Multiple interactive factors in hepatocarcinogenesis

Cancer Letters 346 (2014) 17–23

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Mini-review

Multiple interactive factors in hepatocarcinogenesis Jin Ding ⇑, Hongyang Wang ⇑ International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Hospital/Institute, Second Military Medical University, Shanghai 200433, China National Center for Liver Cancer, Shanghai 200433, China

a r t i c l e

i n f o

Article history: Received 17 October 2013 Received in revised form 8 December 2013 Accepted 10 December 2013

Keywords: Hepatocarcinogenesis Hepatitis virus Inflammation Liver tumor initiating cell

a b s t r a c t Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer and the third most frequent cause of cancer mortality globally. Each year there are approximately 630,000 new cases of HCC in the world and more than half of the new cases occur in China. Major risk factors of HCC include HBV or HCV infection, alcoholic liver disease, and nonalcoholic fatty liver disease. Most of these risk factors lead to chronic hepatitis and cirrhosis, which is present in 80–90% of HCC patients. Hepatocarcinogenesis has been regarded as a multi-stage process involving multiple genetic or environmental factors. Interaction and cross-regulation of distinct factors synergistically contributes to HCC occurrence. A comprehensive knowledge on the multiple factors and their interaction in hepatocarcinogenesis is necessary to improve the effectiveness of HCC intervention. In this review, we will focus on the recent progress made in understanding the mechanisms of hepatocarcinogenesis and discuss some potential issues or challenges in this area. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Hepatocellular carcinoma is the fifth most common cancer in men and the seventh in women worldwide. Most of the cancer burden (85%) is borne in developing countries, with a particularly high prevalence in regions where hepatitis B virus (HBV) infection is endemic: Southeast Asia and sub-Saharan Africa [1]. HCC usually occurs after the age of 40 years and reaches a peak at approximately 70 years of age. Incidence of liver cancer in men is two to four times higher than that in women. Risk factors for HCC primarily include hepatitis virus infection, alcohol intake, and nonalcoholic fatty liver disease (NAFLD). Other less common causes include hereditary hemochromatosis, alpha1-antitrypsin deficiency, autoimmune hepatitis, some porphyrias, and Wilson’s disease. Distribution of these risk factors depends on geographic region and race or ethnic group [1]. Despite the genetic factors, most HCC cases emerge on a background of persistent liver injury and chronic liver inflammation. There is accumulating evidence showing that the viral proteins of hepatitis B and C can directly elicit oncogenic effects or contribute to enhanced risk of hepatocellular transformation in cooperation with the hyperproliferative response induced by chronic inflammation. Therefore, an inflammatory and proliferative tissue microenvironment could be a common feature of the preneoplastic liver regardless of the etiology. ⇑ Corresponding authors. Address: Eastern Hepatobiliary Surgery Institute/Hospital, Second Military Medical University, 225 Changhai Road, 200438 Shanghai, China. Tel.: +86 21 81875361; fax: +86 21 65566851. E-mail addresses: [email protected] (J. Ding), [email protected] (H. Wang). 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.12.024

The lack of effective techniques for HCC early diagnosis and curative therapy lead to the extremely poor prognosis of patients. The morbidity and mortality of HCC are nearly identical because the majority of HCC patients are usually diagnosed at late stage in which it is too far advanced to be cured. Therefore, it is critically important to accelerate the translation of HCC basic research into clinical practice to improve the prevention and therapy of this disease. During the recent years, tremendous efforts by scientists all over the world on HCC study have continually unveiled the molecular mechanism of HCC development.

2. Multiple factors involved in hepatocarcinogenesis 2.1. Genome instability Genetic alteration is the earliest cellular event during carcinogenesis and is frequently detected in numerous cancer types. Given that HBV DNA integration is a critical risk factor for genetic disruptions of HCC, no specific universal genetic alteration has been found in HCC to date (Table 1). Non-specific gene mutations of HCC principally involve p53 and b-catenin. [2–8] Moreover, telomere erosion, chromosome segregation defects and oxidative DNA damage also contribute to HCC genome instability. Recently, whole-genome sequencing technologies have provided extensive and detailed information on specific genetic alterations. Sequencing data have led to new insights into tumor genomes, including somatic single-nucleotide variation (SNV) and genomic rearrangements. Thus, the identification of disease-associated polymorphisms can now be successfully achieved by genome-wide

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association (GWA) analysis. Fujimoto et al. sequenced and analyzed the whole genomes of 27 HCCs, 25 of which were associated with hepatitis B or C virus infection [8]. There was no common somatic mutation identified in the multicentric tumor pairs. However, various chromatin regulators, including ARID1A, ARID1B, ARID2, MLL and MLL3, were mutated in approximately 50% of the tumors [8]. Guichard et al. performed high-resolution copynumber analysis on 125 HCC tumors and identified 135 homozygous deletions, 994 somatic gene mutations and new recurrent alterations in four genes (ARID1A, PPS6KA3, NFE2L2, and IRF2) in HCC [6]. The influence of etiological background, such as HBV infection, on somatic mutation patterns has attracted considerable attention [8]. The sequencing of ten HBV-positive HCC patients with portal vein tumor thrombosis or intrahepatic metastases identified C:G > A:T and T:A > A:T transversions frequently occurred in 331 non-silent mutations [11] and variations at chromosome 8p12 may promote HCC progression in patients with HBV [12]. Huang et al. found that ARID1A, which encodes a component of the SWI/SNF chromatin remodeling complex, was mutated in 14 of 110 (13%) HBV-associated HCCs [11]. It has been gradually accepted that molecular genetic alterations could be far superior to phenotypic surrogate parameters, such as age, gender, tumor size, and liver function, in predicting HCC susceptibility and patient outcomes. Evidence from our lab and others suggested that clonal HBV integration may confer a predisposition of genomic somatic copy number variations (SCNV) and abnormal enrichment of variable number tandem repeats (VNTR). A high rate of G/C-rich repeats has been associated with the transcriptional suppression function related to cell organelle/protein binding/biological regulation/cellular processes during the progression of HCC [13,14]. The amplification of chromosome 11q13.2-13.3 was principally found in approximately 33% of HBV-positive HCC, displaying significant correlation with malignant transformation and poor clinicopathological features. 2.2. Hepatitis virus Although the etiology of HCC has not been fully elucidated, major risk factors, including infection with hepatitis B or C virus, alcoholic liver disease, and nonalcoholic fatty liver disease, account for the vast majority of HCC worldwide. Hepatitis virus infection causes chronic liver injury and subsequent progression to severe fibrosis and cirrhosis. The presence of cirrhosis is a major risk factor for the development of HCC. However, HCC can occur in the absence of cirrhosis, suggesting that both HBV and HCV may be directly involved in hepatocarcinogenesis. Chronic HBV infection

accounts for approximately 50% of all cases of hepatocellular carcinoma and virtually all childhood cases. The HBV genome was frequently detected in chronic hepatitis B carriers and patients with HCC [15,16]. A genome-wide association study identified 1p36.22 as a new susceptibility locus for hepatocellular carcinoma in chronic hepatitis B virus carriers [17]. Most recently, it was reported that genetic variants in STAT4 and HLA-DQ genes confer risk of hepatitis B virus-related hepatocellular carcinoma [18]. The integration of HBV into the host genome induces DNA deletions, translocations and mutations in various chromosome positions. Several HBV factors have also been implicated in hepatocarcinogenesis, including the HBx gene, the pre-S2/S gene and the HBV spliced protein [19]. It is accepted that HBx is indispensable in hepatocarcinogenesis. HBx not only promotes persistent viral infection by enhancing HBV gene expression and replication, but also leads to genome instability through suppression of p53-regulated DNA repair. Moreover, we reported that HBx-mediated transformation of hepatic progenitor cells contributes to hepatocarcinogenesis [20]. Aflatoxin produced by mold of grains has been accepted as an HCC specific carcinogen for a long time [1]. When the HBV carriers were exposed to aflatoxin in diet, their risk for developing HCC is about 60 times higher than unexposed individuals. This increase in cancer risk is much higher than that of people with HBV infection or aflatoxin exposure alone. In contrast to HBV, HCV is an RNA virus that is unable to reverse transcribe to DNA. Various HCV proteins, including the core, envelope and nonstructural protein, have been shown to possess oncogenic properties [21]. It has been reported that proteins encoded by HCV RNA are involved in the manipulation of diverse cellular functions, including apoptosis, proliferation, and ER stress, etc. 2.3. Inflammatory microenvironment Epidemiological studies have established the correlation between chronic infectious diseases and tumors, and it is well known that persistent inflammation increases the cancer risk and facilitates carcinogenesis. HCC predominantly arises from cirrhotic liver where there has been repeated wound-healing response as a result of chronic inflammation [22]. It is widely accepted that an inflammatory microenvironment plays an essential role in the initiation and progression of HCC [23]. The infiltration of inflammatory cells may be caused by necrotic or apoptotic hepatocytes. Various cytokines and chemokines secreted by immune cells and the direct contact between surface proteins on immune cells and hepatocytes remodel the pre-neoplastic microenvironment in liver, promoting genetic mutation and proliferation of both hepatocytes and

Table 1 Mutation frequency of prooncogenes and tumor suppressors in HCC. Gene

Full name

Protein function

Mutation rate (%)

Expected outcome

Reference

TP53 CTNNB1 AXIN1 CDKN2A IGF2R KRAS PIK3CA PTEN ARID1A ARID1B ARID2 S6K1 NFE2L2 ERRFI1 IRF2 MLL3 HNF1A

Tumor protein p53 Beta-catenin Axin 1 Cyclin-dependent kinase inhibitor 2A Insulin-like growth factor 2 receptor Kirsten rat sarcoma viral oncogene homolog Phosphatidylinositol 3-kinase, catalytic, alpha polypeptide Phosphatase and tensin homolog AT rich interactive domain 1A AT rich interactive domain 1B AT rich interactive domain 2 Ribosomal protein S6 kinase Nuclear factor, erythroid 2-like 2 ERBB receptor feedback inhibitor 1 Interferon regulatory factor 2 Myeloid/lymphoid or mixed-lineage leukemia 3 HNF1 alpha

DNA damage response, other Positive regulator of Wnt signaling Negative regulator of Wnt signaling Positive regulator of senescence Growth factor signaling Growth factor signaling Positive regulator of Akt signaling Negative regulator of Akt signaling Chromatin remodeling Chromatin remodeling Chromatin remodeling Growth factor signaling Transcriptional factor/oxidative stress Growth factor signaling Transcriptional factor/TP53 pathway Histone methyltransferase Transcription of liver-specific genes

11.4–35.1 20–40 3–16 7.2 0–13 <2 <3 <5 10–16.8 6.7 5.6–16 9.6 6.4 5.4 4.8 4.2 <3

Loss-off-unction Gain-of-function Loss-of-function Loss-of-function Loss-of-function Gain-of-function Gain-of-function Loss-of-function Loss-of-function Loss-of-function Loss-of-function Unknown Unknown Loss-of-function Loss-of-function Loss-of-function Loss-of-function

[2,3] [2,4] [2] [5,6] [7] [5,6] [5,6] [5,6] [6,8] [8] [9,10] [6] [6,9] [8,9] [6,9] [9] [7]

J. Ding, H. Wang / Cancer Letters 346 (2014) 17–23

cholangiocytes. Tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) are the most essential cytokines produced by activated macrophages in the liver microenvironment. Mice lacking IL-6 develop much less HCC in response to the liver carcinogen diethylnitrosamine (DEN). The gender-based production of IL-6 might account for the much higher HCC load in males [24]. High levels of circulating IL-6 are associated with enhanced HCC risk and may serve as a predictor of the progression from viral hepatitis to HCC in humans. Inflammatory cytokines activate transcription factors such as NFjB, STAT3 and AP-1, which mediate tumorigenesis, metastasis and angiogenesis via induction of numerous down-stream target genes [25,26]. Recent studies revealed that liver damage-mediated inflammation and carcinogenesis is triggered by a complex crosstalk between NF-jB, JNK and STAT3 signaling pathways [27]. Increased COX2 expression was found not only in the liver of patient with chronic hepatitis and cirrhosis, but also in the HCCs of patients. The upregulation of COX2 expression and PGE2 synthesis leads to enhanced EGFR signaling and transformation of epithelial cells [28]. Furthermore, many reports have described the growth inhibitory properties of COX2 inhibitors in experimental HCC [29]. Epigenetic events, which are considered as key mechanisms in the regulation of gene activity states, are also commonly deregulated in HCC. Epigenetic deregulation was also demonstrated to be an underlying mechanism by which inflammation promotes HCC development [30]. It has been documented that inflammatory responses of hepatocytes play decisive roles at different stages of tumor development, including initiation, promotion, and progression. Immune cells that infiltrate tumors engage in an extensive crosstalk with cancer cells and affect immune surveillance and responses to therapy. Therefore, resolving inflammation should be an essential part of HCC prevention and therapy. For example, interferon was reported to prevent HCC recurrence and improve the survival of HCC patients [31]. 2.4. Oncogenic molecules and pathways Hepatocarcinogenesis is a process closely correlated with the disruption of several signaling pathways involved in cell survival, proliferation, differentiation and apoptosis. As shown in Fig. 1, these signaling cascades in hepatoma cells are usually interconnected and are affected by the aberrant expression of proto-oncogenes or loss of tumor suppressor genes [32]. p53 and retinoblastoma (Rb) associated pathway are frequently disrupted in hepatoma cells, resulting in distinct cell-cycle checkpoints or resistance to apoptosis [33,34]. p53 was recognized as one of the most frequently mutated genes in human cancer with a mutation rate of approximately 10–35% in HCCs in different areas [2,3]. In addition, HBx and HCV core protein can repress p53 activity. These results indicate an association between p53, hepatitis virus and hepatocarcinogenesis [35]. Disruption of the Rb regulatory network is common in HCC carcinogenesis. Rb expression was found to be absent in 28% of human HCCs [36]. Simultaneously, other members of the Rb network also exhibit aberrant expression in HCCs. For example, cyclin D1/Cdk4, which phosphorylates and inactivates Rb, is overexpressed in 58% of HCCs [37]. P16 protein, a negative regulator of Rb, is absent in 34% of HCCs [38]. Aberrant Wnt signaling-triggered cancer development is most likely caused by the hyperactivation of b-catenin. The prevalence of b-catenin mutations in human HCCs has been estimated from 20% to 40% and some reports describe a close association of b-catenin mutations with aflatoxin B1 exposure and HCV infection [2,39]. The activation of Akt signaling and impaired expression of PTEN has been reported in over 40% of human HCC [40]. Akt signaling is also responsible for b-catenin activation. Our lab and others have reported that gankyrin, a highly expressed oncoprotein in HCC, regulated these oncogenic pathways during HCC initiation and

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progression [41–43]. We also reported that forced gankyrin expression significantly decreased the level of hepatocyte nuclear factor 4 in both hepatocytes and hepatoma cells [44]. Other key molecules involved in hepatocarcinogenesis include p21/p27, cyclin D1, c-myc and survivin, etc. [45]. These key molecules are usually regulated by the critical signaling pathways in HCC including TGF-b/Smad, Wnt/b-catenin, IKK/NF-jB, etc. Hedgehog (Hh) and Notch signaling play important roles in embryonic development and act as critical regulators of cell differentiation, apoptosis and proliferation. It has been reported that the Notch signaling pathway is activated in human HCCs and could elicit tumor formation in vivo [46,47]. Aberrant activation of Hh signaling has also been implicated in hepatocellular carcinoma [48]. Generally, activation of one signaling pathway may lead to the activation or inactivation of another, and feedback loops of signaling pathways are commonly observed in HCC. Simultaneous blockage of distinct key molecules in oncogenic signaling network might be one of the most promising approaches to conquer HCC. Multi-target drugs, such as sorafenib and gefitinib, have been shown to prolong the survival of HCC patients [49,50].

2.5. Liver tumor-initiating cell Cancer arises from a small, defined subset of undifferentiated cells, termed tumor-initiating cells (T-ICs) or cancer stem cells (CSCs) [51]. These cells are able to self-renew and differentiate into the bulk tumor population during carcinogenesis [52]. Increasing evidence shows that the existence of liver T-ICs is responsible for the heterogeneous and hierarchical organizations of HCCs [53,54]. As shown in Table 2, distinct surface antigen markers have been reported to identify liver T-ICs, which consist of CD133, CD90, EpCAM, CD13, CD24, OV-6, CD44 and ALDH [55–66]. Many chemotherapeutic agents inhibit the proliferation of cancer cells while exhibiting poor effectiveness against T-ICs [67]. Therefore, eradication of liver T-ICs is essential to achieve a stable, long-lasting remission, and even a cure for HCC [68,69]. Specific therapies targeting T-ICs are currently limited. Rodent and human liver cancer stem/progenitor cells display increased expression of receptor tyrosine kinases (RTK). The use of specific RTK inhibitors, including imatinib mesylate and PHA665752, were reported to be effective in HCC intervention [70,71]. Natural compounds such as lupeol have also been demonstrated to be effective dietary phytochemicals that targets liver T-ICs [72]. When T-ICs migrate into blood vessels and become circulating T-ICs, they play a critical role in the metastasis of HCC and the formation of portal vein tumor thrombosis [57,64]. Adult hepatocytes possess stem cell-like properties because they are capable of extensive proliferation upon liver damage and can differentiate into both hepatocyte and biliary lineages under special conditions [73]. Thus, hepatocytes may transform into liver T-ICs through dedifferentiation and initiate hepatocarcinogenesis [74]. Another possible origin of liver T-ICs is transformed liver progenitor cells [75]. Liver progenitor cells usually reside quiescently in the bile ductules and canals of Hering. However, they are known to differentiate into hepatocytes or cholangiocytes and repopulate the liver when the replication of liver parenchymal cells is limited [73]. Liver progenitor cells exposed to pre-neoplastic conditions such as chronic inflammation could be transformed into liver T-ICs [76,77]. Our previous study demonstrated that long-term TGF-b exposure drove the transformation of hepatic progenitor cells into hepatic T-ICs, which contributes to cirrhosis-elicited hepatocarcinogenesis [78]. It is widely accepted that HCC development is attributed to the propagation of liver T-ICs. However, the molecular mechanisms underlying liver T-ICs maintenance and expansion remain unclear. Several studies have demonstrated that a comprehensive signaling network involving TGF-b, JAK/STAT3,

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Fig. 1. Interactive signalings in HCC development.

Table 2 Biomarkers of liver cancer stem cells. Markers

Year

Cell line/clinical sample

Function in CSCs

Mechanism

References

CD133 CD90 EpCAM CD44 CD13 CD24 OV-6 DLK1 ALDH ICAM-1

2007 2008 2009 2008 2010 2011 2008 2012 2008 2013

Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell

Self-renewal, tumorigenicity, chemoresistance, invasiveness Tumorigenicity, metastasis Self-renewal, invasiveness, tumor formation Tumor formation, chemoresistance, metastasis Self-renewal, cell proliferation, tumor formation Tumor formation, self-renewal, chemoresistance, metastasis Tumor formation, chemoresistance Self-renewal, cell proliferation, tumor formation, chemoresistance Tumor formation, cell proliferation Tumor formation, self-renewal, metastasis

Neurotensin/IL-8/CXCL1 signaling Not reported Activation of Wnt signaling Regulation of redox status through xCT ROS-induced DNA damage reduction STAT3-mediated NANOG regulation Activation of Wnt signaling Not reported Not reported Not reported

[55] [56,57] [58] [57,59] [60] [61] [62] [64] [65] [66]

line/sample line/sample line/sample line/sample line/sample line/sample line/sample line line line/sample

Notch, and PI3-K/Akt/mTOR regulates the activation and functions of liver T-ICs [79–81]. 2.6. Systematic factors Epidemiological studies have illustrated that men have a higher prevalence of HCC than women. The ratio of males to females with HCC ranges from 2:1 to 4:1 depending on the regions and lifestyles [82]. The increased incidence has been attributed to men being more vulnerable to hepatitis virus infection or alcohol addiction. Moreover, men have a higher level of androgenic hormone which was accepted as a major factor for sex discrepancy of HCC morbidity. However, recent evidence indicates that it is indeed androgen receptor (AR) instead of androgen that influences hepatocarcinogenesis [83–85]. Hepatic AR directly interacts with the HBV

genome by binding with androgen response elements (AREs) near the viral core promoter to enhance the expression of HBx which in turn cooperates with AR to facilitate tumorigenesis [86]. The induction of the proinflammatory cytokine IL-6 after liver injury is critical for HCC initiation, and estrogen is capable of suppressing the production of IL-6 by kupffer cells. Thus, reduced IL-6 may also explain why women have a lower incidence of HCC than men [24,87]. Recently, a large-scale cDNA transfection screening revealed that a cluster of genes associated with host-cell systemic regulation were involved in HCC development. These genes include transporter genes, which play a crucial role in the nervous system, and some neurotransmitter-related genes [88]. It was recently demonstrated that acetylcholine (ACh) can be synthesized by HCC cells both in vitro and in vivo. ACh interacts with ACh receptors expressed in HCC cells as an autocrine/paracrine system to

J. Ding, H. Wang / Cancer Letters 346 (2014) 17–23

promote cell proliferation and apoptosis resistance. The ACh degradation enzyme acetylcholine esterase (AChE) can adjust the ACh level in HCC cells and cancer microenvironment. Downregulation of AChE led to ACh induction in about 2/3 of HCCs and correlated with poor clinical outcome of patients [89]. These results indicate that components of the nervous system may have a direct effect on HCC development, aside from the systemic regulation of immune system. Obesity and diabetes, in particular type II diabetes, have been proposed to be risk factors for HCC [90–92]. Case-control studies from the USA, Italy, Japan and Taiwan have illustrated the positive association between diabetes and HCC [93–95]. Moreover, diabetic patients with HBV/HCV infection have a higher risk of HCC incidence [96,97]. In addition, the influence of obesity on HCC risk has also been evaluated in cohort studies [98]. In a large prospective cohort study of more than 900,000 individuals followed for a 16-year-period, the morbidity of liver cancer was five times greater for men with the high baseline body mass index (range, 35–40) than men with a normal body mass index [99]. 3. Perspective and challenge Hepatocarcinogenesis is a complicated process involving various factors of both host and environment. Interactions and crossregulation of distinct factors synergistically contribute to the development of HCC [100]. In recent years, gene knockout (KO) mouse models have been employed by numerous groups to dissect HCC pathogenesis. This approach has no doubt provided valuable insights into mechanisms of hepatocarcinogenesis. However, conflicting roles in the regulation of HCC have been reported for the same molecules in distinct animal models [101–103]. These opposing results do not necessarily obscure the understanding of molecular pathogenesis of HCC development. Instead, further experiments carefully designed to decipher the paradoxical roles of oncogenic molecules will lead to a better understanding of hepatocarcinogenesis [103]. Further understanding of hepatocarcinogenesis will ultimately facilitate the prevention of HCC in humans. For example, because hepatitis virus infection is directly linked to HCC onset, the development and application of potent antiviral agents or vaccines is essential for HCC prevention. The first universal HBV vaccination program for newborns began 20 years ago in Taiwan, with infants of mothers at high-risk for HBV infection (HBsAg-positive) receiving both the vaccine and an injection of hepatitis B immune globulin. Since then, the morbidity of HCC in children between 6 and 14 years of age has declined by 65–75% [104]. Genetic heterogeneity and existence of T-ICs result in the poor therapeutic effect of current HCC chemotherapy [105]. Molecular classification and targeted therapy aiming at different hallmark characteristics of HCC will greatly favor the personalized management of HCC. Combinations of targeted drugs and chemotherapy are likely to achieve maximal therapeutic benefits. Comprehensive analysis of the multiple oncogenic factors and their interactive regulation in hepatocarcinogenesis is necessary to improve the effectiveness of HCC intervention. Although recent progress on HCC research offers promise to translate bench-tobed discoveries and shed new light on the prognosis of HCC patients, further studies are still urgently needed. Conflict of Interest The authors declare that they have no conflict of interest. Acknowledgements The authors apologize for not citing many other exciting papers in this minireview and thank Zhong Li, Kun Wu, Le Qu, Daimin

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Xiang (Second Military Medical University) and Gensheng Feng (University of California, San Diego) for their efforts in manuscript preparation. The research work in authors’ laboratory was supported by grants from National Natural Science Foundation of China 81222034 and 81372329; Ministry of science and technology key program 2012ZX10002009 and 2012ZX10002010; Shanghai Pujiang program. References [1] B. Hashem, Current Concepts Hepatocellular Carcinoma, N. Engl. J. Med. 365 (2011) 1118–1127. [2] M.L. Tornesello, L. Buonaguro, F. Tatangelo, G. Botti, F. Izzo, F.M. Buonaguro, Mutations in TP53, CTNNB1 and PIK3CA genes in hepatocellular carcinoma associated with hepatitis B and hepatitis C virus infections, Genomics 102 (2013) 74–83. [3] S.P. Hussain, J. Schwank, F. Staib, X.W. Wang, C.C. Harris, TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer, Oncogene 26 (2007) 2166–2176. [4] B. Cieply, G. Zeng, T. Proverbs-Singh, D.A. 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[18]

[19]

[20]

[21] [22] [23]

[24]

[25] [26] [27]

[28]

[29] [30]

[31]

[32]

[33]

[34]

[35]

[36]

[37] [38]

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