An Update on the Molecular Genetics

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Semin Liver Dis 2004;  77-88
DOI: 10.1055/s-2004-823102
  


An Update on the Molecular Genetics of Hepatocellular Carcinoma
  
Arief Suriawinata1 M.D. , Ruliang Xu2 M.D.  Ph.D.  
1 Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire
2 Assistant Professor, The Lillian and Henry M. Stratton-Hans Popper Department of Pathology, The Mount Sinai Medical Center, New York, New York


  
MOLECULAR GENETICS OF HBV- AND HCV-ASSOCIATED HEPATOCARCINOGENESIS
MOLECULAR GENETICS OF AFLATOXIN-ASSOCIATED HEPATOCARCINOGENESIS
MOLECULAR GENETICS OF WILSON DISEASE AND HEMATOCHROMATOSIS-ASSOCIATED HEREDIATARY HEPATOCARCINOGENESIS
MOLECULAR GENETICS OF PRENEOPLASTIC LESIONS
MOLECULAR GENETICS OF HEPATOCELLULAR CARCINOMA
FUTURE CHALLENGES
REFERENCES
  

  

Hepatocellular carcinoma (HCC) is associated with multiple risk factors and is believed to arise from preneoplastic lesions, usually in the background of cirrhosis. Extensive studies on HCC and its precursors have demonstrated complex and heterogeneous genetic or chromosomal abnormalities along the way from preneoplastic lesions to HCCs. These genetic abnormalities include loss of heterozygosity, microsatellite instability, gene alterations, and aberrant global gene expression profiles. Although some genetic alterations involving the p53 family, Rb family, and Wnt pathways are particularly important in the development of HCCs, the molecular pathogenesis of HCC differs with etiology in some extent. Recent studies using DNA microarray technique have identified some unique gene expression profiles in hepatitis B virus (HBV)- and hepatitis C virus (HCV)-associated HCCs. Gene expression profiling also allows people to distinguish HCCs from normal tissue or preneoplastic lesions and to evaluate metastatic or recurrent potentials. These unique genes or gene products associated with malignant transformation and recurrent or metastatic potentials may serve as molecular markers for early diagnosis, prediction of prognosis, and responsiveness to therapy. To date, information that has accumulated for the past several decades is still incomplete, and we still are faced with a great challenge in deciphering the molecular mechanisms of HCCs.

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KEYWORDS
Hepatocellular carcinoma - hepatocarcinogenesis - molecular genetics - gene expression profile


  

HCC is one of the most frequent neoplasms worldwide.[1][2] The major risk factors include chronic HBV and HCV infection, environmental carcinogens such as aflatoxin B1 (AFB1), alcoholic cirrhosis, and inherited genetic disorders such as hemochromatosis, Wilson disease, α1-antitrypsin deficiency, and tyrosinemia. Among them, HBV, HCV, and AFB1 are responsible for approximately 80% of all HCCs.[2]

HCC nearly always develops in the setting of chronic hepatitis or cirrhosis resulting from the previously mentioned risk factors, in which there is continuous inflammation and regeneration of hepatocytes. In recent years, numerous studies have given rise to information on the genetic alterations or chromosomal aberrations during the processes of inflammation, regeneration, and cirrhosis and in HCCs. The data from research on human HCCs have demonstrated altered genomics, gene products, and molecular interaction between hepatocytes and the specific etiologic agents of HCC leading to the development of HCC.[3][4][5]

It appears that the development of HCCs in general is not a random event. Several lines of evidence suggest that hepatocarcinogenesis may begin in preneoplastic lesions such as macroregenerative nodules (MRN) and low-grade dysplastic (LGD) and high-grade dysplastic (HGD) nodules.[6][7][8][9] Accumulation of genetic alterations or emergence of new alterations in preneoplastic lesions is believed to lead to the development of HCCs. Although some HCCs have the same genetic changes irrespective of their etiology, most HCCs have unique molecular mechanisms related to a specific etiology.[3][10][11] For example, molecular pathways of HBV- and HCV-induced hepatocarcinogenesis involving Rb1, p53, and Wnt families are different from those associated with alcoholism.[10] Figure [1] summarizes the current concept of the multistage hepatocarcinogenesis associated with different risk factors.


Figure 1 Summary of multistage hepatocarcinogenesis associated with different risk factors. (CMAR, cellular adhesion regulatory molecule). (Modified from Staib F, et al. TP53 and liver carcinogenesis. Human Mutation, 21:201-216, 2003. Copyright © 2003 by Wiley-Liss, Inc. Reprinted by permission of Wiley-Liss, Inc, a subsidiary of John Wiley & Sons, Inc.)

Research on molecular genetics and pathogenesis of HCC has become a hot spot in cancer study because of its scientific merits as well as its clinical importance. Despite rapid expansion of information gained from these researches, molecular mechanisms of hepatocarcinogenesis and molecular genetics of HCC remain unclear, in part because of the high complexity of genetic alterations in HCCs and their precursors and too much information to be interpreted rationally. Important progress in this field has been reviewed in several excellent articles during last several years.[11][12][13][14] The purpose of this article is to provide updated information on the molecular genetics of hepatocarcinogenesis, including the known genetic and epigenetic events, and to provide insights to better understand the mechanisms of hepatocarcinogenesis.

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MOLECULAR GENETICS OF HBV- AND HCV-ASSOCIATED HEPATOCARCINOGENESIS
HBV is a Hepadnavirus and has a small double-stranded DNA genome. Although much has been known about HBV DNA integration into hepatocytes, the complex role of HBV in hepatocarcinogenesis through direct and indirect mechanisms is still being debated.[15] Integration of HBV DNA sequences into the host cell genome can activate cellular oncogenes by a cis-acting mechanism in a few HCC cases.[16] Chromosomal instability may also result from HBV DNA integration, as exemplified by frequent chromosomal translocation and deletion or amplification of large chromosomal regions at the site of HBV integration. Indirectly, liver cell injury mediated by cellular immune responses may be sufficient to cause liver cancer by promoting cell death, proliferation, and genetic mutations, which may accumulate in the context of chronic necroinflammatory disease. This hypothesis has been strongly supported by the finding that HBV envelope coding region transgenic mice develop chronic hepatitis and ultimately HCC after stimulation of the immune response by adoptive transfer of activated T lymphocytes.[17] HBV large envelope protein by itself can also elicit hepatotoxicity and hepatitis, which eventually result in hepatic malignancy in transgenic mice. These findings suggest that inflammation alone is sufficient for hepatocarcinogenesis.[18][19]

HBV also encodes a 154-amino acid (16.5-kD) viral protein termed HBx. This protein may act as a transactivator that upregulates a variety of viral and cellular genes and is implicated in oncogenesis.[20][21] In the nucleus, HBx protein may function as a coactivator by interacting with transcription factors and with elements of the basal transcription machinery transcription factor IIB [TFIIB], TATA Box-binding protein [TBP], and RNA polymerase II subunit RPB5).[22][23] In the cytoplasm, HBx protein activates the Src pathway, stimulating the intracellular viral replication process, and the Ras/Raf signaling pathway, leading to the activation of several oncogenes such as c-myc, c-jun, and c-fos.[24][25][26][27] HBx protein interacts with multiple regulatory proteins, such as DNA repair protein UVDDB, p53 tumor suppressor protein, and proteasome subunits.[28][29][30] HBx protein also indirectly interacts with nuclear transcription factor (NF-κβ), which modulates the immune response.[26]

HBV infection is associated notably with a high level of transforming growth factor-β (TGF-β) expression.[31] HBx protein amplifies TGF-β signaling through direct interaction with Smad4, which contributes to hepatic fibrosis. TGF-β expression could be part of a chain event by which HBV contributes to the development of HCC. Expression of HBx protein is generally maintained through the tumorigenic process.[32][33] Although HBx protein may not have transforming activity by itself, its overexpression may sensitize liver cells to other carcinogens and activate cellular oncogenes in a multistep hepatic transformation.[34]

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HBx protein can alter global gene expression patterns in vivo. In freshly isolated normal human hepatocytes expressing HBx protein and in liver samples from patients with HBV chronic active hepatitis, complementary DNA (cDNA) microarray analysis of gene expression profiles has demonstrated that HBx protein dysregulates tumor suppressors (downregulating p53, antigen presenting cell [APC] gene), cell cycle regulators, growth factors and their receptor genes (upregulating cyclin D3, cdk4, insulin-like growth factor IR [IGF-IR], downregulating TGF-β, p21waf1, gas6), cytokines and their receptors (upregulating interferon α/β receptor 2, downregulating interleukin 6 [IL6]), and genes involving in apoptosis (Bcl-XL, Fas, Caspase 4).[35] Further study by serial analysis of gene expression profiling technique has confirmed that HBx protein can upregulate or downregulate many transcripts. These upregulated transcripts include three major classes of genes encoding ribosomal proteins, transcription factors containing zinc-finger motifs, and gene products involved in the protein degradation pathway. These results suggest that HBx protein may function as a major regulator in common cellular pathways that, in turn, regulate protein synthesis, gene transcription, and protein degradation.[36]

HCV is another hepatotropic virus, and its infection is strongly associated with the development of HCC. It is a single-stranded RNA virus, and its genome is never integrated into the genome of hepatocytes.[37] More patients with HCV infection-induced cirrhosis develop HCC than develop HBV, yet there are no known HCV genes that have oncogenic properties. HCV-induced oncogenesis has been shown to result from “abduction” of host intracellular pathways by viral proteins, notably the viral core protein, and by interaction between viral proteins and host immune response. Viral core protein can interact with several intracellular pathways, including indirect activation of TNF-α receptor, the Raf-1 kinase, and NF- κβ pathways resulting in inhibition of TNF-α and Fas-mediated apoptosis. Inhibition of apoptosis induced by TNF-α and modulation of other cytokine activities could prolong survival of infected hepatocytes, allowing them to have more accumulated genetic damages that led to the development of malignancy.[38][39][40]

Although direct induction of HCC by HCV core protein has been demonstrated in transgenic mice,[41] the mechanism of HCV core protein-induced HCC remains unknown in humans.[40] Other HCV proteins, including nonstructural protein NS5A and replicase have also been implicated, but their role in hepatocarcinogenesis is less clear than that of the core protein.[40][42][43][44][45]

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MOLECULAR GENETICS OF AFLATOXIN-ASSOCIATED HEPATOCARCINOGENESIS
AFB1 is a fungal metabolite that contaminates food in certain areas of the world.[46] It is produced by Aspergillus flavus and related fungi that grow on improperly stored foods, such as corn, rice, and peanuts. Dietary exposure to AFB1 is associated with an increased incidence of HCC.[47] In the past two decades, much has been learned about the relationship between DNA damage by AFB1 and the biological end points of mutagenesis and carcinogenesis.[48] AFB1 requires conversion by cytochrome P450 to AFB1 to 8, 9-exo-epoxide to damage DNA. The AFB1 epoxide reacts with guanine to form several adducts. One reasonable hypothesis is that the adducts or secondary DNA lesions lead to heritable genetic changes necessary for malignant transformation. AFB1 exposure is related to high frequency of mutations affecting 249ser of p53 tumor suppressor gene (TSG) (codon 249 G:C to T:A transversion).[48][49] The same study also concludes that toxic effects of AFB1 are more broadly based than just having an effect on p53. The strong association between the 249ser mutation of p53 gene and AFB1 exposure have been confirmed by other studies.[50][51][52] The mutational spectrum of p53 in human HCC in areas of low AFB1 exposure seems to be different from that of 249ser mutation.[50][51] A dose-dependant relationship between p53 mutation load of 249ser mutant cells and the intake of AFB1 in nontumorous liver tissue has been observed.[52]

It appears that there is a synergism between HBV infection and high aflatoxin exposure in hepatocarcinogenesis.[53] AFB1 induces promutagenic N7dG adduct formation through metabolic activation in hepatocytes.[54][55] In the background of viral infection, this product could allow the fixation of the G:C to T:A transversion resulting in 249ser mutation, which may select a hyperproliferative cell clone, leading to neoplastic process.[14] In the areas with high prevalence of AFB1 and HBV infection, this synergistic interaction has highly significant epidemiological and clinical implications.

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MOLECULAR GENETICS OF WILSON DISEASE AND HEMATOCHROMATOSIS-ASSOCIATED HEREDIATARY HEPATOCARCINOGENESIS
HCC is a known complication of Wilson disease and hemochromatosis. In Wilson disease and hereditary hemochromatosis, the excessive absorption and accumulation of the metals result in irreversible damage in human vital organs, including the liver. Both conditions may lead to cirrhosis and, in some cases, to HCC. The relative risk of HCC in hereditary hemochromatosis is 200-fold, whereas in Wilson disease the risk is somewhat lower.[56][57] Iron could be involved in the development of HCC in hereditary hemochromatosis patients irrespective of the presence or absence of cirrhosis.[58] HFE gene mutations in hemochromatosis, except for some C282Y homozygotes, do not increase the risk to develop HCC in patients with cirrhosis.[59][60] It appears, therefore, that the genetics of these conditions does not directly contribute to the development of HCC.

The molecular mechanism in these genetic disorder-associated HCCs relates to the interactions between iron or copper overload and immune response in human, which results in inflammation and generation of free radicals that damage the human DNA and cause genetic alterations. Mutations within the p53 gene with clustering at codon 220 (A-G) may be characteristic of hemochromatosis-associated HCC, as suggested by a study from a group of British patients.[61] In nontumorous hepatocytes from patients with hemochromatosis or Wilson disease, there is high frequency of specific mutation at codon 249 of p53 (G:C to T:A transversion). Mutation at codon 250 (C:G to T:A transition) is also observed in Wilson disease-associated HCC.[56] Oxidative free radicals-induced mutations in some important genes, including TSGs, cell cycle regulator genes, genes associated with DNA repair, and apoptosis, are believed to be main mechanisms of the development of Wilson disease and hemochromatosis-associated HCC.[56][62][63]

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MOLECULAR GENETICS OF PRENEOPLASTIC LESIONS
Hepatocarcinogenesis in humans is a process that may take decades to develop after the initial viral infection or exposure to carcinogens. Similar to colonic carcinogenesis, the development of HCC may undergo a sequence from hyperplasia to dysplasia to carcinoma.[6][64][65] Epidemiological studies have shown that MRN and LGD and HGD are earlier stages in the development of HCC. They are present mainly in cirrhotic livers, in which more than 80% of HCCs occur. These lesions have been shown to exhibit the high apoptotic count and clonality that are distinct from the surrounding cirrhotic nodules.[64][65][66][67][68][69][70] Furthermore, these lesions frequently accompany HCCs spatially and temporally.[4][6][9][64] The progressive accumulation of genetic changes in cirrhotic, dysplastic, and malignant hepatocellular nodules is in keeping with a multistep process of carcinogenesis; within this spectrum, high-grade dysplastic nodules can be considered advanced precursors of HCC.[65][71][72]

There are several significant genetic changes during a long preneoplastic stage leading to HCC, including upregulation of growth factors, aberrant methylation, and microsatellite instability. Upregulation of TGF-α and IGF-2 results from the combined actions of cytokines produced by chronic inflammatory cells in carcinogen-damaged liver, viral transactivation, and the regenerative response of the liver to cell loss.[73] These growth factors are responsible for accelerated hepatocyte proliferation. Clonal expansion of hepatocytes is initiated during regeneration in damaged or cirrhotic livers.[74]

Both hypomethylation and hypermethylation are observed in livers with chronic hepatitis and cirrhosis.[75][76][77][78] DNA methyltransferases (DNMT) are increased in a fraction of livers affected with chronic hepatitis and cirrhosis. Furthermore, both DNMT1 and DNMT3a are strongly upregulated in HCCs, suggesting progression of dysregulation from preneoplastic lesion to HCC.[76][79]

Loss of heterozygosity (LOH) and microsatellite instability occur in hepatocytes in some chronic hepatitis, cirrhosis, and HCC.[80][81] There is minimal genetic abnormality detected in the DNA of cirrhotic nodules. The gain at chromosomal loci 1q is the most common finding, which is also shared by dysplastic nodules and small HCCs.[82] In a recent study performed on microdissected tissue, almost half of cirrhotic nodules showed LOH with informative markers, including allelic chromosomal arms 1q and 14q, TBP, and BRCA1. LOH in some other markers (NF1, 6q, and IGFIIr) were similar between cirrhotic nodules and microscopic HCCs from the same patient. In contrast to HCCs, no cirrhotic nodules showed LOH at p53 and Rb loci.[83] In a similar study using a polymerase chain reaction (PCR)-based LOH assay, LOH was detected in chromosome 8p21.3-p22 in approximate 40% dysplastic nodules and HCCs. LOH on chromosome 11p13 was found in 15.8% of dysplastic nodules and 31.6% of HCCs. D8S261 showed the most frequent LOH (28.6% in dysplastic nodule and 40.0% in HCCs). In dysplastic nodules, there was more LOH of D11S995 (33.3%) but less LOH of D11S907 (7.1%), whereas in HCCs, LOH of D11S907 (44.4%) was found more frequently than was LOH of D11S995 (8.3%).[84] In general, the multiplicity of allelic deletions in affected cell populations is low in chronic hepatitis but rises in dysplastic lesions and is highest in HCCs.[82][85]

Recently, global genetic alterations in the sequence of hyperplasia to dysplasia to carcinoma were explored. A study by cDNA gene expression profiling analysis shows that among 1152 genes (human cDNAs) tested, there are more than 50 genes that are consistently dysregulated in MRNs and dysplastic nodules, compared with normal or surrounding cirrhotic tissue. These dysregulated genes, 29 upregulated and 24 downregulated, include oncogenes (v-akt homolog 2, Kangai 1, and so on), TSGs (WT1, Rb1, and so on), DNA repair genes (DNA excision repair protein ERCC5), genes encoding cell growth factor and cytokines (Flt-3, epidermal growth factor receptor, IGF binding protein 3, and so on), gene encoding adhesion proteins (pax5, and so on), gene family of signal transduction (protein kinase C, α; protein kinase; cAMP dependent, type I, β, and so on), transcriptional factors (tyrosine protein kinase, C-terminal Sre kinase [CSK], and so on), transcription factor and DNA binding, and housekeeping genes.[86] A similar study has demonstrated that these preneoplastic lesions exhibit relatively unique gene expression patterns. They can be classified based on their gene expression profiles and can be distinguished from surrounding normal tissue and HCCs.[13][87] These differences between HCCs and its precursors suggest that many of the cells harboring the early genomic aberrations do not evolve into the malignant phenotype.[14][88] Early genetic changes appear to be insufficient to induce malignant transformation of hepatocytes because HCCs ultimately develop in only a fraction of the livers with chronic hepatitis or cirrhosis.

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MOLECULAR GENETICS OF HEPATOCELLULAR CARCINOMA
The potential causes of structural genetic alterations in HCCs are numerous and varied.[11][14][40][82] Several studies have yielded extensive genetic heterogeneity of HCCs because of their divergent characteristics. However, there remain high coefficients of variation among results from different studies. Individual HCC typically contains multiple genomic and chromosomal alterations; even in small, well-differentiated HCCs there often are simultaneous structural alterations in many genes and chromosomes.[89]

Early cytogenetic studies have revealed that most HCCs are aneuploid and harbor multiple different chromosomal abnormalities, with recurrent deletions of the short arm of chromosome 1.[90][91] Subsequently, during the last decade, extensive studies have used restriction fragment length polymorphism, which allows identification of nonrandom, recurrent DNA copy number losses in HCC on multiple chromosomal arms, including 1p, 4p, 5q, 6q, 8p, 9p, 13q, 16p, 16q, and 17p, and gains on 1q, 6p, 8q, and 17q.[92][93] These findings suggest that a variety of TSGs in these chromosomal arms may be inactivated during the carcinogenesis, which is consistent with the multifactor etiology of HCC. The development of PCR-based approach in recent years has allowed scanning for LOH.[94][95][96] Fine mapping of LOH loci suggests that more than one TSG might be located along the chromosomal arms. LOH has been reported for the p53 locus at 17p13, Rb at 13q14, axin at 16p13, and mannose-6-phosphate and IGF II receptor at 6q27.[95][97][98][99] LOH at chromosome 1p might represent an early event in HCC in tumors that are small and well-differentiated.[100] LOH at chromosomes16p and 17p is frequently associated with advanced tumor stage and poor prognosis and is not detected in early-stage HCCs; therefore, it may be involved in enhancement of tumor aggressiveness.[101][102]

Comparative genomic hybridization (CGH) analysis gives more precise results for the screening of chromosomal aberrations in HCCs than PCR-LOH analysis using randomly selected microsatellite markers. CGH has confirmed DNA copy losses at chromosomal arms affected by LOH. The most frequent DNA copy gains or losses are on chromosomes 1q, 8q, 6p, and 17q, which are also observed in other human malignancies, with chromosome 1q as the commonest aberration in different geographic location.[71][103][104] Comparative analysis between primary HCCs and matched metastatic lesions shows an increased rate of losses at chromosome 8p in metastatic tumors.[105] This is further confirmed by genome-wide microsatellite analysis. Regions with increased allelic imbalance in metastatic lesions are 8p23.3, 8p11.2, 17p11.2 to 13.3, 4q21 to 22, 4q32-qter, 8q24.1, 9p11, 9q31, 11q23.1, 13q14.1 to 31, 13q32-qter, 16p13.3, 16q13, 16q22, and 19p13.1, and these are considered to be related to the metastasis phenotype.[106] The resulting chromosomal aberration profiles are also used as genomic fingerprints to determine tumor clonalities and their relationships; therefore, CGH analysis is also a useful tool to determine a tumor as a recurrent or a second primary.[107]

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Comparative analysis of HBV- and HCV-associated HCCs reveals that gain at 10q is exclusively associated with HCV-positive cases, whereas loss of 4q and 16q and gain of 11q are seen preferentially in HBV-positive cases.[82][108] In HBV, genes involved in inflammation are predominant, whereas in HCV, expression of anti-inflammatory response genes is relatively dominant. Tumors associated with alcoholism have more frequent alterations in the Rb1 and p53 pathways than those caused by HCV infection have.[109] The “aflatoxin-associated” codon 249 mutation is identified only in samples from Asia and Africa, where chronic HBV infection is endemic and the aflatoxin content of the diet is high.[110][111] Hence, the variation and complexity of chromosomal changes or genetic alterations may reflect the heterogeneity of risk factors; variation of histological grade, size, or clinical stage; geographic distribution; and long latency for hepatocarcinogenesis. On the other hand, most chromosomal abnormality appears to be similar between HBV- and HCV-associated HCCs, suggesting that there is a common pathway in both conditions. A recent study has demonstrated that the Rb1, p53, and Wnt pathways are commonly affected in HCCs induced by different etiologies, probably reflecting common pathogenetic mechanisms, in other words, chronic liver injury and cirrhosis.[10]

The frequently deleted chromosome regions by LOH in HCCs contain many TSGs and some oncogenes, including p53, Rb, p16, PTEN, DLC1, and IGF-2R.[10][97][112][113][114][115] Recent studies have suggested that some TSGs in HCCs can be silenced through epigenetic disruption, such as promoter CpG island methylation, during hepatocarcinogenesis. Frequent methylation of some TSGs, such as SOCS-1, GSTP, APC, E-cadherin, and p15, is well-documented. Methylation of these genes is more frequent in HCC than in nontumorous liver or cirrhotic liver. It appears that promoter hypermethylation of TSGs is a common event in the development of HCC and may play an important role in hepatocarcinogenesis.[116][117][118][119]

The p53 gene is the most common gene involved in human carcinogenesis and is activated in response to DNA damage, inducing either cell cycle arrest to permit DNA repair or apoptosis. Loss of p53 function occurs mainly through allelic deletions at chromosome 17p13 and missense mutations in the specific DNA-binding domain.[14][120][121] In HCC, LOH at chromosome 17p13 has been observed in 25 to 60% of tumors.[122] Analysis of nodule-in-nodule type HCC shows that p53 mutation is associated with the progression of HCC from an early to a more advanced stage.[122][123][124] The interval between surgical resection and the recurrence of HCC is significantly longer in patients with the wild-type p53 gene than it is in those with mutations, strongly suggesting a pathological role for the mutant p53 gene in HCC recurrence.[125][126] HBx protein can bind to the C terminus of p53, inhibit its sequence-specific DNA binding and transcriptional activation, and suppress p53-induced apoptosis.[127] HBx protein affects a wide range of p53 functions and thereby contributes to the molecular pathogenesis of HCCs. More recently, p53 has been identified as a downstream target of ZBP-89, a zinc finger transcription factor, which promotes growth arrest through stabilization of the p53 protein. ZBP-89 is colocalized with p53 in the nucleus in about two thirds of all cases positive for the nuclear p53 protein, suggesting that ZBP-89 may play a role in the nuclear accumulation of the p53 protein in a subset of recurrent HCC. With accumulation of p53 protein in the nucleus, tumor cells undergo apoptosis and thus are more susceptible to radiotherapy and chemotherapy. Therefore, colocalization of p53 protein with ZBP-89 may define a subgroup of recurrent HCC that is more sensitive to treatment.[128]

Allelic deletions at chromosome 13q in human cancer have been associated with the inactivation of two TSGs, Rb and BRCA2, located at 13q14 and 13q12 to 13.[82][100][108] In HCC, LOH at the Rb locus has been found in 25 to 48% of cases, and in up to 50% of cases Rb1 is strongly downregulated.[113] Rb gene plays an important role in cell cycle control and can be inactivated by different ways, including mutations in the gene itself, loss of TGF-β responsiveness, and inactivation of p16, p15, or CDK4.[129][130] Among the known TSGs, the inactivation of p16 is reported to be second only to p53 inactivation in human neoplasia. Inactivation of p16 is one of different ways that Rb gene can be inactivated. The p16 protein loss may contribute to both early- and late-stage hepatocarcinogenesis because it is observed in early-stage HCC and occurred approximately twice as often in advanced HCCs as in early stage HCCs.[131] Loss of p16 protein is mainly due to inactivation of p16 by promoter hypermethylation, homozygous deletions, and point mutations, and, in some cases, posttranscriptional inactivation.[131][132][133]

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ABBREVIATIONS
AFB1 aflatoxin B1

CGH comparative genomic hybridization

DNMT DNA methyltransferases

HBV hepatitis B virus

HBX hepatitis B virus X protein

HCC hepatocellular carcinoma

HCV hepatitis C virus

HGD high-grade dysplasia

IGF insulin-like growth factor

LGD low-grade dysplasia

LOH loss of heterozygosity

MRN macroregenerative nodule

TBP TATA Box-bindings protein

TFIIB transcription factor IIB

TGF transforming growth factor

TNF tumor necrosis factor

TSG tumor suppressor genes

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