Immune mediated inflammation caused during chronic HCV infection indirectly triggers hepatocarcinogenesis. Simultaneous with a direct role of HCV in inducement of HCC by altering several cellular pathways involved in metabolism, DNA repair and apoptosis^[14], chronic HCV infection enhances the reactive oxygen species (ROS) which damages the liver cells. At the same time, HCV also induces inflammation by activating hepatic stellate cells (HSCs)^[28]. These HSCs get activated by ROS, growth factors, cytokines, adipokines and chemokines secreted by hepatocytes, Kuffer cells and inflammatory cells^[29]. The progress of disease is increased by cumulative effect of inflammation, ROS, steatosis and IR caused during chronic HCV infection. The activated HSCs, under the effect of fibrogenic cytokines undergo epithelial to mesenchymal trans-differentiation (EMT) into myofibroblast like cells which cause liver fibrosis^[14]. Transforming growth factor beta (TGF-β) cytokine regulates EMT demonstrating its pro-oncogenic functions^[30]. Hepatic fibrosis is closely associated with HCC development. EMT pathway plays a major role in transition of hepatocyte to cancerous cell and process of metastasis known with expression of E-cadherin and Vimentin^[31]. The IR stimulates HSCs and links fibrosis with steatosis. The process of fibrogenesis is regulated by a number of signaling pathways including SMADs, phosphatidylinositide 3-kinases (PI3K), protein kinase (Akt), mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinases (JNKs) pathways. JNK activation by IL1-β cytokine increases fibrogenesis, oncogenesis and cell motility^[32,33]. Thus all these liver alterations finally produce a suitable environment for development of HCC in chronic HCV infection.

It has been observed that HCV genotype-3 induces steatosis in patients with chronic HCV infection^[34]. HCV induces steatosis by increasing lipid synthesis and reducing its secretion and degradation. The structural and nonstructural proteins of HCV directly interfere in lipid synthesis^[35] and very-low-density lipoprotein secretion^[36,37]. These HCV related proteins also inhibit fatty acid oxidation^[38,39] and enhance fatty acid release from adipocytes^[34]. All this finally results in hepatic steatosis. The HCV related proteins are also involved in producing ROS^[40] and glucose homeostasis. HCV interferes with insulin signaling by proteosomal degradation of insulin receptor substrate 1 (IRS-1) and IRS-2 by suppressor of cytokine signaling (SOCS) protein or PI3K/Akt/mTOR pathway. IRS-1 is reported to be inactivated by TGF-α and PI3K/Akt also^[41]. In this manner, the early stage of chronic HCV infection with increasing steatosis and IR creates an environment to help in hepatocarcinogenesis leading to development of HCC.

HCV influences both innate and adaptive immunity. This virus inhibits type 1 Interferon production and CD4⁺ T-cell transformation to Th2, Th17 and regulatory T-cell. This disturbs the function of cytotoxic CD8⁺ T-cells and natural killer (NK) cells^[42-48]. It results in chronic liver inflammation which disturbs tissue homeostasis and promotes pro-carcinogenic environment. Simultaneously, there is an increase in the release of ROS, nitric oxide (NO), cytotoxic cytokines and lipid peroxidation. It also helps in immune escape of neoplastic transformed cells facilitating the development of HCC^[49]. During chronic HCV infection, the inflammatory cytokine like tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-23, IL-6 and lymphotoxins-alpha and beta (LT-α and β) are also increased causing chronic liver inflammation and HCC progression^[49-51]. There is already a report demonstrating an important role of LT-α and LT-β in the development of HCC^[51]. In fact, activation of NF-κB pathway by LTs triggers the hepatocarcinogenesis by increasing production of chemokines and cytokines. In patients with chronic HCV infection, the liver infiltrating T and B-cells not only fail viral clearances but also increase chronic inflammation^[51,52]. Also, an increased number of CD8⁺ is accompanied by reduction in NK and NKT cells which are involved in cancer immune surveillance^[52]. These informations indicate that during chronic HCV infection there is a regular tumor promoting inflammation and impaired anticancer immune scanning, which ultimately facilitates towards HCC.

As described earlier there is high occurrence of steatohepatitis in patients with chronic HCV infection. The accumulation of free fatty acid induces production of ROS and mitochondrial dysfunction and Endoplasmic reticulum (ER) stress. In turn, oxidative stress stimulates lipid peroxidation and increases inflammation in liver tissue. Increased ROS levels have direct effect on fibrosis by increasing collagen 1 expression^[50]. The HCV induced steatosis changes the liver T-cell function. HCV related proteins in the liver develop extensive steatosis which is accompanied by an infiltrate of CD8⁺ T-cell secreting Th2 type cytokine^[53]. A massive liver infiltration by CD8⁺ and NKT cells induces steatosis, inflammation and carcinogenesis^[54]. In HCV infected patients, the risk of HCC development may also be linked with the severity of liver fibrosis. TGF-β is an important cytokine involved in fibrogenesis. Its expression is directly affected by HCV related proteins or oxidative/ER stress and NF-κB pathway activation^[55-58]. This concludes that hepatic fibrosis caused by various mechanisms is a big inducer promoting hepatocarcinogenesis.

There are a number of genes associated with HCV induced HCC. The tumor suppressor gene P53 was the first one noted for its association with development of HCC. Recent studies have shown a subset of genes frequently mutated in HCV patients^[59,60]. Oncogene CTNNB1 which encodes β-catenin protein of WNT-pathway shows a mutation of 30%. WNT ligands activate signal transduction cascade resulting in inhibition of β-catenin degradation complex. It has been observed that WNT pathway gets mutated in HCC, which stabilizes β-catenin. This β-catenin translocates to the nucleus and regulates genes responsible for cell survival and proliferation. NS5A indirectly regulates the WNT pathway through PI3K and activate Akt. Increased β-catenin has been observed in HCV infected cells. The significance of β-catenin with in HCV infected cells is still uncertain^[61]. However, its level is increased mostly in HCC patients. Similarly, reduction in the size of telomere triggers cellular senescence. Activation mutation in the telomerase reverse transcriptase (TERT) promoter gene has been detected in HCC induced by HCV infection in addition to other etiologies^[62-64]. HCV core protein downregulates CDKN2A expression to overcome hepatocyte senescence. Increased telomerase activity, a characteristic of transforming or transformation prone cells, was observed in HCV core- transfected primary human hepatocytes that acquired an immortalized phenotype. In line with this observation, somatic mutation in the TERT promoter that enhance TERT expression were shown to be among the earliest and most prevalent neoplastic events associated with all major etiologies including HCV. Host genetic variants are also associated with a high risk of HCC^[65]. PNPLA3 gene (patatin-like phospholipase domain-containing protein-2) shows a significant association with fatty liver disease in HCV patients having a higher risk of HCC^[66-68]. On a similar pattern, polymorphisms in several other cytokines/receptors genes have been found to be associated with HCC. These are cytokines TNF-α, IL-10, IL-23R and vascular endothelial growth factor (VEGF) etc. genes. Host responds differently to variation in viral genome. For example, HCV genotype 1a and 1b reported to be associated with HCC^[69].

Various studies have demonstrated a dysregulation of epigenetic regulatory genes in HCC^[70]. Histone-lysine N-Methyltransferase enzyme (EZH2) is one such an example which is aberrantly expressed in HCC^[71] and this also targets expression of tumor suppressor miRNAs^[72]. The changes in gene methylation were also related with virus induced tumors^[73]. Various tumor suppressor genes including CDKN2A, GSTP1, RUNX3, APC, SOCS-1 and RASSF1A are highly methylated in HCC caused by HBV and HCV infection^[74]. Epigenetic alterations in HCC may be mediated by changes in miRNAs and long noncoding RNAs. There are several miRNAs which modulate HCV replication in a positive and negative manner^[75].

Structural and nonstructural HCV proteins have a direct role in inducing neoangiogenesis. HCV core promotes angiogenesis by upregulating hypoxia inducible factor 1-alpha which regulates VEGF and cyclooxygenase 2. VEGF is an important endothelium specific growth factor in HCC and for this reason, VEGF level in serum is used as a prognostic factor in HCC^[76]. Angiopoietin-2 is also upregulated by HCV infection^[77].

HCV core protein, which regulates HCV RNA translation and its replication, interacts with component proteins of various cell-signaling pathways. In addition, this protein modulates host immune response, oxidative stress, lipid metabolism and also apoptosis^[78]. In some recent studies on HCV infected patients, core gene has been found to undergo frequent mutations^[79]. The role of core protein in the development of HCC was studied in transgenic mouse model. The information collected from these studies indicate that core gene overexpression results in steatosis in early life with development of adenoma and HCC in later years^[80]. In few other studies, the presence of steatosis in liver induced by core protein could not be related to HCC development^[29,81]. According to recent reports, core protein shows interference with cellular proteins and it is considered as a major risk factor for the progression of HCC^[82]. Of course, the presence of core protein has been associated with its activation of lipogenic pathway in HCC cases^[83]. Core protein often remains associated with lipid droplets in CHC cases and possibly causes steatosis through several mechanisms including peroxisome proliferator-activated receptor alpha and sterol-regulatory element binding protein-1 pathways^[46,81,84].

Similarly, core protein also interacts with ER or mitochondria and induces ER stress by accumulation of ROS^[85]. ROS causes DNA damage and accelerates hepatocarcinogenesis. The effect of HCV core have also been demonstrated on signaling pathways responsible for cell cycle like stimulation of G₁/S transition by increasing the levels of cyclin E/Cdk2^[86] and apoptosis. Core protein interacts with tumor suppressor including P53, P73 and P21^[87] as well as regulator of apoptosis like TNF-α signaling or Bcl-2 members. Core proteins also effects growth and proliferation of cells through activation of signaling pathways like RAF/MAPK (Mitogen activated protein kinase)^[88], Wnt/β-catenin and TGF-β^[39,89]. All these pathways have been reported to be active in HCC. Therefore, these findings about HCV core indicate that this protein has a potential role in cell proliferation and reduction of apoptosis during development of HCC.

The effect of structural proteins E1/E2 was also studied on malignant transformation of hepatocytes. The results indicated these proteins to interfere with Interferon actions by inhibiting dsRNA protein kinase (PKR)^[90,91]. In addition, E2 protein also inhibits activation of T and NK cells^[91] and MAPK/extracellular signal regulated protein kinase pathway including the transcription factor ATF-2 and promotes cell proliferation and cell survival^[92].

NS2 activates cyclin D/CDK4 and induces expression of Cyclin E^[93]. Some studies also supported its role in the inhibition of apoptosis by interference with p53 pathway.

The NS3 transforms mammalian cells but its role in HCC is less clear^[94,95]. This protein interacts with tumor suppressor p53. NS3 protein modulates various signal transduction pathways having transformation potential. NS3 interacts with protein kinase A and inhibits its translocation to nucleus. NS3 also inhibits interferon response factor (IRF-3) mediated induction of type-1 interferon, necessary to escape immune surveillance. NS3/4A interacts with ATM, Check point kinase, preventing DNA repair. This also disturbs endoplasmic reticulum leading to cell death^[96]. Similarly, NS3/4A target adaptor molecules in TLR3 and RIG-1 signal pathway, thereby interfering with activation of IRF-3 transcription factor and promoting proliferation^[97-99]. All these reactions contribute to cancer promoting effect of HCV.

This protein is needed for replication of HCV genome. It forms part of viral replicates complex. Inside the nucleus, NS5A acts as transcription factor activator^[100] and interacts with various signaling pathways including cell cycle/apoptosis, lipid metabolism^[46,101] and also shares some signaling targets with core. It has been reported to interfere with PKR-p38 signaling pathway and inducing aberrant mitosis and chromosomal instability leading to HCC^[102]. NS5A inhibits TGF-β signaling by preventing nuclear translocation of SMAD proteins down regulating tumor suppressor CDKN1A^[103]. On a similar pattern, NS5A inhibits tumor necrosis factor-α (TNF-α) mediated apoptosis^[104]. NS5A acts a transcriptional activator for many genes including p53. NS5A also interacts with pathways like Bcl-2, PI3K, Wnt/β-catenin signal and mTOR for proliferation of cells and inhibition of apoptosis. It has been found that HCV NS5A influences EMT pathway and helps in transition process of epithelial cells to mesenchymal stem cells. NS5A work in cooperation to TGF-β to activate stellate cell causing fibrosis. Also HCV core protein was found to induce EMT in primary hepatocyte by suppressing cytostatic effect via SMAD3^[105,106]. Thus NS5A and core produce cells in tumor mass that are not differentiated and mobile via EMT pathway EMT contributes to liver fibrosis on the line as in lungs, kidney and intestine.

NS5B binds with Rb and promotes its cytoplasmic relocalization and proteasomal degradation^[107,108]. This finally activates E2F responsive genes, which in turn stimulates cell cycle progression^[108].

Above reports demonstrate a clear effect of HCV-related proteins on various pathways engaged in progression of infected cells to malignant cells. These proteins enhance the level of underlying inflammation, oxidative stress, ER stress, steatosis, fibrogenesis and finally cell proliferation. Although it is not possible to emphasis their direct effect in exclusion either on initiation or progression, but there is no doubt that involvement of these proteins at various steps of complex mechanism, helps in progression of carcinogenesis resulting in development of HCC.