**4.1 Hepatocellular injury and the production of a protumorigenic microenvironment**

The original model of hepatocellular injury in NASH was described as a "two-hit" hypothesis, where the first hit, steatosis, sensitizes the hepatocyte to injury and cell death resulting from the "second-hit" of oxidative stress [36]. Although this model is largely seen as overly reductive, it does provide a conceptual framework for hepatocellular injury in NAFLD associated HCC. Lipotoxicity, a state of lipid dysregulation

#### **Figure 1.**

*Stepwise pathogenesis of NAFLD associated HCC. In the setting of environmental and genetic predisposition, the sequalae of metabolic reprograming and hepatocellular injury in NAFLD lead to the creation of a protumorigenic microenvironment and ultimately hepatocarcinogenesis. Created with BioRender.com.*

#### *Non-alcoholic Fatty Liver Disease Associated Hepatocellular Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.106816*

leading to organelle dysfunction and cell death, is often-considered the initial metabolic insult that causes hepatocellular injury in NAFLD [32]. Increased hepatic lipid deposits are a hallmark of NAFLD. Multiple features of metabolic syndrome, including excess dietary free fatty acids (FFAs), excess FFA release from adipose tissue, increased insulin resistance, upregulated de-novo lipogenesis and alterations of the gut microbiota all contribute to a lipid-rich hepatic metabolic state [37]. At increased concentrations in the liver, lipids are directly and indirectly hepatotoxic, promoting proapoptotic and ER-stress pathways, while inducing mitochondrial dysfunction and reactive oxygen species (ROS) production [38]. In addition to lipid induced hepatocellular injury, derangements in other metabolic pathways seen in NAFLD, including bile acid metabolism and iron storage, likely contribute to hepatocyte damage and hepatocarcinogenesis as well [39, 40].

As in many cancers, the innate and adaptive immune system play a Janus-faced role in tumor development: both promoting necroinflammation and therefore carcinogenesis, while also performing antitumor cell killing and immune surveillance [41]. Hepatocellular injury in NAFLD plays a central role in disrupting this balancing act, skewing the immune response to favor tumorigenesis. Hepatocyte death stimulates the immune response through exposure of immune cells to damage associated molecular patterns (DAMPS). Additionally, microbiome changes in NAFLD patients likely contribute to the inflammatory immune phenotype, with the increased "leakiness" gut leading to increased translocation of lipopolysaccharide (LPS) and other pathogen associated molecular patterns (PAMPS) into the portal circulation [42]. Both DAMPS and PAMPS act through Toll-like receptors (TLR) and other pattern recognition receptors to activate liver resident macrophages (Kupffer cells). Activated Kupffer cells (KCs) recruit and stimulate other innate and adaptive immune cell subsets which secrete proinflammatory factors including IL-1β, IL-2, IL-7, IL-12, IL-15, TNFα, and IFNγ, further promoting an immunostimulatory and cytotoxic environment. A major consequence of the immunostimulatory environment is the activation of non-parenchymal hepatic cells including hepatic stellate cells, which increase extracellular matrix deposition and fibrosis. Moreover, stimulated innate cells directly contribute to the abundance of ROS and therefore oxidative DNA damage in the liver due to increased respiratory burst activity [41]. Together these immunostimulatory processes perpetuate hepatic cell death, promote hepatic stellate cell mediated fibrosis, and contribute genotoxic metabolites to the microenvironment, all key drivers of hepatocarcinogenesis. In response to these chronic inflammatory conditions, many immune exhaustion responses are induced, including the expression of immunosuppressive factors (IL-10, TGFβ) and the immune checkpoint PD-L1. While this immunosuppressive response contributes to the reduction of detrimental inflammation, antitumor cytotoxic immune response is inhibited as well, contributing to tumor growth.

In addition to the directly cytotoxic and genotoxic mechanisms described above, the positive feedback loop of hepatocellular injury, necroinflammation and fibrosis indirectly promote tumor progression through induction of angiogenesis. In response to inflammatory stimuli, activated monocytes increase production of VEGF and MMP9, promoting tumor neovascularization, growth, and metastasis [43]. Notably even prior to HCC development, NAFLD patients exhibit increased serologic markers of angiogenesis and increased neovascularization in biopsy samples [44], further contributing to the confluence of protumorigenic factors ultimately leading to tumorigenesis in NAFLD.

The influence of metabolic syndrome can be observed in each of these protumorigenic mechanisms, hepatocellular injury, chronic inflammation, immune exhaustion, and increased neovascularization. Many features of metabolic syndrome including hyperlipidemia, hyperglyceridemia, and obesity directly contribute to steatosis and lipotoxic hepatocellular injury. Adipocytes directly produce multiple inflammatory cytokines (TNFα, IL-6) and proangiogenic factors (VEGF, FDGF), likely contributing to oncogenic chronic inflammation, immune exhaustion, and angiogenesis [45]. Mouse models of NAFLD induced HCC highlight the importance of metabolic syndrome in HCC pathogenesis. In mice with diet ± activity modifications designed to recapitulate conditions common metabolic syndrome, the vast majority (60–89%) of mice develop HCC [46], suggesting a potent role of metabolic syndrome in HCC development.

#### **4.2 Hepatocarcinogenesis and disease progression**

Ultimately the protumorigenic microenvironment results in DNA damage and subsequent mutagenesis. DNA oxidative damage is a major contributor to mutagenesis in NAFLD associated HCC. The DNA oxidative stress marker 8-hydroxy-2′-deoxyguanosine (8-OHdG) in NAFLD associated HCC is increased compared to that of healthy patient livers or tumors from patients with viral and alcohol associated HCC [47]. 8-OHdG is an independent risk factor for hepatocarcinogenesis and therefore highlights the role of oxidative damage in HCC pathogenesis. In addition to genotoxic alterations from DNA oxidative damage, oxidative damage can cause epigenetic changes, which may play a role in HCC carcinogenesis. Epigenetic inactivation of tumor suppressor genes consistent with oxidative DNA damage response have been observed in NAFLD induced HCC patients [48]. Furthermore, alterations in DNA repair pathways may also contribute to genomic instability. Upregulation of DNA-dependent protein kinase, a central member of the error prone DNA repair mechanism nonhomologous end joining (NHEJ), has been observed in NAFLD associated HCC [49]. Together these mechanisms lead to an increased mutagenic state in NAFLD associated HCC.

Although a wide variety of mutations have been documented in NAFLD associated HCC, hotspot genes and mutational signatures have been described. In a cohort of 80 patients with NAFLD associated HCC, the most frequently mutated genes were the telomerase (TERT) promoter (56%); the gene encoding beta-catenin, CTNNB1 (28%); the tumor suppressor, TP53 (18%); and the activin receptor, ACVR2A (10%) [35]. Notably, TERT promotor, CTNNB1 and TP53 are mutated at similar rates in HCC patients en masse regardless of etiology; however, mutations in ACVR2A are more enriched in patients with NAFLD associated HCC compared with that in other etiologies [50]. Transcriptionally, the majority of NAFLD-associated HCC tumors demonstrated upregulation of either the WNT–TGFβ or WNT–βcatenin oncogenic signaling pathways, highlighting the importance of both noncanonical and canonical WNT signaling in NAFLD associated HCC carcinogenesis [35]. Moreover, other transcriptional signatures consistent with underlying pathogenic features of NAFLD associated HCC are enriched in these patients including bile acid metabolism, oxidative stress, and inflammation-related gene signatures. Together these genomic and transcriptomic alterations drive malignant transformation and disease progression.
