Keywords

10.1 Introduction

Hepatocyte death is generally regarded as the initial event in almost all liver diseases , including viral hepatitis, alcoholic and non-alcoholic fatty liver diseases, drug and toxin-induced liver injury, biliary disorders, and autoimmune hepatitis. A number of biologically active mediators, receptors, intracellular signaling pathways, and different cell types contribute to the initiation and progression of liver diseases through hepatocyte death. Some known examples include: direct damage to hepatocytes by alcohol and its metabolites; excessive accumulation of bile acids and saturated fatty acids cause hepatocyte death; in Hepatitis B and C, the virus infection mediates cytotoxic T lymphocyte-mediated hepatocyte death. Hepatocyte death in these diseases will lead to liver inflammation, fibrosis, and ultimately induction of hepatocarcinogenesis . In a clinical setting, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are often measured to determine the extent of hepatocyte injury. Elevated levels reflect the degree of hepatocyte injury as hepatocytes release these enzymes into the blood stream when damaged. Since previous clinical observations determined a strong correlation between ALT levels and liver cancer development, persistent hepatocyte death is suggested to be associated with tumor development (Chen et al. 2011; Lee et al. 2010). These clinical observations are supported by recent studies using animal models that demonstrate hepatocellular death contributes to the triggering of hepatic inflammatory and fibrogenic reaction, and the induction of carcinogenesis. Notably, in contrast to increased cell death that drives tumorigenesis , loss of regular programmed cell death is also associated with malignant transformation and cancer development by escaping the death of malignant cells. This chapter will highlight the underlying molecular mechanisms of hepatocyte death and its contribution to liver inflammation, fibrosis, and tumorigenesis .

10.2 Death Receptor-Mediated Cell Death Signaling

Tumor necrosis factor α (TNFα) is a pleiotropic cytokine, and its most well-characterized receptor is TNF receptor type I (TNFR1). The signaling initiated by the TNFα-TNFR1 interaction activates both nuclear factor κB (NF-κB)-dependent cell survival and death-inducing pathways, known as the best-characterized death receptor signaling that contributes to hepatocyte survival and death (Justus and Ting 2015). Binding of TNFα to TNFR1 leads to Complex I being formed at TNFR1 that consists of TNFR1-associated death domain protein (TRADD) , receptor-interacting serine/threonine-protein kinase 1 (RIPK1) , TNF receptor-associated factor 2 (TRAF2) , and cellular inhibitor of apoptosis 1 and 2 (cIAP1/2 ) (Fig. 10.1). Complex I subsequently induces the generation of K63 polyubiquitin chains on RIPK1 that enable the recruitment of TGF-β activated kinase 1 (TAK1) and associated binding proteins TAK1-binding proteins 2 and 3 (TAB2/3) as well as the IκB kinase (IKK) complex, comprising the regulatory subunit NF-κB essential modulator (NEMO /IKKγ) and two kinases, IKKα and IKKβ. The recruitment and interaction of these complexes is essential for the phosphorylation and degradation of IĸBα, thereby activating NF-ĸB (Justus and Ting 2015).

Fig. 10.1
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IKK/NF-κB , JNK, and apoptosis in TNF receptor signaling . Binding of TNF to the TNF receptor type I (TNFR1) leads to the formation of “complex I” comprising TRADD, TRAF2, and cIAP. K63-ubiquitination of RIP1 recruits and activates TAK1 and IKK complex (IKKα/IKKβ/NEMO ). TAK1 also activate JNK and its mediated AP-1/c-Jun transcription factor . Simultaneously, JNK1 promotes caspase-8-dependent apoptosis through inhibition of c-FLIP. TAK1 phosphorylates and activates IKK complex that leads to phosphorylation, unbiquitination, and degradation of IκBα, resulting in nuclear translocation and activation of NF-κB comprising p50 and p65 subunits. NF-κB inhibits ROS and caspase-8 through SOD2 and c-FLIP transcription, respectively. NEMO physiologically regulates NF-κB activation and simultaneously prevents the formation of the complex IIb (RIPK1, FADD , caspase-8). Complex IIb-mediated hepatocyte apoptosis requires RIPK1 kinase activity. NEMO and RIPK1 protein physiologically inhibit the formation of Complex IIa (TRADD, FADD, caspase-8). Alternatively, when caspase-8 and/or FADD are inhibited, RIP1 and RIP3 are phosphorylated and induce necroptosis

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Previous reports have demonstrated that at least two checkpoints regulate cell death in the TNFR1 signaling pathway (Justus and Ting 2015). One of the checkpoints involves the NF-ĸB-dependent transcription and expression of survival proteins, such as c-FLIP and SOD2, which in turn inhibit cell death by protecting against TRADD/ Fas-associated protein with death domain (FADD )/caspase-8 (Complex IIa)-dependent apoptosis. The other checkpoint occurs early in the pathway at the Complex I level where the ubiquitination of RIPK1 allows for its association with TAK1 and NEMO . This interaction prevents RIPK1 from associating with FADD and caspase-8 and thereby inhibiting the formation of RIKP1/FADD/caspase-8 complex (Complex IIb), preventing apoptosis. This checkpoint does not require NF-ĸB activation and de novo protein synthesis to inhibit cell death pathway (Justus and Ting 2015).

TNF-mediated signaling also contributes to the activation of mitogen-activated protein kinase (MAPK) pathway. Phosphorylation and ubiquitination of TAK1, a MAP kinase kinase kinase, activate MAP kinase kinase 4 (MKK4) and MKK7, which then activate c-Jun-N-terminal Kinase (JNK) , induction of phosphorylation, and activation of the AP-1/c-Jun transcription factor (Liu et al. 2002). Although early TNF-mediated JNK activation is transient and involved in cell survival and proliferation through AP-1, sustained JNK activation participates in TNF-mediated hepatocyte death through reactive oxygen species (ROS) production; likely independent of AP-1 (Wagner and Nebreda 2009; Micheau and Tschopp 2003; Ventura et al. 2004; Karin and Lin 2002; Kamata et al. 2005). TNF-induced JNK activation is negatively regulated by NF-ĸB-mediated survival factors that block both caspase-8-dependent cell death and sustained JNK activation (Liu et al. 2002).

10.3 IKK/NF-ĸB Negatively Regulates Hepatocyte Apoptosis

It has been reported that NEMO -deficient cells are susceptible to TNF-mediated apoptosis (Legarda-Addison et al. 2009). Since NEMO binding to RIPK1 polyubiquitin chains inhibits the interaction of RIPK1 with caspase-8, in the absence of NEMO Complex IIb formation is promoted, thereby inducing apoptosis. IKKα/IKKβ kinases directly phosphorylate RIPK1 at the level of Complex I, thereby inhibiting the formation of Complex IIb and its mediated hepatocyte apoptosis, which is independently of NF-ĸB (Dondelinger et al. 2015; Koppe et al. 2016). In mice with deletion of NEMO in hepatocytes (NEMOLPC-KO mice ), their livers become more sensitive to LPS- and TNFα-induced liver injury and spontaneously develops inflammation, steatosis, fibrosis, and hepatocellular carcinoma (Luedde et al. 2007; Ehlken et al. 2014). NEMO prevents spontaneous hepatocyte death by two distinct mechanisms: One is the function through NF-ĸB-mediated transcription of anti-apoptotic survival proteins, and the other is to inhibit RIPK1-mediated hepatocyte apoptosis by the NF-ĸB-independent manner (Kondylis et al. 2015). Since NEMOLPC-KO mice with bearing mutated RIPK1D138N of which kinase activity is absent showed less hepatocyte apoptosis, NEMO negatively regulates RIPK1 kinase activity to prevent hepatocyte apoptosis (Kondylis et al. 2015) (Fig. 10.1). Notably, RIPK1 also has a scaffold function that normally inhibits RIPK1 kinase-independent pro-apoptotic pathway through TRADD/FADD /caspase-8 complex (Complex IIa) (Fig. 10.1). Indeed, when whole RIPK1 protein is deleted, NEMOLPC-KO mice showed severe liver injury through formation of Complex IIa (Kondylis et al. 2015). Both FADD and Caspase-8 are required for forming the complex associated with either RIPK1 or TRADD. Importantly, TRADD-mediated alternative hepatocyte apoptosis pathway will not be activated until both RIPK1 and NEMO are inhibited.

10.4 JNK Activation and Hepatocyte Death

In hepatocytes, TNF signaling also activates JNK-dependent pathway, induction of phosphorylation, and activation of the AP-1/c-Jun transcription factor (Liu et al. 2002). TNF-induced JNK activation is negatively regulated by NF-ĸB-mediated survival factors that block both caspase-8-dependent cell death and sustained JNK activation (Liu et al. 2002). Sustained JNK activation promotes TNF-mediated hepatocyte death through ROS production but independent of AP-1 (Kamata et al. 2005) (Fig. 10.1). The liver expresses two JNK isoforms, JNK1 and JNK2. Compared to JNK1, JNK2 plays crucial roles in TNF-mediated hepatocyte apoptosis through caspase-8 activation, Bid cleavage, and mitochondrial cytochrome c release (Wang et al. 2006). In concanavalin A (ConA)-induced T cell-mediated liver injury , both JNK1 and JNK2 play a role (Maeda et al. 2003).

Mitochondrial Bcl-XL , Mcl-1 , and Sab (SH3BP5) are substrates for JNK. JNK translocation to the outer membrane of mitochondria plays a role in TNF-mediated hepatocyte death (Win et al. 2011). JNK phosphorylates mitochondrial Sab , which promotes translocation of MKK4, JNK, and Bax to mitochondrial outer membrane, thereby inducing mitochondrial ROS formation, sustained JNK activation, and hepatocyte death (Win et al. 2011). JNK also contributes to the cleavage of mitochondrial Bid, a BH3 only protein , through caspase-8 (Wang et al. 2006; Ni et al. 2009), which induces Bid translocation to mitochondria, cytochrome-c release, and caspase-9/caspase-3 activation, thereby promoting hepatocyte death (Wang et al. 2006; Takamura et al. 2007).

JNK is also involved in saturated fatty acids- and endoplasmic reticulum stress-induced hepatocyte death (Seki et al. 2012).

A variety of JNK-dependent biological functions are mediated through AP-1/c-Jun activation. In TNF-mediated hepatocyte apoptosis, c-Jun- and transcription-independent JNK-mediated signaling pathways are more important (Schwabe et al. 2004; Czaja 2003). AP-1/c-Jun may have protective effects against hepatocyte death (Eferl et al. 2003).

10.5 RIP3 and Regulation of Necroptosis and Hepatocyte Death

Necroptosis is defined as a programmed necrotic death, which differs from necrosis characterized by swelling of cells and cell organelles along with plasma membrane rupture. Necroptosis requires activation of both RIPK1 and RIPK3 , which are normally inhibited through caspase-8 and/or FADD by cleaving RIPK1 and RIPK3. When caspase-8 and/or FADD are inhibited (this may occur in some diseases, such as viral infection), necroptosis (as a back-up cell death form) is induced through activating RIPK1 and RIPK3 (Fig. 10.1). RIPK3-mediated necroptosis contributes to hepatocyte death in acetaminophen (APAP)-induced liver injury, ethanol-mediated liver injury, and the development of methionine choline-deficient diet-induced non-alcoholic steatohepatitis (NASH) (Deutsch et al. 2015; Roychowdhury et al. 2013; Gautheron et al. 2014). However, recent studies demonstrated contradictory results that high-fat diet-induced non-alcoholic fatty liver disease (NAFLD) and insulin resistance were exacerbated in RIPK3 null mice (Roychowdhury et al. 2016; Gautheron et al. 2016). Further studies are needed for determining the role of RIP3K and necroptosis in liver disease.

10.6 Inflammasome Activation and Pyroptosis

Inflammasome and caspase-1 activation is required for IL-1β and IL-18 processing in macrophages. Notably, inflammasome and caspase-1 activation also contributes to “Pyroptosis,” a type of programmed cell death, which is distinct from apoptosis and necrosis (Fink and Cookson 2005). Pyroptosis is characterized by pore formation in cell membrane, membrane disruption, and cell swelling, and shows positivity for PI and TUNEL staining. NLRP3D303N mutation is responsible for human cryopyrin-associated periodic syndrome, and causes excessive activation of inflammasome through NLRP3 (Wree et al. 2014a). A recent study showed mice with mutated NLRP3 developing spontaneous liver injury, inflammation, and fibrosis along with hepatocyte pyroptosis (Wree et al. 2014a). These mice are also sensitive to high nutrient stress and overt NASH development (Wree et al. 2014b). These findings indicate that hepatocyte pyroptosis is associated with liver inflammation and fibrosis.

10.7 Hepatocyte Death-Induced Inflammation through DAMPs

Hepatic inflammation is believed to be the first response after liver injury, which is associated with driving fibrogenesis and hepatocarcinogenesis . Dead or dying hepatocytes produce or release so-called damage-associated molecular patterns (DAMPs ), which induce inflammation by recruiting and/or stimulating immune cells, production of inflammatory cytokines, and further perpetuating inflammation as well as hepatocyte injury. Hepatocyte-derived DAMPs include nucleotides, nuclear proteins, mitochondrial proteins and DNAs, lipids, and cytokines (Matzinger 2002; Chen and Nunez 2010; Rock et al. 2011; Kono and Rock 2008). DAMPs are generally released after hepatocyte necrosis and necroptosis due to cell membrane rupture, while other DAMPs are also known to leak out from apoptotic hepatocytes. Hepatocyte-derived DAMPs reported previously are listed in Table 10.1. A nuclear protein high-mobility group box 1 (HMGB1) is an early mediator released from dead hepatocytes, which can trigger inflammation in the setting of ischemia-reperfusion injury and APAP-induced liver injury, but not LPS, TNF, and Fas-mediated liver injury (Tsung et al. 2005; Huebener et al. 2015) (Fig. 10.2). HMGB1 mediates ischemia-reperfusion liver injury through toll-like receptor 4 (TLR4 ) (Tsung et al. 2005). In APAP-induced liver injury, RAGE is more important than TLR4 through recruiting neutrophils (Huebener et al. 2015). Hepatocytes contain a large amount of ATP, a key factor for energy generation, which is released from dying hepatocytes. As a DAMP, extracellular ATP promotes liver inflammation through binding to its receptor P2X7 in APAP-induced liver injury (Hoque et al. 2012). During hepatocyte death, mitochondria damage also occurs. Since mitochondria contain ATP, formyl-peptides, and mitochondrial DNA, extracellularly leaked mitochondrial components recruit neutrophils, promotion of sterile inflammation and injury in the liver (McDonald et al. 2010). Notably, denatured host DNA and mitochondrial DNA activate TLR9, promoting liver inflammation in APAP-induced liver injury and in NAFLD (Imaeda et al. 2009; Garcia-Martinez et al. 2016) (Fig. 10.2). Kupffer cells are the major source of IL-1β and IL-18 that require processing to be active forms for secreting extracellularly. Inflammasome activation is a main mechanism for the activation of IL-1β and IL-18. Hepatocyte-derived DAMPs , such as ATP, uric acids, and oxidized mitochondrial DNA, are involved in NLRP3 inflammasome activation in APAP-induced liver injury, alcoholic and non-alcoholic steatohepatistis (Petrasek et al. 2015; Wan et al. 2016; Iracheta-Vellve et al. 2015) (Hoque et al. 2012; Shimada et al. 2012). Inflammatory cytokines , such as IL-1α and IL-33, are also released after hepatocyte death (Sakurai et al. 2008; McHedlidze et al. 2013). IL-1α released after carcinogen- and hepatotoxin-induced death mediates IL-6 production, promoting liver inflammation and hepatocyte proliferation (Sakurai et al. 2008). Hepatocyte-derived IL-33 promotes type 2 immune response by producing IL-13 from innate lymphoid cell type 2 (ILC2) in parasite infection (McHedlidze et al. 2013).

Table 10.1 Hepatocyte-derived DAMPs and their receptors and actions
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Fig. 10.2
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Hepatocyte death and cellular crosstalk in liver inflammation, fibrosis , and carcinogenesis. In acute and chronic liver disease, hepatocyte apoptosis, necroptosis, and necrosis occur. Apoptotic hepatocytes release apoptotic bodies that are phagocytosed by Kupffer cells and hepatic stellate cells (HSCs ). Necrotic cells release DAMPs, such as HMGB1, mitochondrial DNA, ATP, IL-1α, IL-33, uric acids, and adenosine. The DAMPs regulates Kupffer cells and HSC activation for liver inflammation and fibrosis. Hepatocyte apoptosis may participate in tumorigenesis through compensatory proliferation

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Viral hepatitis caused by hepatitis B or C virus induces hepatocyte apoptosis. Since hepatitis B and C virus themselves are minimal cytopathic, apoptosis is mainly caused by viral specific cytotoxic T cells and natural killer (NK) cells that eliminate infected hepatocytes (Malhi et al. 2010). T and NK cell-mediated hepatocyte killing is mediated by Fas and TRAIL-mediated signaling (Malhi et al. 2010). Anti-viral immunity does not always successfully eradicate all viral particles. In this setting, viral infection will be sustained and becomes chronic, which is characterized by persistent hepatocyte death and inflammation, ultimately induction of HSC activation and liver fibrosis/cirrhosis.

10.8 Hepatocyte Death as a Trigger for Liver Fibrosis

Liver fibrosis is an alternative repair response against liver injury and results in liver dysfunction, portal hypertension, and hepatocarcinogenesis . Activated hepatic stellate cells (HSCs ) are the major contributors of the development of liver fibrosis by producing extracellular matrix, such as collagen fibers. Dead hepatocyte-released DAMPs directly stimulate HSC activation and/or indirectly activate HSC through activating other non-parenchymal cells, such as Kupffer cells , NK cells, T cells, B cells, and dendritic cells. Early studies showed that hepatocyte-derived apoptotic bodies engulfed by Kupffer cells and HSCs , induce HSC activation (Canbay et al. 2003a, b). Previous human cohorts demonstrated that fibrosis after viral hepatic injury is well correlated with elevated blood ALT levels, suggesting a possible link between hepatocyte death and liver fibrosis in human liver fibrosis (Fattovich et al. 2008; Wiese et al. 2014). Hepatocyte apoptosis has been proven to be an early event in a rapidly fibrosis progression in patients transplanted for hepatitis C (Meriden et al. 2010). In addition, there is a positive correlation of active caspase-3 and 7, and plasma-fragmented cytokeratin-18 (CK-18) levels (which is cleaved by caspase-3 from full-length CK-18) with the stage of hepatic fibrosis in NASH patients (Feldstein et al. 2003, 2009). A number of animal studies have also demonstrated that mice-specific deletion of anti-apoptotic molecules in hepatocytes enhances TNF-mediated hepatocyte death or spontaneously develops hepatocyte death. These anti-apoptotic molecules include NEMO , TAK1, Bcl-xL , and Mcl-1 (Luedde et al. 2007; Inokuchi et al. 2010; Takehara et al. 2004; Vick et al. 2009). In these mice, continuous hepatocyte apoptosis along with elevated ALT leads to liver fibrotic response.

As mentioned above, DAMPs released from dying cells trigger inflammation, linking to fibrosis. HMGB1 promotes HSC proliferation and migration through TLR4 (Wang et al. 2013). Interestingly, TLR9 activation by denatured host nucleic DNA and mitochondrial DNA is capable of stimulating HSCs in upregulation of fibrogenic genes (TGF-β and collagen) but limiting chemotaxis (Watanabe et al. 2007; Garcia-Martinez et al. 2016) (Fig. 10.2). Adenosine is released from dying hepatocytes, which makes higher adenosine levels in local liver injured sites. Adenosine is derived from the dephosphorylation of adenosine tri-, di, and monophosphates, and also from the degradation of nucleic acids through the uric acid pathway. Adenosine upregulates TGF-β and collagen α1(I) levels in HSCs but inhibits PDGF plus ATP-induced HSC chemotaxis via the A2a receptor (Hashmi et al. 2007). This system can hold HSCs when they migrate to an injured site with upregulation of fibrogenic molecules. Phagocytosis of dead hepatocyte-derived apoptotic bodies by HSCs induces fibrogenic response, such as upregulation of α-smooth muscle actin, TGF-β, and collagen α1(I) expression (Canbay et al. 2003b) (Fig. 10.2).

TGF-β is a potent fibrogenic cytokine that directly activates HSCs and stimulates collagen production. TGF-β also participates in hepatocyte apoptosis that indirectly promotes HSC activation and fibrosis in NASH and TAK1LPC-KO mice (Yang et al. 2013, 2014).

Parasite infection-induced hepatocyte death releases IL-33 that stimulates ILC2, producing IL-13 (McHedlidze et al. 2013). IL-13 then stimulates HSCs through IL-4Rα and STAT6 and contributes to parasite-mediated HSC activation and liver fibrosis.

In contrast to hepatocyte death that generally promotes fibrosis, death in HSC is implicated in fibrosis regression and resolution. When continuous fibrotic stimuli are removed, fully activated HSCs undergo apoptosis and fibrosis regression or resolution may occur (Kisseleva and Brenner 2006). NK cells play an important role in HSC killing through IFN-γ (Jeong et al. 2011). After full activation, some HSCs become senescent, which halt collagen production. Senescent HSCs become more susceptible to death through NK cells, and exogenous IL-22 treatment promotes HSC senescent and accelerates HSC apoptosis (Kong et al. 2012).

10.9 Hepatocyte Death as an Initiator of Hepatocarcinogenesis

Unlike other organs, the liver has the most powerful self-repairing capacity after tissue injury. After more than 50% of hepatectomy or acute liver damage by hepatotoxin exposure, liver may regenerate to original mass and recover its normal function Unfortunately, this event does not always occur in the setting of chronic liver disease. The complications of prolonged hepatocellular injury include fibrosis, which is abnormal wound-healing response, and become suitable “soil” for carcinogenesis. Hepatitis, fibrosis, and cirrhosis are well-established risk factors for hepatocellular carcinoma (HCC ). Clinically, more than 80% of HCC patients have liver fibrosis/cirrhosis before being diagnosed with HCC (Fattovich et al. 2004). It suggests that long-term hepatocyte damage promotes malignant transformation of hepatocytes.

Through animal models, with pure cell death type and known mechanism, it is suggested that hepatocyte death itself and compensatory proliferation after massive liver injury are both the key factors for hepatocarcinogenesis . Mice with hepatocytes deficient in IKKβ, a crucial factor for NF-κB activation, cause massive hepatocyte death after exposure to chemical carcinogen diethylnitrosamine (DEN) (Maeda et al. 2005). These mice also showed more compensatory hepatocyte proliferation and hepatocarcinogenesis than wild-type counterparts. Mice with hepatocytes deficient in NEMO or TAK1, another regulator for NF-κB activation, showed spontaneous hepatocarcinogenesis in conjunction with hepatocyte injury and compensatory liver cell proliferation (Luedde et al. 2007; Inokuchi et al. 2010). Since death receptor-mediated hepatocyte apoptosis is associated with liver phenotype of aforementioned mutant mice, the deletion of FADD prevents the development of HCC in these mice. Hepatocarcinogenesis in NEMOLPC-KO mice is mediated by inhibiting NF-κB activation and promoting RIPK1 kinase activity-mediated hepatocyte apoptosis (Kondylis et al. 2015). However, since RIPK1 molecule also has a scaffold function for NF-κB activation, whole RIPK1 deletion does not reduce hepatocarcinogenesis in NEMOLPC-KO mice (Kondylis et al. 2015). In NEMOLPC-KO mice and TAK1LPC-KO mice , additional deletion of FADD or caspase-8 prevents hepatocyte apoptosis and hepatocarcinogenesis but additional RIPK3 deletion did not suppress hepatocyte injury and carcinogenesis, suggesting that hepatocyte apoptosis, but not necrosis/necroptosis, is an essential mechanism for hepatocarcinogenesis (Luedde et al. 2007; Vucur et al. 2013; Kondylis et al. 2015) (Fig. 10.2). One human study demonstrated that the downregulation of NEMO expression is correlated with the prognosis and survival of HCC patients (Aigelsreiter et al. 2012).

Deficient in anti-apoptotic proteins Mcl-1 and Bcl-xL in hepatocytes causes spontaneous hepatocyte apoptosis that results in the development of HCC (Takehara et al. 2004; Weber et al. 2010). In Mcl-1LPC-KO mice , additional deletion of Bak (a downstream molecule of Mcl-1 towards apoptosis) reduces HCC development (Hikita et al. 2012). These findings also support the mechanism that hepatocyte apoptosis is associated with HCC development. Notably, these genetic animal models might not recapitulate human HCC conditions since in most clinical settings external injuries caused by virus, toxin, alcohol, and lipids are associated with HCC formation.

microRNAs (miRNAs) are small, noncoding RNA molecules that negatively regulate the expression of corresponding mRNA. Hepatocytes predominantly express miR-122, which accounts for 70% of all miRNAs in hepatocyte. When hepatocytes are damaged, miR-122 is released to blood circulation then blood miR-122 levels increase significantly. miR-122 plays dominant roles as a tumor suppressor and a negative regulator for lipid metabolism as mice deficient in miR-122 spontaneously develop hepatic steatosis, fibrosis, and carcinogenesis (Tsai et al. 2012). Although evidence that released miR-122 plays a role in the development of liver inflammation and tumorigenesis is still lacking, released miR-122 may influence liver pathophysiology.

It is strongly suggested that apoptosis in healthy hepatocyte is associated with promoting tumorogenesis, while impaired death or programmed death of cancer or dysplastic cells may lead or enhance tumor formation and progression. Targeted ablation of Fas gene causes liver hyperplasia in mice (Adachi et al. 1995). In HCC , Fas expression is lower in human HCC tissue than non-cancerous tissue (Higaki et al. 1996). Another well-known example is NF-κB, which acts as both a promoter and a suppressor of HCC. In Mdr2 knockout mice model , overexpression of mutated IκBα in hepatocytes inhibits inducible NF-κB activation and reduces liver tumor development compared with mice with normal NF-κB signaling . In this setting, NF-κB functions as a tumor promoter (Pikarsky et al. 2004).

10.10 Conclusions

A number of animal models have suggested a strong link between cell death and carcinogenesis. Several human studies also suggest this link but the direct molecular mechanism is still unclear. Mutations, such as p53, β-catenin, and TERT promoter, are often observed in human HCC . However, some mouse models enable developing HCC spontaneously without treatment with carcinogen. It is hypothesized that chronic hepatocyte damage induces extensive compensatory proliferation that increases the incidence of mutations on tumor suppressor gene(s) and eventually develops dysplastic cells or transformed cancer cells. Another hypothesis is that in some hepatocytes that escaped from cell death stimuli apoptotic signals directly affect mutations in tumor suppressor gene(s) . Notably, cancer cells usually escape from the natural selection guard system by specific mutations, such as p53 mutation (Guichard et al. 2012). In summary, hepatocyte death is clearly associated with promoting liver inflammation and fibrosis whereas in hepatocarcinogenesis hepatocyte death has dual roles: On one hand, hepatocyte death signal functions as a tumor promoter. On the other hand, hepatocyte death acts as a tumor suppressor by eliminating cells with gene mutations.