**5. Roles of somatic mutation during the process of HCC evolution**

The spontaneous rate of somatic mutations is not high enough to trigger the evolution process. HBV participates in the alteration of the host genome, both directly and indirectly. First, HBV can cause somatic mutations by directly integrating into the human genome. Second, mutant HBV contributes to the maintenance of non-resolving inflammation, that induces long-term up-regulation of APOBECs [7]. Somatic mutations can be classified according to their effects on *Cancer Evo-Dev*. A small proportion of the mutations can lead to advantageous phenotypes that are positively selected during the evolution process and thus are called "driver" mutations. The remaining mutations are "passengers" that contribute very little to carcinogenesis [4]. Due to survival competition and the positive selection of the inflammatory microenvironment, driver mutations accumulate sufficiently to promote malignant transformation. The distribution, combination, and dynamic patterns of driver mutations reflex the pressure of microenvironmental selection and growth advantage of cell subsets. As HCC has many etiological causes and experiences a long evolutionary process, the somatic mutation spectrum is most heterogeneous [6, 83]. The driver somatic mutations affect multiple functions, like signaling pathways, EMT, and energy metabolism.

*Hepatitis B Virus (HBV) - Induced Hepatocarcinogenesis, a Founding Framework of Cancer… DOI: http://dx.doi.org/10.5772/intechopen.99838*

### **5.1 Somatic mutations alter "stem-ness" signaling pathways**

Based on the investigations of whole-exome sequencing, it is found that the somatic mutation in HCC evolution mainly altering six cancer related pathways: signaling pathway related with telomere maintenance, Wnt/b-catenin pathway, P53 and cell cycle pathway, oxidative stress pathway, epigenome modifiers, RAS/RAF/ mitogen-activated protein kinase pathway, and PI3K/AKT/mTOR pathways [84]. Among them, the somatic mutation related to telomeres pathway is most frequent. Telomerase is activated in more than 90% HCC patients. Somatic mutation within the promoter of telomerase reverse transcriptase (*TERT*) is the major cause with the prevalence ranging from 54–60%. The second cause is the HBV integration in the *TERT* promoter, which is observed in 10–15% of HCC patients. Interestingly, the mutation of catenin beta 1 (*CTNNB1*) is more frequent in hepatitis C virus induced HCC, indicated a different way of *Cancer Evo-Dev* [85, 86]*.* The frequencies of mutation in other hot genes range from 5–20%. Although the spectrums and frequencies of altered genes vary greatly among individuals, they are usually clustered to pathways or functional groups that are closely related to stem-ness and embryonic characteristics. In this regard, global mutation rates of functionally related genes are added together to define the mutation rate of a given signaling pathway. Mutation rates of Wnt/β-catenin, p53/cell cycle control, JAK/STAT, and PI3k/mTOR pathways range from 12–72%. Similar outstanding outcomes are also observed in functional gene groups of chromatin remodeling and telomere maintenance. Therefore, it is promising to use combo somatic mutations as predictive and prognostic biomarkers just like gene signatures [19].

#### **5.2 Somatic mutations affect HCC evolution through regulating EMT**

APOBECs can promote gene demethylation and remove epigenetic memory to stabilize the pluripotent state in embryonic stem cells through deaminating 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) [87, 88]. EMT is a landmark event of *Cancer Evo-Dev*, which is driven by transcription factors, like ZEB1, ZEB2, SNAI1, and SNAI2. AID, a member of the APOBECs family, is upregulated by inflammatory signals and induces demethylation of the promoters of ZEB1, ZEB2, SNAI1, and SNAI2. Silencing AID leads to increased methylation of CpG island proximal to the promoters of these EMT regulators, thus inhibits EMT and invasion of cells [89]. AIDinduced, CpG methylation-dependent mutagenesis is proven to be a common feature of cancer evolution [90]. Therefore, it is reasonable to postulate that re-expression of embryonic factors in cancers might result from epigenetic reprogramming caused by APOBECs family, that is upregulated by proinflammatory factors.

#### **5.3 Somatic mutations reprogram energy metabolism**

To support the rapid growth of malignant cells, tumor tissues prefer to use glycolysis for energy production, even in the presence of oxygen. Glucose is more easily to be metabolized to lactate in tumor tissues than in normal tissues. This pattern of energy metabolism was identified in 1920 and was termed as Warburg effect [91]. Warburg effect in TAMs promotes vascular network formation, augments extravasation of tumor cells out of blood vessels, and induces higher levels of EMT at inflammatory foci within the tumor [92]. In the microenvironment with both hypoxia and hypoglycemia, stem cell-, angiogenic-, and EMT-biomarkers, as well as glycoprotein-P content and invasiveness of cancer cells are enhanced [93]. Thus, we believe that the Warburg effect promotes the evolutionary process of cancer under both hypoxia and hypoglycemia conditions. The Warburg effect can provide

essential energy for cell survival in a hostile microenvironment, furthermore, glycolysis generates the raw material for DNA synthesis of progeny cells. HBV infection and somatic mutation are both the possible origin of Warburg phenotype. In HBV-HCC, the major pattern of single nucleotide variants in mitochondrial DNA (mtDNA) is C > T, that is the character of APOBEC induced mutation. This kind of mutation mainly occurs in the D-loop region of mtDNA and promotes the proliferation, invasion, and metastasis of HCC cells [94]. Pyruvate kinase M2 (PKM2), an alternatively spliced variant of the pyruvate kinase gene that is preferentially expressed during embryonic development and in cancer cells, alters the final rate-limiting step of glycolysis, resulting in the cancer-specific Warburg effect [95]. Besides the Warburg effect, HCC cells also enhance other patterns of energy metabolism during evolution. For example, the inactivating mutation of ribosomal S6 kinase 2 (RSK2) can support cholesterol metabolism in HCC [96].

#### **5.4 HBV integration**

HBV integration is a kind of somatic mutation that is specific to the HBVinduced *Cancer Evo-Dev.* Although the HCC in an individual can be monoclonal, HBV integration is common in most clones, indicating it is the early driver event for HCC evolution [83]. The HBV integration can be detected in 85–90% of HBV-HCC patients [97]. Moreover, the prevalence of HBV integration is 60–75% in HCCs from patients with occult HBV infection, indicating the HBV integration contributes to the occult HBV infection induced HCC [98, 99]. Approximately five thousand HBV integration events have been reported and more than half of them locate in the intergenic regions. Only the HBV integration events within thirteen genes are repeated in diverse studies [1]. *TERT*, mixed-lineage leukemia 4 (*MLL4*), fibronectin 1 (*FN1*), cyclin E1 (*CCNE1*), and cyclin A2(*CCNA2*) are the top five most frequently integrated genes [85, 100–105]. The X and core genes of HBV are the regions that most frequently insert into the human genome [103, 105]. Cis-activation of host genes is an important mechanism by which HBV integration promotes HCC evolution. The highest frequency of HBV integration is observed in the promoter region of *TERT* [85, 100–105]. The HBV integration within the *TERT* promoter leads to an increased mRNA level of *TERT*, that is significantly associated with a poor prognosis of HCC [103, 105]. MLL4 is the second most frequently integrated gene and the HBV integration mainly locate in the introns and exons [85, 105]. Since *MLL* gene family has methyltransferase activity, the HBV integration within *MLL4* may promote HCC evolution in an epigenetic way. As the third most frequently integrated gene, *FN1* is reported to create a microenvironment promoting metastasis of lung cancer [106]. Most HBV integration events within *FN1* are detected in the adjacent tissues of HCC, indicating these mutations may contribute to the microenvironment of the early stage of HCC evolution [85, 101]. HBV integration is associated with an increased expression of *CCNE1*, that is reported to promote hepatic inflammation and hepatocarcinogenesis [107]. The HBV-*CCNA2* chimeric transcript encodes a chimeric protein promoting cell cycle progression [108]. Besides affecting the expression or function of coding genes, HBV integration within the region of long interspersed nuclear elements (LINEs) can generate HBx-LINE1 chimeric transcript acting as long non-coding RNA (lncRNA). This lncRNA increases the activity of the Wnt pathway through decrease the level of miR-122 [104]. The DNA fragment with HBV integration can be used as a circulating biomarker of HCC recurrence. The HBV-host chimera DNA can be detected in more than 90% of HCC patients before surgery. After the surgery, HBV-host chimera DNA can still be detected in 20% of HCC patients, which may come from the mutant hepatocytes at the early stage of evolution and are significantly associated with HCC recurrence [109]. Thus,

#### *Hepatitis B Virus (HBV) - Induced Hepatocarcinogenesis, a Founding Framework of Cancer… DOI: http://dx.doi.org/10.5772/intechopen.99838*

most HBV integration occurs randomly. The integration mutations that endow the hepatocytes with survival advantage will have the opportunity of accumulation.

As mentioned above, hepatocarcinogenesis involves the co-evolution of HBV and transformed cells. The interaction between somatic mutation and HBV mutation occurs during this process. The deletion, duplication, and translocation are observed near the insertion site of integrated HBV fragments [84]. The frequency of HBV mutation is positive associated with the level of HBV integration. The prevalence of HBx mutation is significantly higher in patients with HBV integration in *TERT* promoter (35%) than in patients without these integration events (19.8%) [83]. There are studies reporting the selective expression of mutant HBx and preS2 genes in the tumor tissues from patients with occult HBV infection [110]. These pieces of evidence support that the integration and selection of mutant HBV fragments play important roles in the HCC evolution.
