1.2 Chronic liver damage caused by viral infection and environmental factors

Chronic liver damage caused by viral infection and environmental factors (such as alcohol or metabolic syndrome) can result in increased risk for HCC. The cirrhotic liver is a permissive factor for HCC due to the large regenerative activity repetitive damage-regeneration cycles or formation of dysplastic nodules (International Consensus Group for Hepatocellular Neoplasia, 2009). Thus, the cirrhotic liver may be considered a pre-neoplastic “cancer field” comprised of genetically abnormal but non-neoplastic tissue that is at high risk for malignant transformation (Figure 1) (Braakhuis et al., 2003). Understanding the molecular mechanisms of HCV-induced hepatocarcinogenesis will aid in the development of improved therapeutic modalities (Crippin et al., 2002).

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FIGURE 1

Ten hallmarks for cancer development via HCV. Ten hallmarks of cancer are triggered by HCV, including 1) genetic changes, 2) angiogenesis, 3) defective tumor immunity, 4) immortalization, 5) metabolic reprogramming, 6) excessive and prolonged inflammation, 7) migration/invasion/metastasis, 8) evading cell cycle arrest, 9) anti-cell death and 10) compensatory regeneration/proliferation. HCV core protein, E1, E2, NS3, NS5A, and NS5B are involved in progression of HCV-induced HCC.

HCV-associated cirrhosis can result in HCC by ultimately promoting tumor-initiating stem-like cells (TIC) formation since deposition of extracellular matrix (ECM), including collagen and laminin, promotes tumor-prone microenvironment. These TICs can develop into several different types of liver cancer, e.g., HCC and cholangiocarcinoma (CC). TICs are resistant to conventional chemotherapy and immunotherapy and persist as recurrent tumors or circulating tumor cells. TICs share key features with embryonic stem cells (ESCs) present in preimplantation blastocyst stage embryos, including the expression of a core pluripotency-associated transcription factor (TF) network (Kim et al., 2010; Ikushima et al., 2011). Liver progenitor cells have asymmetric cell division process. In untransformed stem cells, self-renewal occurs through asymmetric cell division, in which one daughter cell retains the multipotent progenitor status of its parent while the other cell commits to a specialized cell fate. In contrast to ESCs, TICs fail to control the self-renewing mode of cell division. As asymmetric cell division mechanism is disrupted in HCCs, TICs exhibit a loss of this intrinsic asymmetry without regulated differentiated daughter cells, leading to the ectopic implementation of stem cell gene expression programs in both progeny cells. This leads to subsequent unchecked expansion of the progenitor cell pool (Cicalese et al., 2009; Knoblich, 2010; Martin-Belmonte and Perez-Moreno, 2012). Thus, understanding the mechanism of genesis of TICs paves the way for novel therapeutic approaches, including cell fate-determinant molecule NUMB, which is p53-MDM2 associated proteins. This key cell fate determinant molecules are targeted by interacting protein TBC1D15 in TICs (Feldman et al., 2013).

2.2 Variations in TP53, CTNNB1, ARID1A, and non-coding regions

Mutations in tumor-suppressor genes or proto-oncogenes or the activity of growth factors during chronic HCV infection transforms hepatocytes, cholangiocytes, and liver progenitor cells (Simonetti et al., 1992). Whole genome and exome analysis demonstrated that TP53, CTNNB1, and chromatin modulators, including ARID1A and ARID2, are the most frequently mutated coding genes in HCC (Shibata and Aburatani, 2014; Totoki et al., 2014; Fujimoto et al., 2016). Loss-of-function ARID1A mutations are correlated with poor prognosis, sorafenib resistance, HCC invasion and metastasis (Nhieu et al., 1999). The most frequently mutated driver genes in human alcohol-associated HCCs, but not in dysplastic macronodules (Guichard et al., 2012), are in the chromatin remodeling complex (ARID1A) (8%–38%), β-Catenin/Wnt (activating mutations in exon 3 of CTNNB1), and TP53 (30%–65%). CTNNB1 point mutations occurred in serine residues (Ser 33, 37, and 45) and through the destruction complex (GSK3β and CSNK1A1). The more frequent mutations in non-coding regions are found in the TERT promoter and TFPI2. Long intergenic noncoding RNAs, including NEAT1 and MALAT1, are also frequently mutated (Fujimoto et al., 2016). Genomic gain of function causes focal amplifications in cancer-related genes such as VEGFA and FGF3/4/19/CCND1, which is associated with a good response to the multi-kinase inhibitor sorafenib (Arao et al., 2013; Llovet, 2014). Point mutations and also STV breakpoints in HCC tissues are detected in cancer-driver genes, including TERT, ARID1A, ARID2, and PTEN, (Fujimoto et al., 2016). Therefore, both nucleotide variants and STVs of chromosomes are detected in hepatitis virus-related HCCs. These diver mutations direct self0renewal ability of TICs.

The take-away message is that tumor driver gene mutations in TP53, CTNNB1, ARID1A make hepatocytes susceptible for HCC development. These driver mutation are targeted and translated into therapies, including ICG-001 for CTNNB1 mutation (Delgado et al., 2014; Lin et al., 2016) and Adenovirus expressing functional p53 (Anderson et al., 1998). The status of current related therapeutic strategies showed promising responses for ICG-001 therapy and Ad-p53 (Anderson et al., 1998).

2.3 Induction of mutator phenotype

HCV-associated HCC has a 3–5 times higher mutation rate than associated non-tumor liver tissues. Furthermore, the ratio of amino acid replacement and silent mutations in the tumors, but not in the neighboring non-tumor tissues, was significantly higher than the ratio expected in the absence of selection of growth advantage of premalignant cells (Machida et al., 2004a) since chronic hepatocellular turnover could select for cells with genetic or epigenetic changes that confer growth advantages allowing clonal expansion. For example, HCV infection induced a mutator phenotype, which involves enhanced mutations in many somatic genes, including immunoglobulin (Ig) genes, proto-oncogenes and tumor suppressor genes (Machida et al., 2004a). HCV infection promotes error-prone DNA polymerase expression and increases mutation frequencies by induction of ROS and reactive nitrogen species and by inhibition of DNA repair mechanisms (Machida et al., 2004a). Therefore, HCV-induced mutations contribute to the eventual selection and amplification of certain deleterious mutations of proto-oncogenes or tumor suppressor genes in tumors. Accordingly HCV-associated oncogenesis is characterized by a long latency period due to the need for multiple hits.

These mutator phenotype can be great target for immune checkpoint inhibitor since high genetic instability is good prognosis maker for immune checkpoint inhibitor. Indeed, immune checkpoint inhibitor Anti-PD-1 therapy got FDA approval after landmark clinical trial (El-Khoueiry et al., 2017).

2.4 Reactive oxygen species

Viral or immune-mediated reactive oxygen species (ROS) induce oxidative stress. ROS-associated oxidative DNA damage (such as 8-oxo-dG) promotes DNA mutagenesis, leading to oncogenic transformation in chronic hepatitis C (Figure 2). For example, the HCV core protein promotes formation of intracellular ROS, both in vitro (Okuda et al., 2002) and in vivo (Korenaga et al., 2005), via its localization to the mitochondria and inhibition of electron transport (Korenaga et al., 2005). The electron flow that interacts with oxygen molecules results in formation ROS prior to reaching the cytochrome oxidase complex. HCV infection induces ROS (Choi and Ou, 2006), which leads to oxidative DNA damages and lipid peroxidation in HCV-infected cells (Machida et al., 2006). Consequently pretreatment of HCV-infected cells with ROS inhibitors prevented mitochondrial damage and the production of ROS (Machida et al., 2006). ROS with NO (Machida et al., 2004b) induces steatosis (through lipid peroxides) and oncogenesis (through DNA mutations and STAT3 activation), leading to acute hepatocyte damage (via production of STAT3) (Machida et al., 2006). Individual liver cells undergo disease evolution to reproduce HCV-associated HCC (Salk et al., 2010). Activated inflammatory cells (CTL or NK) also release ROS and nitrogen (RNS) species and induce lipid peroxidation (Bartsch and Nair, 2006), leading to a pro-carcinogenic microenvironment. Viral particle was consisted with structural proteins, nucleocapside Core, Envelop genes E1 and E2 and p7. Non-structural proteins are consisted of Serine protease NS2 and NS3, protease cofactor NS4A, NS4B, NS5A, and RNA-dependent RNA polymerase NS5B (Holmes and Chung, 2016). The expression of some HCV proteins, in particular structural nucleocapside protein core and non-structural viral protein NS5A, may also contribute directly to the induction of oxidative stress (Figure 2).

The take-away message is that HCV-associated ROS production promotes DNA damages and inhibits DNA repair, leading to mutator phenotypes. These HCV-associated mutaor phenotype can be targeted by vitamin treatment. However current clinical reports are not optimistic for HCC patients.

7.2 Development of HCC in HCV patients with SVR and the role of TICs

Direct-acting antiviral agents might promote tumour occurrence in patients with cirrhosis, or recurrence in patients with presumed cure of hepatocellular carcinoma while DAA significantly reduces viral load. In view of the potential clinical implications, this controversy calls for a thorough and expeditious consideration of the hypothetical oncogenic activity of novel HCV drugs (Llovet and Villanueva, 2016).

Due to the “new” antiviral drugs available for HCV treatment, the incidence of HCC has changed. However, some patients who achieved sustained viral response develop HCC.

7.3 Toll-like receptor signaling

Mouse hepatocytes that express HCV-NS5A in liver upregulate the expression of Toll-like receptor 4 (TLR4) and develop liver tumors containing NANOG positive, tumor-initiating stem-like cells (TICs). The TLR signaling pathway is often upregulated in chronic liver diseases, especially since many different liver cell types express TLRs (Testro and Visvanathan, 2009). Hepatocytes express TLR1 through TLR9. Stellate cells express TLR2, 3, and 4. Bile duct epithelium expresses TLR2, 3, 4, and 5. Kupffer cells express TLR2, 3, and 4. Chronic alcohol consumption is associated with activation of TLR1, 2, and 6–9, which further increases the TNF-α response to LPS in mice (Testro and Visvanathan, 2009). Human monocytes exposed to ethanol for a week develop hypersensitivity to LPS through decreased IRAK-M expression, which activates mitogen-activated protein kinase (MAPK) and NF-κB following TLR4 signaling. This leads to activation of NF-κB, AP-1, and ERK (Mandrekar et al., 2009) and associated inflammatory response.

8.2 Epigenetic changes to sustain proliferation

Epigenetics is stable alterations in gene expression without genetic modifications in the sequence (Herceg, 2007). Epigenetic and genetic mechanisms silences cellular genes leading to transformation in human cancers, including HCC (Vaissiere et al., 2008). Contribution of different epigenetic factors, including genomic DNA methylation, histone modifications, and miRNA regulation, contribute to HCC dissemination, invasion, and metastasis. The reversal of deregulated epigenetic changes is emerging treatment of HCC (Nishida and Goel, 2011). High-throughput screening provides targeting inflammation-epigenome cross-talk in HCC to discover novel epigenetic targets (Herceg and Paliwal, 2011). Epigenetic Mechanism promotes the HBV/HCV-Related HCC tumorigenesis (Rongrui et al., 2014)

8.3 LncRNAs in HCV-associated HCC

Non-coding RNAs [LncRNA and microRNA (miRNA)] are dysregulated in HCV-induced liver carcinogenesis via regulation of gene expression. LncRNAs expression (LINC01419, AK021443, UCA1, and WRAP53) are increased in HCV-related HCCs compared to non-cancerous tissues (dysplasia) while lncRNA AF070632 is decreased in advanced HCC samples compared with early HCC. These lncRNAs are associated with Child-Pugh score. LINC01419 and AK021443 were mostly involved in cell cycle progression, whereas AF070632 regulates cofactor binding, oxidation-reduction and carboxylic acid catabolic process (Zhang et al., 2015). Two lncRNAs, including urothelial carcinoma associated-1 (UCA1) and WD repeat containing, antisense to TP53 (WRAP53) are upregulated in serum. UCA1 and WRAP53 (+) HCC patients had a decreased recurrence-free survival (RFS) and increased cumulative hazards. WRAP53 was an independent prognostic factor of RFS (Kamel et al., 2016). Some of these lncRNAs were dysregulated predominantly in one specific hepatitis virus-related HCC, including PCAT-29 in HBV-related HCC, aHIF and PAR5 in HCV-related HCC, and Y3 in HDV-related HCC. DBH-AS1, hDREH and hPVT1 were differentially expressed in HCC of different viral etiology (Zhang et al., 2016).

LncRNA and miRNAs have been associated with HCC (Wong et al., 2018). Due to this review being focused on TICs, more details about TICs and non-coding RNAs are described (such as (Huang et al., 2018; Machida, 2020; Rojas et al., 2022): More details about TICs and non-coding RNAs are included such as: (Huang et al., 2018; Machida, 2020; Rojas et al., 2022). In Table 1 HCV-associated HCC are involved in alterations of lncRNAs [(Hou and Bonkovsky, 2013; Lange et al., 2013; Zhang et al., 2015; Zhang et al., 2016; Fu et al., 2016; Shi et al., 2016; Hai et al., 2017; Wong et al., 2018; Sur et al., 2018; Toraih et al., 2018; Wang et al., 2018; Zhang et al., 2018; Cheng et al., 2019; Refai et al., 2019; Wang et al., 2019; Wu et al., 2019; Yang et al., 2019; Zhao et al., 2019; Zheng et al., 2019; Zhong et al., 2019; El-Khazragy et al., 2020; Ferrasi et al., 2020; Lorini et al., 2020; Machida, 2020; Mohyeldeen et al., 2020; Morishita et al., 2020; Oura et al., 2020; Unfried and Fortes, 2020; Jing et al., 2021; Sabry et al., 2021; Wong and Wong, 2021; Yao et al., 2021; Wang et al., 2022; Wei et al., 2022)]. Upregulated lncRNAs include NORAD (LINC00657), HCP5, lnc-HOTAIR (HOX antisense intergenic RNA), CASC11, HEIM, eosinophil granule ontogeny transcript (EGOT), lncRNA SEMA3B-AS1 [SEMA3B Antisense RNA 1 (Head To Head)], TPT-1S, LINC01189. In contrast, TPT1-AS1, LINC01152, aHIF and PWAR5 (PAR5) are downregulated.

TABLE 1

LncRNAs linked to HCV-associated HCC.

lncRNA	Classification	Size (kb)	Tissues	Expression	Function	Reference
BC017743	Unknown	2.3	Liver	Up	Tumor suppressor region	Zhang et al. (2016)
BC043430	Unknown	1.9	Liver	Up	Tumor suppressor region	Zhang et al. (2016)
LINC01152	Unknown	3.1	Liver	Down	Unknown	Zhang et al. (2016)
aHIF	Unknown	1.0	Liver	Down	Poor prognostic outcomes	Zhang et al. (2016)
PWAR5 (PAR5)	Unknown	~3.6	Liver	Down	Poor prognostic outcomes	Zhang et al. (2016)
AF070632	Unknown	~1.9	Liver	Down	LncRNA-protein interaction,supress angiogenesis,potential biomarker and therapeutic target	Zhang et al. (2015)
AK021443	Unknown	~1.6	Liver	Up	Cell cycle regulation, Proliferation	Zhang et al. (2015)
LINC01419	Unknown	~5.1	Liver	Up	Cell cycle regulation, Proliferation	Zhang et al. (2015)
UCA1	Unknown	~7.3	Liver, bladder, gastric, ovary, esophagus	Up	LncRNA-miRNA interaction, Proliferation	Kamel et al. (2016)
WRAP53	Antisense	~1.8	Liver	Up	Unknown, Biomarker	Kamel et al. (2016)
NORAD (LINC00657)	Unknown	5.3	Liver		Impairs Wee1 Expression, molecular decoy for PUMILIO proteins (PUM1/PUM2)	Sur et al. (2018)
HCP5	Unknown		Liver	Up	rs2244546 in HCP5 as a novel tagging SNP, a Hybrid HLA Class I Endogenous Retroviral Gene	Lange et al. (2013)
lnc-HOTAIR ((HOX antisense intergenic RNA)	Unknown	2.2	Liver	Up	master regulator of chromatin dynamics and cancer, Predict genotype 4 following direct-acting antivirals therapy	El-Khazragy et al. (2020)
CASC11	Unknown	0.52	Liver	Up	inhibiting miRNA-188-5p, ~2.1 kb upstream of c-Myc	Cheng et al. (2019)
HAND2-AS1	Antisense		Liver, cervical cancer, osteosarcoma	Up	downregulating RUNX2 expression, represses cervical cancer progression by interaction with transcription factor E2F4, represses HIF1α-mediated energy metabolism	Kang et al. (2018); Jing et al. (2021)
PLAC2	Unknown		Liver	Down	Upregulates p53	Zheng et al. (2019)
SAMMSON	Unknown		Liver	Up	negatively regulates miR-9-3p, 30 kb downstream from MITF	Mohyeldeen et al. (2020)
HEIM	Unknown		Liver	Up	serum and exosomes as biomarker in the HCV-related HCC	Zhang et al. (2018)
eosinophil granule ontogeny transcript (EGOT)	Unknown		Liver, Head and neck squamous cell carcinomas (HNSCCs) a	Up	Increases the Expression of HMGA2 via Down-Regulating miR-33a-5p	Wu et al. (2019)
CASC2	Unknown		Liver	CASC2 Down	CASC2 was downregulated in HCC/HCV patients	Refai et al. (2019)
TUG1	Unknown		Liver	Up	TUG1 was overexpressed in relation to HCV and the control group, Tug1 lncRNA locus is essential for male fertility	Refai et al. (2019)
LINC01189	Unknown	1.4	Liver	Up	cell proliferation and chemoresistance through hsa-miR-155-5p	Yao et al. (2021)
lncRNA SEMA3B-AS1 (SEMA3B Antisense RNA 1 (Head To Head))	Antisense		Liver	Up	SEMA3B-AS1 in HCC tissues was inversely correlated with microRNA (miR)-718 and positively correlated with PTEN	Zhong et al. (2019)
MALAT1	Unknown	6.7	Liver	Up	Represent a putative non-invasive prognostic biomarker	Toraih et al. (2018)
TPT1-AS1	Antisense		Liver	Down	Suppresses HCC Cell Proliferation Downregulating CDK	Mohyeldeen et al. (2020)
NEAT1	Unknown	3.7 kb	Liver	Up	accurately differentiated between HCC patients and healthy controls, recruiting and binding to PRC2	Mohyeldeen et al. (2020)
TUG	Unknown	7.1 kb	Liver	Up	accurately differentiated between HCC patients and healthy controls, recruiting and binding to PRC2	Mohyeldeen et al. (2020)
Linc-p21	Unknown		Liver	Up	Unknown, Biomarker	Lim et al. (2019)
H19	Unknown		Liver	Up	Unknown, Biomarker	Lim et al. (2019)
LET	Unknown		Liver	Up	Unknown, Biomarker	Lim et al. (2019)
HULK	Unknown		Liver	Up	IGF2BP1 regulation, Biomarker	Lim et al. (2019)
HOTAIR	Unknown		Liver	Up	Guide of epigenetic repressors	Wong et al. (2018)
HOTTIP	Unknown		Liver	Up	Guide of epigenetic activators	Wong et al. (2018)

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8.4 MicroRNAs

miRNA targets hundred mRNAs, miRs are diagnostic markers and therapeutic target for personalized therapy. miRNAs are differentially expressed in liver cancer and are related to different stages of liver carcinogenesis, supporting the diagnosis and prognosis tools of miRNAs in HCC patient. miRNAs promote or inhibits carcinogenesis via activation of oncogenes and/or suppression of tumor suppressors (Chen, 2005). HCV requires liver-specific miR-122 for replication (Jopling et al., 2005). Sequestering miR-122 in patients leads to a dose-dependent decrease in HCV viremia in phase 2a trial (Janssen et al., 2013). Mice lacking miR-122 have high tumor incidence (Tsai et al., 2012). The miR-122 abundance is reduced in human advanced fibrosis (Trebicka et al., 2013) and in therapresistant HCV patients (Sarasin-Filipowicz et al., 2009). Recruitment of miR-122 to the HCV genome depletes this important liver-specific miR-122. HCV sequesters anti-tumorigenic miR-122 to promote HCC development.

miR-21, miR-17, miR-222, miR-224, and miR-221 are increased in liver cancer (Borel et al., 2012) (Ladeiro et al., 2008) while miR-200, let-7, miR-29, miR-123, miR-122, miR-199a, and miR-199 b are decreased in HCCs (Hou et al., 2011; Huang and He, 2011; Anwar and Lehmann, 2015). miR-199a/b-3p prevents the p21-stimulated kinase 4/Raf/MEK/ERL pathway and suppresses HCC.

Down-regulation of miR-199a/b is associated with poor prognosis and low survival rate (Li et al., 2011). Increased miR-224 is associated with malignancy aggression, deteriorated liver function, and poor prognosis (Wang et al., 2008; Zhuang and Meng, 2015).

8.5 Histone modifications

Induction of HCV proteins or the infection of HCC cells with HCV cell culture (HCVcc) suppresses histone H4 methylation/acetylation and histone H2AX phosphorylation for HCC development, indicating that HCV-induced overexpression of PP2Ac are associated with HCC via deregulation of epigenetic histone modifications (Duong et al., 2010). HCV infection upregulates histone deacetylation (HDAC) activity through affecting hepcidin expression, a key suppressor of iron availability (Miura et al., 2008). The induced HCV oxidative stress leads to suppression of hepcidin expression by increased HDAC function. HCV increases histone deacetylation (HDAC) activity through negative regulator of iron availability (hepcidin expression) (Miura et al., 2008). Furthermore, antiviral agents IFN with epigenetic drugs (such as DNMT inhibitors or HDAC inhibitors) counteract epigenome changes with cytokines (Muller, 2006).

HCV caused epigenetic alteration mainly occurred on DNA repair-related genes. Induction of HCV proteins or the infection of HCC cells with HCVcc inhibits histone H4 methylation/acetylation and histone H2AX phosphorylation and inhibited DNA damage repair, indicating that HCV-induced overexpression of PP2Ac promotes hepatocarcinogenesis via dysregulation of epigenetic histone modifications (Duong et al., 2010).

10.1 Compensatory regeneration and proliferation

Chronic liver inflammation causes liver injury with compensatory hepatocellular proliferation which indirectly promotes HCC. Regenerative pathways promote dedifferentiation and proliferation to replace damaged tissue in inflammatory liver disease and hepatocellular necrosis associated with chronic hepatitis C infection.

For example, the Wnt/β-catenin pathway is directly altered by HCV. Initially, NS5A promotes β-catenin stabilization through inactivation of GSK3-β, which normally promotes β-catenin degradation (Street et al., 2005; Park et al., 2009). Addiotional study shows that NS5A directly interacts with β-catenin and stabilizes β-catenin (Milward et al., 2010). Similarly GSK3-β is also a target of hepatitis B virus X-protein which results in stabilization of β-catenin. HCV interacts with the TGF-β pathway leading to cytostatic response or fibrogenic responses. HCV NS5A blocks TGF-β signaling through direct interaction with its receptor, TGF-β receptor I (TβR-I) (Choi and Hwang, 2006). Antagonism of TGF-β signalingpromotes liver damage, fibrosis and cancer. Furthemore, HCV core variant isolated from HCC, but not from surrounding liver tissue blocks TGF-β signaling through interactions with SMAD3 (Pavio et al., 2005).

Cells within such a precancerous field contain mutations that predispose progression to a cancerous phenotype. What is the impact of chronic hepatocyte turnover on tumorigenesis? Perhaps apoptosis of infected hepatocytes,either by immune- or virus-mediated mechanisms with compensatory hepatocellular proliferation, promotes carcinogenesis during decades of chronic HCV infection.

The association of liver injury with HCV infection is depicted in Figure 4. Core protein activates cellular oncoproteins and NF-κB cell signaling pathways and causes p53 and pRb inactivation to initiate genomic instability and uncontrollable cellular proliferations (Smirnova et al., 2006). HCV nonstructural proteins NS5A and NS3 alter host expression and promote liver cell proliferation, leading to HCC development (Jeong et al., 2012). NS5A suppresses immune responses, inactivates tumor suppressors, inhibits apoptosis, and disrupts liver homeostas, thus leading a to a primary liver.

The take-away message is that HCV promotes HCC growth by compensatory regeneration and proliferation. Compensatory regeneration and proliferation were targeted and translated into therapies. the status of current related therapeutic strategies showed sorafenib and regorafenib inhibits compensatory regeneration and proliferation.

We thank Stanley M Tahara (USC) for critical comments and editorial assistance.