direct-acting antiviral agents.
      • Conti F.
      • Buonfiglioli F.
      • Scuteri A.
      • Crespi C.
      • Bolondi L.
      • Caraceni P.
      • et al.
      Early occurrence and recurrence of hepatocellular carcinoma in HCV-related cirrhosis treated with direct-acting antivirals.
      • Pereira Guedes T.
      • Fragoso P.
      • Lemos C.
      • Garrido M.
      • Silva J.
      • Falcão D.
      • et al.
      Long-term follow-up of advanced liver disease after sustained virological response to treatment of hepatitis C with direct-acting antivirals: outcomes from a real-world Portuguese cohort.
      • Verna E.C.
      • Morelli G.
      • Terrault N.A.
      • Lok A.S.
      • Lim J.K.
      • Di Bisceglie A.M.
      • et al.
      DAA therapy and long-term hepatic function in advanced/decompensated cirrhosis: real-world experience from HCV-TARGET cohort.

      Host metabolic cross-talk with HCV-mediated steatosis – the role of hyperinsulinemia and hyperglycaemia

      As a result of peripheral IR, increased pancreatic insulin secretion, and reduced insulin clearance (due to the progressive development of steatosis), hyperinsulinemia occurs. Muscle-IR also increases glucose levels since it reduces muscle uptake of glucose and conversion to glycogen, therefore favouring hyperglycaemia.
      • Azzu V.
      • Vacca M.
      • Virtue S.
      • Allison M.
      • Vidal-Puig A.
      Adipose tissue-liver cross talk in the control of whole-body metabolism: implications in nonalcoholic fatty liver disease.
      Elevated circulating levels of glucose (that activate carbohydrate response element-binding protein [ChREBP]) and insulin (that promote sterol regulatory element-binding protein [SREBP]-1c) have profound metabolic effects on lipid uptake, DNL and lipid remodelling in the liver, resulting in increased hepatic fat influx.
      • Azzu V.
      • Vacca M.
      • Virtue S.
      • Allison M.
      • Vidal-Puig A.
      Adipose tissue-liver cross talk in the control of whole-body metabolism: implications in nonalcoholic fatty liver disease.
      Increased DNL has indeed been described by transcriptomic and/or lipidomic studies in both patients with NAFLD
      • Luukkonen P.K.
      • Sädevirta S.
      • Zhou Y.
      • Kayser B.
      • Ali A.
      • Ahonen L.
      • et al.
      Saturated fat is more metabolically harmful for the human liver than unsaturated fat or simple sugars.
      • Sanders F.W.B.
      • Acharjee A.
      • Walker C.
      • Marney L.
      • Roberts L.D.
      • Imamura F.
      • et al.
      Hepatic steatosis risk is partly driven by increased de novo lipogenesis following carbohydrate consumption.
      • Puri P.
      • Wiest M.M.
      • Cheung O.
      • Mirshahi F.
      • Sargeant C.
      • Min H.K.
      • et al.
      The plasma lipidomic signature of nonalcoholic steatohepatitis.
      • Azzu V.
      • Vacca M.
      • Kamzolas I.
      • Hall Z.
      • Leslie J.
      • Carobbio S.
      • et al.
      Suppression of insulin-induced gene 1 (INSIG1) function promotes hepatic lipid remodelling and restrains NASH progression.
      and CHC,
      • d’Avigdor W.M.H.
      • Budzinska M.A.
      • Lee M.
      • Lam R.
      • Kench J.
      • Stapelberg M.
      • et al.
      Virus genotype-dependent transcriptional alterations in lipid metabolism and inflammation pathways in the hepatitis C virus-infected liver.
      • Wu J.M.
      • Skill N.J.
      • Maluccio M.A.
      Evidence of aberrant lipid metabolism in hepatitis C and hepatocellular carcinoma.
      • Mirandola S.
      • Realdon S.
      • Iqbal J.
      • Gerotto M.
      • Dal Pero F.
      • Bortoletto G.
      • et al.
      Liver microsomal triglyceride transfer protein is involved in hepatitis C liver steatosis.
      and this has been shown to facilitate virus entry into hepatocytes.
      • Yang W.
      • Hood B.L.
      • Chadwick S.L.
      • Liu S.
      • Watkins S.C.
      • Luo G.
      • et al.
      Fatty acid synthase is up-regulated during hepatitis C virus infection and regulates hepatitis C virus entry and production.
      ,
      • Tsutsumi T.
      • Suzuki T.
      • Shimoike T.
      • Suzuki R.
      • Moriya K.
      • Shintani Y.
      • et al.
      Interaction of hepatitis C virus core protein with retinoid X receptor alpha modulates its transcriptional activity.
      ,
      • Lerat H.
      • Kammoun H.L.
      • Hainault I.
      • Mérour E.
      • Higgs M.R.
      • Callens C.
      • et al.
      Hepatitis C virus proteins induce lipogenesis and defective triglyceride secretion in transgenic mice.
      Despite the fact that it has been established that SREBP1-/ChREBP1-mediated transcriptional programmes are activated in NAFLD and CHC, it should be noted that it is still debated whether these pathways are maladaptive responses that cause damage, or whether they represent a protective mechanism favouring lipid remodelling (e.g. the formation of monounsaturated fatty acids) and aimed at maintaining hepatic health/homeostasis when the liver is metabolically challenged by an abnormal calorie load.
      • Azzu V.
      • Vacca M.
      • Kamzolas I.
      • Hall Z.
      • Leslie J.
      • Carobbio S.
      • et al.
      Suppression of insulin-induced gene 1 (INSIG1) function promotes hepatic lipid remodelling and restrains NASH progression.
      ,
      • Vacca M.
      • Allison M.
      • Griffin J.L.
      • Vidal-Puig A.
      Fatty acid and glucose sensors in hepatic lipid metabolism: implications in NAFLD.
      • Knebel B.
      • Haas J.
      • Hartwig S.
      • Jacob S.
      • Köllmer C.
      • Nitzgen U.
      • et al.
      Liver-specific expression of transcriptionally active SREBP-1c is associated with fatty liver and increased visceral fat mass.
      • Denechaud P.D.
      • Bossard P.
      • Lobaccaro J.M.A.
      • Millatt L.
      • Staels B.
      • Girard J.
      • et al.
      ChREBP, but not LXRs, is required for the induction of glucose-regulated genes in mouse liver.
      • Benhamed F.
      • Denechaud P.D.
      • Lemoine M.
      • Robichon C.
      • Moldes M.
      • Bertrand-Michel J.
      • et al.
      The lipogenic transcription factor ChREBP dissociates hepatic steatosis from insulin resistance in mice and humans.
      HCV also suppresses insulin receptor substrates (IRS-1 and IRS-2
      • Shintani Y.
      • Fujie H.
      • Miyoshi H.
      • Tsutsumi T.
      • Tsukamoto K.
      • Kimura S.
      • et al.
      Hepatitis C virus infection and diabetes: direct involvement of the virus in the development of insulin resistance.
      ,
      • Kawaguchi T.
      • Yoshida T.
      • Harada M.
      • Hisamoto T.
      • Nagao Y.
      • Ide T.
      • et al.
      Hepatitis C virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3.
      ,
      • Miyamoto H.
      • Moriishi K.
      • Moriya K.
      • Murata S.
      • Tanaka K.
      • Suzuki T.
      • et al.
      Involvement of the PA28gamma-dependent pathway in insulin resistance induced by hepatitis C virus core protein.
      ), thereby directly causing hepatic IR.
      • Yamaguchi A.
      • Tazuma S.
      • Nishioka T.
      • Ohishi W.
      • Hyogo H.
      • Nomura S.
      • et al.
      Hepatitis C virus core protein modulates fatty acid metabolism and thereby causes lipid accumulation in the liver.
      ,
      • Abid K.
      • Pazienza V.
      • de Gottardi A.
      • Rubbia-Brandt L.
      • Conne B.
      • Pugnale P.
      • et al.
      An in vitro model of hepatitis C virus genotype 3a-associated triglycerides accumulation.
      ,
      • Hourioux C.
      • Patient R.
      • Morin A.
      • Blanchard E.
      • Moreau A.
      • Trassard S.
      • et al.
      The genotype 3-specific hepatitis C virus core protein residue phenylalanine 164 increases steatosis in an in vitro cellular model.
      However, hepatic IR is known to be “selective” (partial), as some pathways still remain insulin sensitive and DNL is not impacted. This phenomenon, yet to be fully understood, occurs because of the complex nature of the insulin signalling pathway: glucose metabolism is controlled mainly by AKT2-mediated inhibition of forkhead box O1 (FOXO1) function (AKT2 signalling is impaired by IR).
      • Matsumoto M.
      • Pocai A.
      • Rossetti L.
      • Depinho R.A.
      • Accili D.
      Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor Foxo1 in liver.
      ,
      • Dong X.C.
      • Copps K.D.
      • Guo S.
      • Li Y.
      • Kollipara R.
      • DePinho R.A.
      • et al.
      Inactivation of hepatic Foxo1 by insulin signaling is required for adaptive nutrient homeostasis and endocrine growth regulation.
      On the other hand, SREBP1-mediated lipogenesis remains activated because it is controlled by mTORC1 (which is less responsive to IR
      • Khamzina L.
      • Veilleux A.
      • Bergeron S.
      • Marette A.
      Increased activation of the mammalian target of rapamycin pathway in liver and skeletal muscle of obese rats: possible involvement in obesity-linked insulin resistance.
      ). The IR-mediated overactivation of FOXO1 in fatty liver worsens hyperglycaemia; however, FOXO1 also promotes IRS-2 expression
      • Qu S.
      • Altomonte J.
      • Perdomo G.
      • He J.
      • Fan Y.
      • Kamagate A.
      • et al.
      Aberrant Forkhead box O1 function is associated with impaired hepatic metabolism.
      further favouring a permissive mechanism that keeps DNL active in the face of ongoing steatosis.
      • Rametta R.
      • Mozzi E.
      • Dongiovanni P.
      • Motta B.M.
      • Milano M.
      • Roviaro G.
      • et al.
      Increased insulin receptor substrate 2 expression is associated with steatohepatitis and altered lipid metabolism in obese subjects.
      ,
      • Valenti L.
      • Rametta R.
      • Dongiovanni P.
      • Maggioni M.
      • Fracanzani A.L.
      • Zappa M.
      • et al.
      Increased expression and activity of the transcription factor FOXO1 in nonalcoholic steatohepatitis.
      Intriguingly, insulin also governs a positive feedback loop mediated by phosphatase and tensin homolog (PTEN): PTEN is a negative regulator of the PI3K/AKT pathway that is suppressed by insulin signalling itself
      • Liu J.
      • Visser-Grieve S.
      • Boudreau J.
      • Yeung B.
      • Lo S.
      • Chamberlain G.
      • et al.
      Insulin activates the insulin receptor to downregulate the PTEN tumour suppressor.
      and DNL products (e.g. monounsaturated fatty acids that promote miR-21)
      • Vinciguerra M.
      • Veyrat-Durebex C.
      • Moukil M.A.
      • Rubbia-Brandt L.
      • Rohner-Jeanrenaud F.
      • Foti M.
      PTEN down-regulation by unsaturated fatty acids triggers hepatic steatosis via an NF-kappaBp65/mTOR-dependent mechanism.
      ,
      • Vinciguerra M.
      • Sgroi A.
      • Veyrat-Durebex C.
      • Rubbia-Brandt L.
      • Buhler L.H.
      • Foti M.
      Unsaturated fatty acids inhibit the expression of tumor suppressor phosphatase and tensin homolog (PTEN) via microRNA-21 up-regulation in hepatocytes.
      to partially preserve insulin intracellular signalling in hepatocytes at the expense of worsening the hepatic lipid content and steatohepatitis.
      • Vinciguerra M.
      • Veyrat-Durebex C.
      • Moukil M.A.
      • Rubbia-Brandt L.
      • Rohner-Jeanrenaud F.
      • Foti M.
      PTEN down-regulation by unsaturated fatty acids triggers hepatic steatosis via an NF-kappaBp65/mTOR-dependent mechanism.
      The HCV core protein (genotype 3a) also suppresses PTEN via miR-21, therefore leading to the formation of large lipid droplets and enhancing virus secretion.
      • Clément S.
      • Peyrou M.
      • Sanchez-Pareja A.
      • Bourgoin L.
      • Ramadori P.
      • Suter D.
      • et al.
      Down-regulation of phosphatase and tensin homolog by hepatitis C virus core 3a in hepatocytes triggers the formation of large lipid droplets.
      • Peyrou M.
      • Clément S.
      • Maier C.
      • Bourgoin L.
      • Branche E.
      • Conzelmann S.
      • et al.
      PTEN protein phosphatase activity regulates hepatitis C virus secretion through modulation of cholesterol metabolism.
      • Clément S.
      • Sobolewski C.
      • Gomes D.
      • Rojas A.
      • Goossens N.
      • Conzelmann S.
      • et al.
      Activation of the oncogenic miR-21-5p promotes HCV replication and steatosis induced by the viral core 3a protein.
      PTEN is also a well-described tumour suppressor
      • Lee Y.R.
      • Chen M.
      • Pandolfi P.P.
      The functions and regulation of the PTEN tumour suppressor: new modes and prospects.
      : PTEN inhibition in CHC and NAFLD might thus have a strong impact on both hepatocellular carcinogenesis
      • Stiles B.
      • Wang Y.
      • Stahl A.
      • Bassilian S.
      • Lee W.P.
      • Kim Y.J.
      • et al.
      Liver-specific deletion of negative regulator Pten results in fatty liver and insulin hypersensitivity [corrected].
      ,
      • Horie Y.
      • Suzuki A.
      • Kataoka E.
      • Sasaki T.
      • Hamada K.
      • Sasaki J.
      • et al.
      Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas.
      and HCC clinical outcomes.
      • Rahman M.A.
      • Kyriazanos I.D.
      • Ono T.
      • Yamanoi A.
      • Kohno H.
      • Tsuchiya M.
      • et al.
      Impact of PTEN expression on the outcome of hepatitis C virus-positive cirrhotic hepatocellular carcinoma patients: possible relationship with COX II and inducible nitric oxide synthase.
      In conclusion, IR/T2DM and steatosis are independently associated with fibrosis and liver disease progression in patients with CHC in cross-sectional and longitudinal studies
      • Negro F.
      Abnormalities of lipid metabolism in hepatitis C virus infection.
      ,
      • Hui J.M.
      • Sud A.
      • Farrell G.C.
      • Bandara P.
      • Byth K.
      • Kench J.G.
      • et al.
      Insulin resistance is associated with chronic hepatitis C virus infection and fibrosis progression [corrected].
      ; the co-occurrence of CHC and systemic metabolic disturbances might increase the risk of CHC progression and HCC.
      • Hung C.H.
      • Wang J.H.
      • Hu T.H.
      • Chen C.H.
      • Chang K.C.
      • Yen Y.H.
      • et al.
      Insulin resistance is associated with hepatocellular carcinoma in chronic hepatitis C infection.
      Unlike genotype 3 (where steatosis is mainly driven by direct virus-mediated effects), the association of obesity with fibrogenesis is more evident in patients carrying HCV genotype 1 where steatosis is more linearly influenced by systemic metabolic unbalances.
      • Hui J.M.
      • Sud A.
      • Farrell G.C.
      • Bandara P.
      • Byth K.
      • Kench J.G.
      • et al.
      Insulin resistance is associated with chronic hepatitis C virus infection and fibrosis progression [corrected].
      ,
      • Leandro G.
      • Mangia A.
      • Hui J.
      • Fabris P.
      • Rubbia-Brandt L.
      • Colloredo G.
      • et al.
      Relationship between steatosis, inflammation, and fibrosis in chronic hepatitis C: a meta-analysis of individual patient data.

      The precancerous niche in CHC

      The majority of HCC arises on the background of progressive fibrosis and/or cirrhosis which are known drivers of hepatocarcinogenesis. However, HCC can also develop (albeit at a much lower rate) in transgenic HCV-HCC murine models
      • Bach N.
      • Thung S.N.
      • Schaffner F.
      The histological features of chronic hepatitis C and autoimmune chronic hepatitis: a comparative analysis.
      and in patients with CHC in the absence of significant chronic inflammation and/or cirrhosis.
      • Desai A.
      • Sandhu S.
      • Lai J.P.
      • Sandhu D.S.
      Hepatocellular carcinoma in non-cirrhotic liver: a comprehensive review.
      In the next section we will discuss how HCV infection, CHC progression and metabolic influences interact to favour liver disease progression.

      Viral influences

      HCV is an RNA virus that is predominantly restricted to the cytoplasm and subsequently is unable to integrate into the host genome and exert direct mutagenic effects.
      • Negro F.
      Abnormalities of lipid metabolism in hepatitis C virus infection.
      However, HCV proteins have been implicated in promoting cell proliferation and survival, epithelial-mesenchymal transition, and angiogenesis, through modulation of various signalling cascades including: Raf1/MAPK,
      • Polivka J.
      • Janku F.
      Molecular targets for cancer therapy in the PI3K/AKT/mTOR pathway.
      Wnt/β-catenin pathway,
      • Thompson M.D.
      • Monga S.P.S.
      WNT/beta-catenin signaling in liver health and disease.
      transforming growth factor-β (TGF-β) signaling, PI3K/PTEN,
      • Chen C.Y.
      • Chen J.
      • He L.
      • Stiles B.L.
      PTEN: tumor suppressor and metabolic regulator.
      and insulin signalling/AKT,
      • Hers I.
      • Vincent E.E.
      • Tavaré J.M.
      Akt signalling in health and disease.
      thus contributing directly to HCC development. Furthermore, CHC leads to genome-wide epigenetic changes that persist in patients even after SVR,
      • Perez S.
      • Kaspi A.
      • Domovitz T.
      • Davidovich A.
      • Lavi-Itzkovitz A.
      • Meirson T.
      • et al.
      Hepatitis C virus leaves an epigenetic signature post cure of infection by direct-acting antivirals.
      ,
      • Hamdane N.
      • Jühling F.
      • Crouchet E.
      • El Saghire H.
      • Thumann C.
      • Oudot M.A.
      • et al.
      HCV-induced epigenetic changes associated with liver cancer risk persist after sustained virologic response.
      sustaining metabolic dysregulation and inflammation following viral eradication.

      Lipotoxicity

      Dysregulation of hepatic metabolism leads to the build-up of toxic lipids and intermediates in hepatocytes (“lipotoxicity”) resulting in cell dysfunction and injury. CHC is associated with up to a 5-fold increase in liver reactive oxygen species (ROS) levels.
      • Smirnova O.A.
      • Ivanova O.N.
      • Bartosch B.
      • Valuev-Elliston V.T.
      • Mukhtarov F.
      • Kochetkov S.N.
      • et al.
      Hepatitis C virus NS5A protein triggers oxidative stress by inducing NADPH oxidases 1 and 4 and cytochrome P450 2E1.
      ,
      • Okuda M.
      • Li K.
      • Beard M.R.
      • Showalter L.A.
      • Scholle F.
      • Lemon S.M.
      • et al.
      Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein.
      HCV core proteins can directly increase ROS levels through increased Nox1 and Nox4 production of superoxide,
      • Ivanov A.V.
      • Bartosch B.
      • Smirnova O.A.
      • Isaguliants M.G.
      • Kochetkov S.N.
      HCV and oxidative stress in the liver.
      ,
      • de Mochel N.S.R.
      • Seronello S.
      • Wang S.H.
      • Ito C.
      • Zheng J.X.
      • Liang T.J.
      • et al.
      Hepatocyte NAD(P)H oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection.
      and also indirectly by inducing mitochondrial dysfunction,
      • Ivanov A.V.
      • Bartosch B.
      • Smirnova O.A.
      • Isaguliants M.G.
      • Kochetkov S.N.
      HCV and oxidative stress in the liver.
      ,
      • Kim S.J.
      • Syed G.H.
      • Khan M.
      • Chiu W.W.
      • Sohail M.A.
      • Gish R.G.
      • et al.
      Hepatitis C virus triggers mitochondrial fission and attenuates apoptosis to promote viral persistence.
      • Ripoli M.
      • D’Aprile A.
      • Quarato G.
      • Sarasin-Filipowicz M.
      • Gouttenoire J.
      • Scrima R.
      • et al.
      Hepatitis C virus-linked mitochondrial dysfunction promotes hypoxia-inducible factor 1α-mediated glycolytic adaptation.
      • Kim S.J.
      • Syed G.H.
      • Siddiqui A.
      Hepatitis C virus induces the mitochondrial translocation of Parkin and subsequent mitophagy.
      endoplasmic reticulum stress
      • Asselah T.
      • Bièche I.
      • Mansouri A.
      • Laurendeau I.
      • Cazals-Hatem D.
      • Feldmann G.
      • et al.
      In vivo hepatic endoplasmic reticulum stress in patients with chronic hepatitis C.
      • Merquiol E.
      • Uzi D.
      • Mueller T.
      • Goldenberg D.
      • Nahmias Y.
      • Xavier R.J.
      • et al.
      HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response.
      • Tardif K.D.
      • Mori K.
      • Siddiqui A.
      Hepatitis C virus subgenomic replicons induce endoplasmic reticulum stress activating an intracellular signaling pathway.
      as well as impairing antioxidant mechanisms (glutathione and small metabolite antioxidants).
      • Yadav D.
      • Hertan H.I.
      • Schweitzer P.
      • Norkus E.P.
      • Pitchumoni C.S.
      Serum and liver micronutrient antioxidants and serum oxidative stress in patients with chronic hepatitis C.
      • Moriya K.
      • Miyoshi H.
      • Shinzawa S.
      • Tsutsumi T.
      • Fujie H.
      • Goto K.
      • et al.
      Hepatitis C virus core protein compromises iron-induced activation of antioxidants in mice and HepG2 cells.
      ROS overproduction also occurs when mitochondrial fatty acid oxidation supplies excessive respiratory substrates causing stalling of the respiratory fluxes: saturation of the oxidative system in steatosis leads to mitochondrial dysfunction and promotion of extra-mitochondrial (i.e. microsomal and peroxisomal) oxidation.
      • Pessayre D.
      • Fromenty B.
      NASH: a mitochondrial disease.
      ,
      • Sanyal A.J.
      • Campbell-Sargent C.
      • Mirshahi F.
      • Rizzo W.B.
      • Contos M.J.
      • Sterling R.K.
      • et al.
      Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities.
      Also, fatty acid Ω-oxidation, a minor pathway of fatty acid oxidation usually inhibited by insulin in the healthy liver, is upregulated by IR and leads to long-chain dicarboxylic acid production, further worsening mitochondrial dysfunction; long-chain dicarboxylic acids (and very-long-chain fatty acids) can then be partially metabolised by peroxisomal β-oxidation, further inducing ROS production.
      • Pessayre D.
      • Fromenty B.
      NASH: a mitochondrial disease.
      ,
      • Sanyal A.J.
      • Campbell-Sargent C.
      • Mirshahi F.
      • Rizzo W.B.
      • Contos M.J.
      • Sterling R.K.
      • et al.
      Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities.
      Multiple reports suggest that HCV worsens these events as it drives mitochondrial dysfunction.
      • Yamaguchi A.
      • Tazuma S.
      • Nishioka T.
      • Ohishi W.
      • Hyogo H.
      • Nomura S.
      • et al.
      Hepatitis C virus core protein modulates fatty acid metabolism and thereby causes lipid accumulation in the liver.
      ,
      • Abid K.
      • Pazienza V.
      • de Gottardi A.
      • Rubbia-Brandt L.
      • Conne B.
      • Pugnale P.
      • et al.
      An in vitro model of hepatitis C virus genotype 3a-associated triglycerides accumulation.
      ,
      • Hourioux C.
      • Patient R.
      • Morin A.
      • Blanchard E.
      • Moreau A.
      • Trassard S.
      • et al.
      The genotype 3-specific hepatitis C virus core protein residue phenylalanine 164 increases steatosis in an in vitro cellular model.
      ,
      • Gerresheim G.K.
      • Roeb E.
      • Michel A.M.
      • Niepmann M.
      Hepatitis C virus downregulates core subunits of oxidative phosphorylation, reminiscent of the warburg effect in cancer cells.
      ,
      • Meoni G.
      • Lorini S.
      • Monti M.
      • Madia F.
      • Corti G.
      • Luchinat C.
      • et al.
      The metabolic fingerprints of HCV and HBV infections studied by Nuclear Magnetic Resonance Spectroscopy.
      Lipotoxicity drives cell death and promotes the chronic activation of wound healing responses, thereby promoting CHC progression and a precancerous niche in CHC (Fig. 3).
      DAA treatment can result in weight gain in a proportion of patients with CHC and also causes metabolic changes associated with HCC development.
      Figure thumbnail gr3
      Fig. 3Potential mechanisms of liver tumorigenesis in HCV-infected patients.
      In patients with chronic hepatitis C, hepatocellular carcinogenesis is promoted by a tight interplay between systemic metabolic/pro-inflammatory effects and direct/indirect actions of HCV in the liver where it i) boosts lipotoxicity, oxidative stress, DNA damage, cell death and senescence; ii) promotes wound healing; therefore promoting tumorigenesis and a precancerous microenvironment favouring hepatocellular carcinoma development. RNS, reactive nitrogen species; ROS, reactive oxygen species.

      Inflammation and immunosurveillance

      Oxidative stress, metabolic dysfunction and lipotoxicity can have a profound effect on the immune system in response to both viral infection and also cancerous cells. Effective clearance of HCV infection typically requires the coordinated function of multiple arms of the immune system, including the innate immune system (interferons and natural killer [NK] and NKT cells), as well as the adaptive immune response specific to a given pathogen (CD4+ and CD8+ T cells).
      • Dustin L.B.
      Innate and adaptive immune responses in chronic HCV infection.
      For the majority of patients, these mechanism are unable to eradicate the virus, leading to chronic infection and CHC. The immunological mechanisms (immunosurveillance) that identify and clear HCV-infected cells are similar to those that detect and destroy malignant cells.
      • Dunn G.P.
      • Bruce A.T.
      • Ikeda H.
      • Old L.J.
      • Schreiber R.D.
      Cancer immunoediting: from immunosurveillance to tumor escape.
      Immunosurveillance can be impaired by a number of HCV-dependent mechanisms that do not completely resolve following DAA treatment
      • Hengst J.
      • Falk C.S.
      • Schlaphoff V.
      • Deterding K.
      • Manns M.P.
      • Cornberg M.
      • et al.
      Direct-acting antiviral-induced hepatitis C virus clearance does not completely restore the altered cytokine and chemokine milieu in patients with chronic hepatitis C.
      as well as by systemic metabolic influences that confer increased risk of HCC.
      • Pfister D.
      • Núñez N.G.
      • Pinyol R.
      • Govaere O.
      • Pinter M.
      • Szydlowska M.
      • et al.
      NASH limits anti-tumour surveillance in immunotherapy-treated HCC.
      ,
      • Negro F.
      Residual risk of liver disease after hepatitis C virus eradication.
      Cytotoxic CD8+ T cells are the predominant cell type responsible for clearing HCV-infected hepatocytes. During HCV infection, effector CD8+ T cells become progressively exhausted due to persistent antigen exposure and as a result exhibit reduced antiviral activity.
      • Osuch S.
      • Metzner K.J.
      • Caraballo Cortés K.
      Reversal of T Cell exhaustion in chronic HCV infection.
      ,
      • Luxenburger H.
      • Neumann-Haefelin C.
      • Thimme R.
      • Boettler T.
      HCV-specific T cell responses during and after chronic HCV infection.
      This T-cell exhaustion does not resolve following DAA treatment.
      • Vranjkovic A.
      • Deonarine F.
      • Kaka S.
      • Angel J.B.
      • Cooper C.L.
      • Crawley A.M.
      Direct-acting antiviral treatment of HCV infection does not resolve the dysfunction of circulating CD8+ T-cells in advanced liver disease.
      The reduced activity of T cells in CHC may predispose to HCC, as T cells are thought to orchestrate the clearance of senescent pre-malignant hepatocytes.
      • Kang T.W.
      • Yevsa T.
      • Woller N.
      • Hoenicke L.
      • Wuestefeld T.
      • Dauch D.
      • et al.
      Senescence surveillance of pre-malignant hepatocytes limits liver cancer development.
      T-cell exhaustion is also a feature of HCC, with T cells exhibiting impaired cytotoxic and anti-tumoural activity.
      • Wherry E.J.
      • Kurachi M.
      Molecular and cellular insights into T cell exhaustion.
      Furthermore, the anti-tumoural activity of CD8+ T cells is impaired as a result of the systemic effects of obesity
      • Ringel A.E.
      • Drijvers J.M.
      • Baker G.J.
      • Catozzi A.
      • García-Cañaveras J.C.
      • Gassaway B.M.
      • et al.
      Obesity shapes metabolism in the tumor microenvironment to suppress anti-tumor immunity.
      which may in part explain why patients with NASH may be less responsive to immunotherapy, although this is a recent observation that requires further validation.
      • Pfister D.
      • Núñez N.G.
      • Pinyol R.
      • Govaere O.
      • Pinter M.
      • Szydlowska M.
      • et al.
      NASH limits anti-tumour surveillance in immunotherapy-treated HCC.
      CD4+ T cells are required to maintain antiviral CD8+ T-cell responses and are lost in CHC.
      • Gerlach J.T.
      • Diepolder H.M.
      • Jung M.C.
      • Gruener N.H.
      • Schraut W.W.
      • Zachoval R.
      • et al.
      Recurrence of hepatitis C virus after loss of virus-specific CD4(+) T-cell response in acute hepatitis C.
      HCV causes CD4+ T-cell dysfunction, reducing the cytokine production by T helper 1 cells that is required for antiviral immunity.
      • McMahan R.H.
      • Golden-Mason L.
      • Nishimura M.I.
      • McMahon B.J.
      • Kemper M.
      • Allen T.M.
      • et al.
      Tim-3 expression on PD-1+ HCV-specific human CTLs is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity.
      These effects are not limited to HCV-reactive T cells
      • McMahan R.H.
      • Golden-Mason L.
      • Nishimura M.I.
      • McMahon B.J.
      • Kemper M.
      • Allen T.M.
      • et al.
      Tim-3 expression on PD-1+ HCV-specific human CTLs is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity.
      suggesting that HCV infection may limit the effectiveness of other CD4+ T-cell roles including supporting CD8 T cell-mediated clearance of pre-malignant hepatocytes. Metabolic dysfunction can compound this with hepatic lipotoxicity reducing antigen-specific CD4+ T cell-dependent senescence surveillance of pre-malignant hepatocytes.
      • Ma C.
      • Kesarwala A.H.
      • Eggert T.
      • Medina-Echeverz J.
      • Kleiner D.E.
      • Jin P.
      • et al.
      NAFLD causes selective CD4(+) T lymphocyte loss and promotes hepatocarcinogenesis.
      Furthermore, in CHC, CD4+ regulatory T cells, which suppress the immunosurveillance activities of CD8+ T cells
      • Langhans B.
      • Nischalke H.D.
      • Krämer B.
      • Hausen A.
      • Dold L.
      • van Heteren P.
      • et al.
      Increased peripheral CD4+ regulatory T cells persist after successful direct-acting antiviral treatment of chronic hepatitis C.
      and NK cells,
      • Langhans B.
      • Alwan A.W.
      • Krämer B.
      • Glässner A.
      • Lutz P.
      • Strassburg C.P.
      • et al.
      Regulatory CD4+ T cells modulate the interaction between NK cells and hepatic stellate cells by acting on either cell type.
      expand and remain elevated even after DAA-induced SVR.
      • Langhans B.
      • Nischalke H.D.
      • Krämer B.
      • Hausen A.
      • Dold L.
      • van Heteren P.
      • et al.
      Increased peripheral CD4+ regulatory T cells persist after successful direct-acting antiviral treatment of chronic hepatitis C.
      Regulatory T cells are also increased in patients with NASH and are reported to be critical for NASH progression and HCC initiation in mice.
      • Wang H.
      • Zhang H.
      • Wang Y.
      • Brown Z.J.
      • Xia Y.
      • Huang Z.
      • et al.
      Regulatory T-cell and neutrophil extracellular trap interaction contributes to carcinogenesis in non-alcoholic steatohepatitis.
      ,
      • Dywicki J.
      • Buitrago-Molina L.E.
      • Noyan F.
      • Davalos-Misslitz A.C.
      • Hupa-Breier K.L.
      • Lieber M.
      • et al.
      The detrimental role of regulatory T cells in nonalcoholic steatohepatitis.
      NK cells have also been implicated in the clearance of HCV-infected hepatocytes.
      • Rosen H.R.
      • Golden-Mason L.
      Control of HCV infection by natural killer cells and macrophages.
      In CHC, decreased NK cell levels, altered NK cell subset distribution, activation marker expression and function are observed and not completely resolved following DAA treatment.
      • Rosen H.R.
      • Golden-Mason L.
      Control of HCV infection by natural killer cells and macrophages.
      ,
      • Golden-Mason L.
      • McMahan R.H.
      • Kriss M.S.
      • Kilgore A.L.
      • Cheng L.
      • Dran R.J.
      • et al.
      Early and late changes in natural killer cells in response to ledipasvir/sofosbuvir treatment.
      To date, the presence, regulation and function of NK cells during NAFLD remains controversial, with reports of both increased and decreased numbers as well as activation in patients who are obese or have NAFLD.
      • Martínez-Chantar M.L.
      • Delgado T.C.
      • Beraza N.
      Revisiting the role of natural killer cells in non-alcoholic fatty liver disease.
      NK cells are able to kill malignant cells directly through cytotoxic perforin-mediated lysis or indirectly through modulation of other immune cells. Both of these functions may be impacted by HCV infection and metabolic dysfunction.
      Therefore, HCV infection and steatosis not only increase the likelihood of developing pre-malignant hepatocytes, but can also impair the host’s long-term ability to clear these potentially malignant cells effectively.

      Senescence

      Oxidative stress in patients with CHC drives chronic injury in the liver, leading to increased compensatory proliferation of hepatocytes which, over multiple rounds of cell division, will risk telomere shortening and senescence (Fig. 3).
      • Sekoguchi S.
      • Nakajima T.
      • Moriguchi M.
      • Jo M.
      • Nishikawa T.
      • Katagishi T.
      • et al.
      Role of cell-cycle turnover and oxidative stress in telomere shortening and cellular senescence in patients with chronic hepatitis C.
      ,
      • Wandrer F.
      • Han B.
      • Liebig S.
      • Schlue J.
      • Manns M.P.
      • Schulze-Osthoff K.
      • et al.
      Senescence mirrors the extent of liver fibrosis in chronic hepatitis C virus infection.
      Hepatic ROS levels,
      • Ko E.
      • Seo H.W.
      • Jung G.
      Telomere length and reactive oxygen species levels are positively associated with a high risk of mortality and recurrence in hepatocellular carcinoma.
      HCV progression
      • Isokawa O.
      • Suda T.
      • Aoyagi Y.
      • Kawai H.
      • Yokota T.
      • Takahashi T.
      • et al.
      Reduction of telomeric repeats as a possible predictor for development of hepatocellular carcinoma: convenient evaluation by slot-blot analysis.
      • Kojima H.
      • Yokosuka O.
      • Imazeki F.
      • Saisho H.
      • Omata M.
      Telomerase activity and telomere length in hepatocellular carcinoma and chronic liver disease.
      • Paradis V.
      • Youssef N.
      • Dargère D.
      • Bâ N.
      • Bonvoust F.
      • Deschatrette J.
      • et al.
      Replicative senescence in normal liver, chronic hepatitis C, and hepatocellular carcinomas.
      and NASH
      • Nakajima T.
      • Nakashima T.
      • Okada Y.
      • Jo M.
      • Nishikawa T.
      • Mitsumoto Y.
      • et al.
      Nuclear size measurement is a simple method for the assessment of hepatocellular aging in non-alcoholic fatty liver disease: comparison with telomere-specific quantitative FISH and p21 immunohistochemistry.
      • Ping F.
      • Li Z.Y.
      • Lv K.
      • Zhou M.C.
      • Dong Y.X.
      • Sun Q.
      • et al.
      Deoxyribonucleic acid telomere length shortening can predict the incidence of non-alcoholic fatty liver disease in patients with type 2 diabetes mellitus.
      • Loo T.M.
      • Kamachi F.
      • Watanabe Y.
      • Yoshimoto S.
      • Kanda H.
      • Arai Y.
      • et al.
      Gut microbiota promotes obesity-associated liver cancer through PGE2-mediated suppression of antitumor immunity.
      are associated with telomere shortening/mutations (that are also common in HCC
      • Ningarhari M.
      • Caruso S.
      • Hirsch T.Z.
      • Bayard Q.
      • Franconi A.
      • Védie A.L.
      • et al.
      Telomere length is key to hepatocellular carcinoma diversity and telomerase addiction is an actionable therapeutic target.
      and, more broadly, in cancer
      • Loo T.M.
      • Kamachi F.
      • Watanabe Y.
      • Yoshimoto S.
      • Kanda H.
      • Arai Y.
      • et al.
      Gut microbiota promotes obesity-associated liver cancer through PGE2-mediated suppression of antitumor immunity.
      ).
      Induction of senescence due to telomere shortening is tumour suppressive if cleared by immunosurveillance mechanisms.
      • Kang T.W.
      • Yevsa T.
      • Woller N.
      • Hoenicke L.
      • Wuestefeld T.
      • Dauch D.
      • et al.
      Senescence surveillance of pre-malignant hepatocytes limits liver cancer development.
      However, chronic liver disease can result in an enormous senescence burden, with up to 80% of hepatocytes being senescent, which correlates not only with hepatic inflammation but also with the stage of fibrosis.
      • Aravinthan A.D.
      • Alexander G.J.M.
      Senescence in chronic liver disease: is the future in aging?.
      Failure to clear these senescent cells can result in significant genomic instability, ultimately leading to the formation of cancer clones.
      • Maciejowski J.
      • de Lange T.
      Telomeres in cancer: tumour suppression and genome instability.
      Furthermore, senescence can promote a precancerous niche through the production of senescence-associated secretory phenotype (SASP) which can promote adjacent cells to become senescent.
      • Acosta J.C.
      • Banito A.
      • Wuestefeld T.
      • Georgilis A.
      • Janich P.
      • Morton J.P.
      • et al.
      A complex secretory program orchestrated by the inflammasome controls paracrine senescence.
      ,
      • Baker D.J.
      • Wijshake T.
      • Tchkonia T.
      • LeBrasseur N.K.
      • Childs B.G.
      • van de Sluis B.
      • et al.
      Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders.
      The SASP is also highly proinflammatory,
      • Coppé J.P.
      • Desprez P.Y.
      • Krtolica A.
      • Campisi J.
      The senescence-associated secretory phenotype: the dark side of tumor suppression.
      recruiting immune cells that can cause further hepatocyte telomere damage and shortening,
      • Lagnado A.
      • Leslie J.
      • Ruchaud-Sparagano M.H.
      • Victorelli S.
      • Hirsova P.
      • Ogrodnik M.
      • et al.
      Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner.
      and establishing a vicious cycle of senescence and inflammation (Fig. 4). The effect of HCV infection on the induction or components of SASP is unclear, however, current evidence suggests that HCV infection promotes senescence indirectly through ROS-driven hepatocyte injury.
      • Sekoguchi S.
      • Nakajima T.
      • Moriguchi M.
      • Jo M.
      • Nishikawa T.
      • Katagishi T.
      • et al.
      Role of cell-cycle turnover and oxidative stress in telomere shortening and cellular senescence in patients with chronic hepatitis C.
      Figure thumbnail gr4
      Fig. 4The vicious cycles of senescence in chronic liver disease.
      Herein, we propose a model to explain how senescence might contribute to metabolic dysfunction and hepatic inflammation, and thereby a vicious cycle favouring chronic hepatitis C progression and hepatocellular carcinoma. ER, endoplasmic reticulum; SASP, senescence-associated secretory phenotype.
      Senescence also has a profound effect on systemic
      • Khosla S.
      • Farr J.N.
      • Tchkonia T.
      • Kirkland J.L.
      The role of cellular senescence in ageing and endocrine disease.
      and hepatic metabolism resulting in a worsening of steatosis.
      • Ogrodnik M.
      • Miwa S.
      • Tchkonia T.
      • Tiniakos D.
      • Wilson C.L.
      • Lahat A.
      • et al.
      Cellular senescence drives age-dependent hepatic steatosis.
      Fat accumulation in senescent hepatocytes was associated with mitochondrial dysfunction and impaired lipid metabolism.
      • Ogrodnik M.
      • Miwa S.
      • Tchkonia T.
      • Tiniakos D.
      • Wilson C.L.
      • Lahat A.
      • et al.
      Cellular senescence drives age-dependent hepatic steatosis.
      As a result of enhanced lipotoxicity, further liver injury, compensatory proliferation and ultimately replicative senescence establish a vicious cycle that leads to a worsening of steatosis (Fig. 4). Telomeric dysfunction may also play a wider role in the development of metabolic disease: mice with hyper-long telomeres display less metabolic aging, including lower levels of DNA damage, lower body weight, lower cholesterol and LDL, and also improved glucose and insulin tolerance compared to their wild-type counterparts.
      • Muñoz-Lorente M.A.
      • Cano-Martin A.C.
      • Blasco M.A.
      Mice with hyper-long telomeres show less metabolic aging and longer lifespans.
      Short-term cellular senescence might be an attempt to limit cancer development by engaging immunosurveillance mechanisms, however, the long-term metabolic consequence of this may ultimately lead to an increased risk of HCC. In the future, treatment with senolytics might suppress these vicious cycles of senescence, inflammation and metabolic dysfunction to reduce steatosis and progression to fibrosis and HCC after SVR.
      After successful DAA treatment in advanced CHC, hepatic manifestations and established comorbidities continue to injure the liver and HCC risk remains elevated.

      Fibrogenesis

      Liver fibrosis stage is the strongest predictive factor for liver-related (and all-cause) mortality, independent of any other histological features
      • Dyson J.
      • Jaques B.
      • Chattopadyhay D.
      • Lochan R.
      • Graham J.
      • Das D.
      • et al.
      Hepatocellular cancer: the impact of obesity, type 2 diabetes and a multidisciplinary team.
      and the presence of fibrosis promotes a precancerous liver microenvironment.
      • Baglieri J.
      • Brenner D.A.
      • Kisseleva T.
      The role of fibrosis and liver-associated fibroblasts in the pathogenesis of hepatocellular carcinoma.
      ,
      • Affo S.
      • Yu L.X.
      • Schwabe R.F.
      The role of cancer-associated fibroblasts and fibrosis in liver cancer.
      Fibrogenesis is mediated by activation of hepatic stellate cells that differentiate into collagen-producing myofibroblasts in response to liver injury. Accumulating evidence suggests that fibrosis and activated myofibroblasts contribute to the development of HCC through multiple pathways (reviewed extensively elsewhere
      • Baglieri J.
      • Brenner D.A.
      • Kisseleva T.
      The role of fibrosis and liver-associated fibroblasts in the pathogenesis of hepatocellular carcinoma.
      ,
      • Affo S.
      • Yu L.X.
      • Schwabe R.F.
      The role of cancer-associated fibroblasts and fibrosis in liver cancer.
      ). Briefly, fibrosis leads to the stiffening of the liver microenvironment which can promote malignant transformation of hepatocytes as well as tumour cell proliferation and invasion.
      • Baglieri J.
      • Brenner D.A.
      • Kisseleva T.
      The role of fibrosis and liver-associated fibroblasts in the pathogenesis of hepatocellular carcinoma.
      ,
      • Affo S.
      • Yu L.X.
      • Schwabe R.F.
      The role of cancer-associated fibroblasts and fibrosis in liver cancer.
      Activated myofibroblasts release a plethora of growth factors and cytokines, such as TGF-β, platelet-derived growth factor and hepatocyte growth factor, that suppress immune surveillance and promote tumour angiogenesis, proliferation and invasion.
      • Baglieri J.
      • Brenner D.A.
      • Kisseleva T.
      The role of fibrosis and liver-associated fibroblasts in the pathogenesis of hepatocellular carcinoma.
      ,
      • Affo S.
      • Yu L.X.
      • Schwabe R.F.
      The role of cancer-associated fibroblasts and fibrosis in liver cancer.
      HCV infection primarily promotes fibrogenesis indirectly through chronic liver injury. However, there is some evidence that HCV-infected hepatocytes upregulate the key fibrogenic factor TGF-β through the generation of ROS as well as activation of p38 MAPK, JNK, ERK1/2 and NF-κB-dependent pathways.
      • Lin W.
      • Tsai W.L.
      • Shao R.X.
      • Wu G.
      • Peng L.F.
      • Barlow L.L.
      • et al.
      Hepatitis C virus regulates transforming growth factor beta1 production through the generation of reactive oxygen species in a nuclear factor kappaB-dependent manner.
      Furthermore, TGF-β is also upregulated in vitro by HCV in an endoplasmic reticulum stress-dependant manner involving ROS, JNK and IRE1 signalling pathways
      • Chusri P.
      • Kumthip K.
      • Hong J.
      • Zhu C.
      • Duan X.
      • Jilg N.
      • et al.
      HCV induces transforming growth factor β1 through activation of endoplasmic reticulum stress and the unfolded protein response.
      ; however, whether this mechanism occurs in patients is yet to be confirmed. TGF-β signalling plays a critical role in the progression of fibrosis in NASH,
      • Anderson N.
      • Borlak J.
      Molecular mechanisms and therapeutic targets in steatosis and steatohepatitis.
      as well as promoting epithelial-mesenchymal transition which enables tumour cells to invade and metastasise.,
      • Gough N.R.
      • Xiang X.
      • Mishra L.
      TGF-β signaling in liver, pancreas, and gastrointestinal diseases and cancer.
      Systemic metabolic influences can promote fibrosis and myofibroblast activation. To enable activation, hepatic myofibroblasts undergo significant metabolic reprogramming, including upregulation of DNL, glycolysis and oxidative phosphorylation.
      • Smith-Cortinez N.
      • van Eunen K.
      • Heegsma J.
      • Serna-Salas S.A.
      • Sydor S.
      • Bechmann L.P.
      • et al.
      Simultaneous induction of glycolysis and oxidative phosphorylation during activation of hepatic stellate cells reveals novel mitochondrial targets to treat liver fibrosis.
      • Mejias M.
      • Gallego J.
      • Naranjo-Suarez S.
      • Ramirez M.
      • Pell N.
      • Manzano A.
      • et al.
      CPEB4 increases expression of PFKFB3 to induce glycolysis and activate mouse and human hepatic stellate cells, promoting liver fibrosis.
      • Bates J.
      • Vijayakumar A.
      • Ghoshal S.
      • Marchand B.
      • Yi S.
      • Kornyeyev D.
      • et al.
      Acetyl-CoA carboxylase inhibition disrupts metabolic reprogramming during hepatic stellate cell activation.
      Myofibroblast activation can be further altered by systemic factors upregulated in metabolic disease: i) myofibroblasts express the insulin receptor and, in response to both insulin and IGF1 (insulin-like growth factor 1), upregulate TGF-β and CTGF (connective tissue growth factor) release, exacerbating fibrogenesis in the context of hyperinsulinemia and NASH
      • Cai C.X.
      • Buddha H.
      • Castelino-Prabhu S.
      • Zhang Z.
      • Britton R.S.
      • Bacon B.R.
      • et al.
      Activation of insulin-PI3K/Akt-p70S6K pathway in hepatic stellate cells contributes to fibrosis in nonalcoholic steatohepatitis.
      • Svegliati-Baroni G.
      • Ridolfi F.
      • Di Sario A.
      • Casini A.
      • Marucci L.
      • Gaggiotti G.
      • et al.
      Insulin and insulin-like growth factor-1 stimulate proliferation and type I collagen accumulation by human hepatic stellate cells: differential effects on signal transduction pathways.
      • Paradis V.
      • Perlemuter G.
      • Bonvoust F.
      • Dargere D.
      • Parfait B.
      • Vidaud M.
      • et al.
      High glucose and hyperinsulinemia stimulate connective tissue growth factor expression: a potential mechanism involved in progression to fibrosis in nonalcoholic steatohepatitis.
      ; ii) myofibroblasts also express functional leptin receptors: increased circulating levels of leptin have been described in both obesity
      • Myers M.G.
      • Leibel R.L.
      • Seeley R.J.
      • Schwartz M.W.
      Obesity and leptin resistance: distinguishing cause from effect.
      and CHC
      • Manolakopoulos S.
      • Bethanis S.
      • Liapi C.
      • Stripeli F.
      • Sklavos P.
      • Margeli A.
      • et al.
      An assessment of serum leptin levels in patients with chronic viral hepatitis: a prospective study.
      and can promote myofibroblast proliferation, collagen secretion and suppression of apoptosis,
      • Marra F.
      Leptin and liver tissue repair: do rodent models provide the answers?.
      thereby potentially worsening CHC progression. In a chronic pro-fibrotic niche led by HCV, steatosis, lipotoxicity and adipokines can worsen the chronic activation of wound healing responses and potentiate the mechanisms that lead to HCC development and progression.

      HCC in CHC: Systemic metabolic changes and steatosis fuel tumour development and progression

      The aforementioned metabolic impairment associated with CHC might also directly contribute to cancer
      • Gallagher E.J.
      • LeRoith D.
      Hyperinsulinaemia in cancer.
      : obesity, hyperinsulinemia and diabetes are associated with a greater risk of developing a number of cancers, including colorectal, breast, endometrial, pancreatic, ovarian, liver and gastric cancer.
      • Davila J.A.
      • Morgan R.O.
      • Shaib Y.
      • McGlynn K.A.
      • El-Serag H.B.
      Diabetes increases the risk of hepatocellular carcinoma in the United States: a population based case control study.
      • Avgerinos K.I.
      • Spyrou N.
      • Mantzoros C.S.
      • Dalamaga M.
      Obesity and cancer risk: emerging biological mechanisms and perspectives.
      • Stone T.W.
      • McPherson M.
      • Gail Darlington L.
      Obesity and cancer: existing and new hypotheses for a causal connection.
      • Deng T.
      • Lyon C.J.
      • Bergin S.
      • Caligiuri M.A.
      • Hsueh W.A.
      Obesity, inflammation, and cancer.
      Indeed hyperinsulinemia has a direct effect on cell proliferation and HCC behaviour. First, in the context of fatty liver and hyperinsulinemia, transformed hepatocytes that escape apoptosis/necrosis are exposed (like the surrounding liver parenchyma) to intense metabolic pressure (imposed by hyperglycaemia, hyperinsulinemia, lipotoxicity, and oxidative stress) that will preserve partial intracellular insulin signalling (providing selective advantages to pre-malignant hepatocytes).
      • Shintani Y.
      • Fujie H.
      • Miyoshi H.
      • Tsutsumi T.
      • Tsukamoto K.
      • Kimura S.
      • et al.
      Hepatitis C virus infection and diabetes: direct involvement of the virus in the development of insulin resistance.
      ,
      • Kawaguchi T.
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      • Harada M.
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      • Nagao Y.
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      • et al.
      Hepatitis C virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3.
      ,
      • Miyamoto H.
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      • Moriya K.
      • Murata S.
      • Tanaka K.
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      • et al.
      Involvement of the PA28gamma-dependent pathway in insulin resistance induced by hepatitis C virus core protein.
      ,
      • Aytug S.
      • Reich D.
      • Sapiro L.E.
      • Bernstein D.
      • Begum N.
      Impaired IRS-1/PI3-kinase signaling in patients with HCV: a mechanism for increased prevalence of type 2 diabetes.
      In other words, transformed cells find avenues to improve insulin signalling and glucose uptake to fulfil their metabolic needs.
      • Samuel V.T.
      • Shulman G.I.
      Mechanisms for insulin resistance: common threads and missing links.
      Indeed, HCC features the activation of insulin signalling
      • Sakurai Y.
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      • Obata A.
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      Role of insulin receptor substrates in the progression of hepatocellular carcinoma.
      • Hall Z.
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      • Scott E.
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      • et al.
      Lipid remodeling in hepatocyte proliferation and hepatocellular carcinoma.
      • Chettouh H.
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      • Wendum D.
      • Clapéron A.
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      • et al.
      Mitogenic insulin receptor-A is overexpressed in human hepatocellular carcinoma due to EGFR-mediated dysregulation of RNA splicing factors.
      and the main downstream effectors of the insulin receptor (the PI3K-AKT-mTOR and the RAS-MAPK pathways) are also known drivers of cell proliferation
      • Shintani Y.
      • Fujie H.
      • Miyoshi H.
      • Tsutsumi T.
      • Tsukamoto K.
      • Kimura S.
      • et al.
      Hepatitis C virus infection and diabetes: direct involvement of the virus in the development of insulin resistance.
      ,
      • Kawaguchi T.
      • Yoshida T.
      • Harada M.
      • Hisamoto T.
      • Nagao Y.
      • Ide T.
      • et al.
      Hepatitis C virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3.
      ,
      • Miyamoto H.
      • Moriishi K.
      • Moriya K.
      • Murata S.
      • Tanaka K.
      • Suzuki T.
      • et al.
      Involvement of the PA28gamma-dependent pathway in insulin resistance induced by hepatitis C virus core protein.
      ,
      • Aytug S.
      • Reich D.
      • Sapiro L.E.
      • Bernstein D.
      • Begum N.
      Impaired IRS-1/PI3-kinase signaling in patients with HCV: a mechanism for increased prevalence of type 2 diabetes.
      and survival
      • Sakurai Y.
      • Kubota N.
      • Takamoto I.
      • Obata A.
      • Iwamoto M.
      • Hayashi T.
      • et al.
      Role of insulin receptor substrates in the progression of hepatocellular carcinoma.
      ,
      • Celton-Morizur S.
      • Merlen G.
      • Couton D.
      • Margall-Ducos G.
      • Desdouets C.
      The insulin/Akt pathway controls a specific cell division program that leads to generation of binucleated tetraploid liver cells in rodents.
      ,
      • Wang F.
      • Bank T.
      • Malnassy G.
      • Arteaga M.
      • Shang N.
      • Dalheim A.
      • et al.
      Inhibition of insulin-like growth factor 1 receptor enhances the efficacy of sorafenib in inhibiting hepatocellular carcinoma cell growth and survival.
      in hepatocytes and HCC cells, as well as being promoters of angiogenesis.
      • Tovar V.
      • Alsinet C.
      • Villanueva A.
      • Hoshida Y.
      • Chiang D.Y.
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      • et al.
      IGF activation in a molecular subclass of hepatocellular carcinoma and pre-clinical efficacy of IGF-1R blockage.
      Second, both proliferating hepatocytes and HCC cells similarly rewire cell metabolism, reprogramming (glucose/lipid/cholesterol) biosynthetic pathways to generate specific lipid species (e.g. phosphatidylcholine enriched in monounsaturated fatty acid) to fuel cell division and create new membranes, or to limit oxidative stress, thereby ensuring proliferation and survival.
      • Hall Z.
      • Chiarugi D.
      • Charidemou E.
      • Leslie J.
      • Scott E.
      • Pellegrinet L.
      • et al.
      Lipid remodeling in hepatocyte proliferation and hepatocellular carcinoma.
      ,
      • Lally J.S.V.
      • Ghoshal S.
      • DePeralta D.K.
      • Moaven O.
      • Wei L.
      • Masia R.
      • et al.
      Inhibition of acetyl-CoA carboxylase by phosphorylation or the inhibitor ND-654 suppresses lipogenesis and hepatocellular carcinoma.
      ,
      • Lupberger J.
      • Croonenborghs T.
      • Roca Suarez A.A.
      • Van Renne N.
      • Jühling F.
      • Oudot M.A.
      • et al.
      Combined analysis of metabolomes, proteomes, and transcriptomes of hepatitis C virus-infected cells and liver to identify pathways associated with disease development.
      The upregulation of DNL and the formation of complex lipids, potentiated by obesity, are therefore adaptive metabolic responses that may fuel HCC cells, as shown by multiple lines of evidence: i) the upregulation of DNL and fatty acid desaturation has been widely described in HCC (including HCV-associated HCC) and is considered a prognostic factor for patient survival
      • Yang W.
      • Hood B.L.
      • Chadwick S.L.
      • Liu S.
      • Watkins S.C.
      • Luo G.
      • et al.
      Fatty acid synthase is up-regulated during hepatitis C virus infection and regulates hepatitis C virus entry and production.
      ,
      • Wu J.M.
      • Skill N.J.
      • Maluccio M.A.
      Evidence of aberrant lipid metabolism in hepatitis C and hepatocellular carcinoma.
      ,
      • Calvisi D.F.
      • Wang C.
      • Ho C.
      • Ladu S.
      • Lee S.A.
      • Mattu S.
      • et al.
      Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma.
      • Liu G.
      • Liu G.
      • Cui X.
      • Xu Y.
      Transcriptomic data analyses reveal a reprogramed lipid metabolism in HCV-derived hepatocellular cancer.
      • Lien E.C.
      • Westermark A.M.
      • Zhang Y.
      • Yuan C.
      • Li Z.
      • Lau A.N.
      • et al.
      Low glycaemic diets alter lipid metabolism to influence tumour growth.
      ; ii) HCC growth/malignancy can be stratified on the basis of acetate metabolism and acetyl-CoA synthase 1 expression
      • Björnson E.
      • Mukhopadhyay B.
      • Asplund A.
      • Pristovsek N.
      • Cinar R.
      • Romeo S.
      • et al.
      Stratification of hepatocellular carcinoma patients based on acetate utilization.
      ; iii) the ablation/inhibition of key DNL enzymes (e.g. fatty acid synthase, or acetyl-coenzyme A carboxylase [ACC]) protects mice from HCC.
      • Lally J.S.V.
      • Ghoshal S.
      • DePeralta D.K.
      • Moaven O.
      • Wei L.
      • Masia R.
      • et al.
      Inhibition of acetyl-CoA carboxylase by phosphorylation or the inhibitor ND-654 suppresses lipogenesis and hepatocellular carcinoma.
      ,
      • Hu J.
      • Che L.
      • Li L.
      • Pilo M.G.
      • Cigliano A.
      • Ribback S.
      • et al.
      Co-activation of AKT and c-Met triggers rapid hepatocellular carcinoma development via the mTORC1/FASN pathway in mice.
      Moreover, the fact that several genes encoding metabolic enzymes involved in fatty acid uptake (e.g. fatty acid binding protein 4/5) strongly correlate with cancer survival confirms how crucial lipid supply is for HCC biology.
      • McKillop I.H.
      • Girardi C.A.
      • Thompson K.J.
      Role of fatty acid binding proteins (FABPs) in cancer development and progression.
      As such, lifestyle interventions (e.g. low carbohydrate diets) or therapies improving systemic IR and/or directly targeting DNL (such as metformin or ACC inhibitors), are attractive and show efficacy in rodent models of HCC
      • Lally J.S.V.
      • Ghoshal S.
      • DePeralta D.K.
      • Moaven O.
      • Wei L.
      • Masia R.
      • et al.
      Inhibition of acetyl-CoA carboxylase by phosphorylation or the inhibitor ND-654 suppresses lipogenesis and hepatocellular carcinoma.
      ,
      • Bhalla K.
      • Hwang B.J.
      • Dewi R.E.
      • Twaddel W.
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      Metformin prevents liver tumorigenesis by inhibiting pathways driving hepatic lipogenesis.
      and, more generally, in cancer.
      • Lien E.C.
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      • Zhang Y.
      • Yuan C.
      • Li Z.
      • Lau A.N.
      • et al.
      Low glycaemic diets alter lipid metabolism to influence tumour growth.
      However, robust clinical evidence of the efficacy of such agents in the prevention of de novo carcinogenesis or of HCC progression is lacking.
      All the aforementioned phenomena are intrinsically linked and, through the process of amplifying each other, might provide (part of) the basis for the high prevalence of HCC in HCV. In patients with HCV genotype 3 these processes will be mainly driven by the virus and should be partially reversed by antiviral treatment; in patients with HCV genotypes 1/2, the hepatic/systemic metabolic disturbances will persist, ultimately conferring an increased risk of cancer (unless pre-existing obesity, IR and diabetes are appropriately managed).
      Last, it should be kept in mind that diabetes is a major driver of hepatocellular carcinogenesis with a 2–3-fold increase in the risk of HCC independent of the presence of other major HCC risk factors including HCV infection
      • Davila J.A.
      • Morgan R.O.
      • Shaib Y.
      • McGlynn K.A.
      • El-Serag H.B.
      Diabetes increases the risk of hepatocellular carcinoma in the United States: a population based case control study.
      : this suggests that systemic metabolic disturbances might play a greater role than HCV per se in the development of HCC in patients with CHC.

      Strategies aimed at reversing metabolic dysfunction and cancer prevention

      As already described, a subset of patients with CHC remain at risk of liver-related morbidity despite SVR. In particular, metabolic risk factors appear to play a dominant role in this persisting risk. Lifestyle interventions such as exercise and diet could therefore be effective in modifying the risk of progression of chronic liver disease, preventing fibrotic progression and the development of HCC.
      • Baumeister S.E.
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      Association between physical activity and risk of hepatobiliary cancers: a multinational cohort study.
      • Turati F.
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      • Polesel J.
      • Bravi F.
      • Rossi M.
      • Talamini R.
      • et al.
      Mediterranean diet and hepatocellular carcinoma.
      • Zhang X.
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      • Chu E.S.
      • Fu K.
      • Lau H.C.H.
      • Wang Y.X.
      • et al.
      Dietary cholesterol drives fatty liver-associated liver cancer by modulating gut microbiota and metabolites.
      Several large cohort studies (although not specific to HCV) have demonstrated an inverse association between physical activity rates and risk of HCC.
      • Baumeister S.E.
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      • Aleksandrova K.
      • Jochem C.
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      • Gunter M.J.
      • et al.
      Association between physical activity and risk of hepatobiliary cancers: a multinational cohort study.
      ,
      • Chun H.S.
      • Park S.
      • Lee M.
      • Cho Y.
      • Kim H.S.
      • Choe A.R.
      • et al.
      Association of physical activity with the risk of hepatocellular carcinoma in patients with chronic hepatitis B.
      ,
      • Luo X.
      • Yang W.
      • Ma Y.
      • Simon T.G.
      • Meyerhardt J.A.
      • Chan A.T.
      • et al.
      Physical activity and risk of hepatocellular carcinoma among U.S. Men and women.
      Mechanistic studies have revealed that exercise modulates key signalling and metabolic pathways involved in proliferation and angiogenesis.
      • Piguet A.C.
      • Saran U.
      • Simillion C.
      • Keller I.
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      • Reeves H.L.
      • et al.
      Regular exercise decreases liver tumors development in hepatocyte-specific PTEN-deficient mice independently of steatosis.
      • Arfianti A.
      • Pok S.
      • Barn V.
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      Exercise retards hepatocarcinogenesis in obese mice independently of weight control.
      • Bianchi A.
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      • Hall Z.
      • Lemos H.
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      • Paish H.
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      Moderate exercise inhibits age-related inflammation, liver steatosis, senescence, and tumorigenesis.
      • Saran U.
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      • Simillion C.
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      Anti-tumoral effects of exercise on hepatocellular carcinoma growth.
      A case control study reported that the Mediterranean diet appears to be protective against HCC, especially in CHC.
      • Turati F.
      • Trichopoulos D.
      • Polesel J.
      • Bravi F.
      • Rossi M.
      • Talamini R.
      • et al.
      Mediterranean diet and hepatocellular carcinoma.
      The mechanistic link between diet and HCC risk is likely multifactorial; the effect of limiting consumption of foods with a high (refined) carbohydrate content is known to be beneficial in i) reducing the steatotic burden and the progression of liver disease
      • Neuschwander-Tetri B.A.
      Pharmacologic management of nonalcoholic steatohepatitis.
      ; ii) decreasing the metabolic pressure on cell proliferation induced by insulin signalling, and iii) preventing the intracellular metabolic changes observed in proliferating/HCC cells. Moreover, as has also been proposed in NASH, reports suggest that dietary cholesterol intake is associated with an increased risk of disease progression in patients with CHC and advanced fibrosis or cirrhosis.
      • Zhang X.
      • Coker O.O.
      • Chu E.S.
      • Fu K.
      • Lau H.C.H.
      • Wang Y.X.
      • et al.
      Dietary cholesterol drives fatty liver-associated liver cancer by modulating gut microbiota and metabolites.
      ,
      • Ioannou G.N.
      The role of cholesterol in the pathogenesis of NASH.
      ,
      • Enjoji M.
      • Yasutake K.
      • Kohjima M.
      • Nakamuta M.
      Nutrition and nonalcoholic Fatty liver disease: the significance of cholesterol.
      A systematic evaluation of the benefit of pharmacological interventions (aimed at treating specific features of MetS) in reducing the risk of fibrotic progression and HCC is in its infancy, especially in CHC; however, there is a growing body of evidence to suggest lipophilic statins, metformin and aspirin may be beneficial.
      • Chen H.P.
      • Shieh J.J.
      • Chang C.C.
      • Chen T.T.
      • Lin J.T.
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      • et al.
      Metformin decreases hepatocellular carcinoma risk in a dose-dependent manner: population-based and in vitro studies.
      • Ma S.
      • Zheng Y.
      • Xiao Y.
      • Zhou P.
      • Tan H.
      Meta-analysis of studies using metformin as a reducer for liver cancer risk in diabetic patients.
      • Simon T.G.
      • Duberg A.S.
      • Aleman S.
      • Chung R.T.
      • Chan A.T.
      • Ludvigsson J.F.
      Association of aspirin with hepatocellular carcinoma and liver-related mortality.
      • Simon T.G.
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      • Aleman S.
      • Hagstrom H.
      • Nguyen L.H.
      • Khalili H.
      • et al.
      Lipophilic statins and risk for hepatocellular carcinoma and death in patients with chronic viral hepatitis: results from a nationwide Swedish population.
      • Simon T.G.
      • King L.Y.
      • Zheng H.
      • Chung R.T.
      Statin use is associated with a reduced risk of fibrosis progression in chronic hepatitis C.
      Lipophilic statins such as simvastatin and atorvastatin modulate cholesterol synthesis and have been shown to reduce the risk of CHC-HCC in a large prospective propensity score matched cohort study,
      • Simon T.G.
      • Duberg A.S.
      • Aleman S.
      • Hagstrom H.
      • Nguyen L.H.
      • Khalili H.
      • et al.
      Lipophilic statins and risk for hepatocellular carcinoma and death in patients with chronic viral hepatitis: results from a nationwide Swedish population.
      as well as reducing the risk of fibrotic progression in a prospective study.
      • Simon T.G.
      • King L.Y.
      • Zheng H.
      • Chung R.T.
      Statin use is associated with a reduced risk of fibrosis progression in chronic hepatitis C.
      Metformin has been shown to be well tolerated and safe in patients with CHC and T2DM, and it may also reduce the risk of liver-related mortality and HCC development beyond its effect on diabetic control;
      • Chen H.P.
      • Shieh J.J.
      • Chang C.C.
      • Chen T.T.
      • Lin J.T.
      • Wu M.S.
      • et al.
      Metformin decreases hepatocellular carcinoma risk in a dose-dependent manner: population-based and in vitro studies.
      ,
      • Ma S.
      • Zheng Y.
      • Xiao Y.
      • Zhou P.
      • Tan H.
      Meta-analysis of studies using metformin as a reducer for liver cancer risk in diabetic patients.
      ,
      • Zhou Y.Y.
      • Zhu G.Q.
      • Liu T.
      • Zheng J.N.
      • Cheng Z.
      • Zou T.T.
      • et al.
      Systematic review with network meta-analysis: antidiabetic medication and risk of hepatocellular carcinoma.
      ,
      • Harris K.