Aberrant Promoter Methylation Profiles of Tumor Suppressor Genes in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most fatal human malignancies, but the molecular mechanisms of hepatocarcinogenesis remain unclear. Although p53 mutations are frequently observed in Asian HCC, it is not a common event in Western HCC. Recent studies suggest that tumor suppressor genes (TSGs) can also be silenced through epigenetic disruption, such as promoter CpG island methylation, during carcinogenesis. To further understand the molecular mechanism of hepatocarcinogenesis, we have investigated the promoter methylation status of nine TSGs (SOCS-1, GSTP, APC, E-cadherin, RAR-β, p14, p15, p16, and p73) in 51 cases of HCC using methylation-specific polymerase chain reaction. We found that 82% of HCCs had methylation of at least one TSG promoter. The most frequently methylated TSGs in HCC were: SOCS-1 (65%), GSTP (54%), APC (53%), E-cadherin (49%), and p15 (49%). Methylation of SOCS-1, GSTP, APC, E-cadherin, and p15 was more frequent in HCC than in nontumor liver (P < 0.05). Methylation of SOCS-1, GSTP, and p15 was also significantly more frequent in HCC than cirrhotic liver (P < 0.05). Although methylation of one or two genes could be seen in both nontumor and cirrhotic livers, 53% of the HCC cases had three or more TSG promoters methylated, in comparison to 0% in nontumor liver and 13% in cirrhosis (P = 0.001). Methylation of SOCS-1, APC, and p15 was more frequently seen in hepatitis C virus-positive HCC than hepatitis C virus/hepatitis B virus-negative HCC. Our data suggest that promoter hypermethylation of TSGs is a common event in HCC and may play an important role in hepatocarcinogenesis. Hepatocellular carcinoma (HCC) is one of the most fatal human malignancies, but the molecular mechanisms of hepatocarcinogenesis remain unclear. Although p53 mutations are frequently observed in Asian HCC, it is not a common event in Western HCC. Recent studies suggest that tumor suppressor genes (TSGs) can also be silenced through epigenetic disruption, such as promoter CpG island methylation, during carcinogenesis. To further understand the molecular mechanism of hepatocarcinogenesis, we have investigated the promoter methylation status of nine TSGs (SOCS-1, GSTP, APC, E-cadherin, RAR-β, p14, p15, p16, and p73) in 51 cases of HCC using methylation-specific polymerase chain reaction. We found that 82% of HCCs had methylation of at least one TSG promoter. The most frequently methylated TSGs in HCC were: SOCS-1 (65%), GSTP (54%), APC (53%), E-cadherin (49%), and p15 (49%). Methylation of SOCS-1, GSTP, APC, E-cadherin, and p15 was more frequent in HCC than in nontumor liver (P < 0.05). Methylation of SOCS-1, GSTP, and p15 was also significantly more frequent in HCC than cirrhotic liver (P < 0.05). Although methylation of one or two genes could be seen in both nontumor and cirrhotic livers, 53% of the HCC cases had three or more TSG promoters methylated, in comparison to 0% in nontumor liver and 13% in cirrhosis (P = 0.001). Methylation of SOCS-1, APC, and p15 was more frequently seen in hepatitis C virus-positive HCC than hepatitis C virus/hepatitis B virus-negative HCC. Our data suggest that promoter hypermethylation of TSGs is a common event in HCC and may play an important role in hepatocarcinogenesis. Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world and among the most fatal of human neoplasms, but the molecular mechanisms of hepatocarcinogenesis are primarily unknown. Recent genetic studies have indicated that both the p53 and pRb pathways may be involved in hepatocarcinogenesis. It has been shown that point mutations of the p53 tumor suppressor gene (TSG) were frequently seen in HCCs in Chinese and African populations.1Bressac B Kew M Wands J Ozturk M Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa.Nature. 1991; 350: 429-431Crossref PubMed Scopus (1293) Google Scholar, 2Hollstein MC Wild CP Bleicher F Chutimataewin S Harris CC Srivatanakul P Montesano R p53 mutations and aflatoxin B1 exposure in hepatocellular carcinoma patients from Thailand.Int J Cancer. 1993; 53: 51-55Crossref PubMed Scopus (105) Google Scholar, 3Hsu IC Metcalf RA Sun T Welsh JA Wang NJ Harris CC Mutational hotspot in the p53 gene in human hepatocellular carcinomas.Nature. 1991; 350: 427-428Crossref PubMed Scopus (1474) Google Scholar However, p53 mutation is not a frequent event in American and European HCCs.4Boix-Ferrero J Pellin A Blesa R Adrados M Llombart-Bosch A Absence of p53 gene mutations in hepatocarcinomas from a Mediterranean area of Spain. A study of 129 archival tumour samples.Virchows Arch. 1999; 434: 497-501Crossref PubMed Scopus (30) Google Scholar, 5Challen C Lunec J Warren W Collier J Bassendine MF Analysis of the p53 tumor-suppressor gene in hepatocellular carcinomas from Britain.Hepatology. 1992; 16: 1362-1366Crossref PubMed Scopus (103) Google Scholar, 6Kazachkov Y Khaoustov V Yoffe B Solomon H Klintmalm GB Tabor E p53 abnormalities in hepatocellular carcinoma from United States patients: analysis of all 11 exons.Carcinogenesis. 1996; 17: 2207-2212Crossref PubMed Scopus (27) Google Scholar This discrepancy may result from different ethic backgrounds and/or different etiologies, such as hepatitis B virus (HBV) and hepatitis C virus (HCV) infections or toxin exposure. Both HBV and HCV infections are thought to be involved in hepatocarcinogenesis because chronic hepatitis and cirrhosis associated with either HBV or HCV infection precede most HCCs. It has been shown that HBV encodes a X-gene product, HBx, that not only can activate the JAK/STAT signaling pathway,7Lee YH Yun Y HBx protein of hepatitis B virus activates Jak1-STAT signaling.J Biol Chem. 1998; 273: 25510-25515Crossref PubMed Scopus (214) Google Scholar but can also interact with p53 and impair the function of wild-type p53.8Kew MC Increasing evidence that hepatitis B virus X gene protein and p53 protein may interact in the pathogenesis of hepatocellular carcinoma.Hepatology. 1997; 25: 1037-1038Crossref PubMed Scopus (13) Google Scholar The core protein of HCV has also been shown to modulate gene transcription, cell proliferation, and cell death.9Forrester K Lupold SE Ott VL Chay CH Band V Wang XW Harris CC Effects of p53 mutants on wild-type p53-mediated transactivation are cell type dependent.Oncogene. 1995; 10: 2103-2111PubMed Google Scholar However, the exact mechanisms underlying virus-associated hepatocarcinogenesis are still unclear. It has recently been proposed that aberrant methylation of CpG islands, which are CpG dinucleotide rich areas located mainly in the promoter regions of many genes, serves as an alternative mechanism for inactivation of TSGs in cancer.10Herman JG Baylin SB Promoter-region hypermethylation and gene silencing in human cancer.Curr Top Microbiol Immunol. 2000; 249: 35-54Crossref PubMed Scopus (155) Google Scholar, 11Herman JG Jen J Merlo A Baylin SB Hypermethylation-associated inactivation indicates a tumor suppressor role for p15INK4B.Cancer Res. 1996; 56: 722-727PubMed Google Scholar, 12Baylin SB Belinsky SA Herman JG Aberrant methylation of gene promoters in cancer—concepts, misconcepts, and promise.J Natl Cancer Inst. 2000; 92: 1460-1461Crossref PubMed Scopus (130) Google Scholar, 13Baylin SB Herman JG DNA hypermethylation in tumorigenesis: epigenetics joins genetics.Trends Genet. 2000; 16: 168-174Abstract Full Text Full Text PDF PubMed Scopus (1428) Google Scholar, 14Baylin SB Herman JG Graff JR Vertino PM Issa JP Alterations in DNA methylation: a fundamental aspect of neoplasia.Adv Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar It has been demonstrated that many TSGs can be functionally silenced through such aberrant promoter methylation. Recent studies have revealed that aberrant methylation of TSG promoters is frequently found in several common malignancies, such as colon, lung, breast, and prostate cancers.15Esteller M Corn PG Baylin SB Herman JG A gene hypermethylation profile of human cancer.Cancer Res. 2001; 61: 3225-3229PubMed Google Scholar, 16Herman JG p16(INK4): involvement early and often in gastrointestinal malignancies.Gastroenterology. 1999; 116: 483-485Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 17Herman JG Civin CI Issa JP Collector MI Sharkis SJ Baylin SB Distinct patterns of inactivation of p15INK4B and p16INK4A characterize the major types of hematological malignancies.Cancer Res. 1997; 57: 837-841PubMed Google Scholar Frequent promoter methylation of p16, p15, and GSTP genes has also been observed in the majority of HCCs in Chinese and Japanese populations.18Wong IH Lo YM Yeo W Lau WY Johnson PJ Frequent p15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients.Clin Cancer Res. 2000; 6: 3516-3521PubMed Google Scholar, 19Yoshikawa H Matsubara K Qian GS Jackson P Groopman JD Manning JE Harris CC Herman JG SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity.Nat Genet. 2001; 28: 29-35PubMed Google Scholar, 20Tchou JC Lin X Freije D Isaacs WB Brooks JD Rashid A De Marzo AM Kanai Y Hirohashi S Nelson WG GSTP1 CpG island DNA hypermethylation in hepatocellular carcinomas.Int J Oncol. 2000; 16: 663-676PubMed Google Scholar However, the frequency of TSG methylation in Western HCC has not been studied as extensively. To elucidate the molecular mechanisms of hepatocarcinogenesis in this population, we have studied methylation of nine TSGs involved in multiple cellular signaling transduction pathways in 51 cases of HCC using a multiplex methylation-specific polymerase chain reaction (MSP) method, and have compared methylation of these genes in HCC to nonmalignant liver and cirrhotic liver. Our study indicates that TSG promoter methylation is a frequent event in Western HCC. Fifty-one cases of HCC, including 25 cases of radical or partial hepatectomy and 26 cases of fine needle aspiration biopsy (FNA), were collected from The Johns Hopkins Hospital pathology archives. Fifteen cases of cirrhotic liver tissues remote from HCC lesions and 14 cases of nontumor liver tissues adjacent to either a hepatic adenoma or a focal nodular hyperplasia were also obtained from surgical resection specimens. The HBV and HCV infection status was tested serologically by enzyme immunoassay before this study and was documented through the pathology laboratory database. This study was reviewed by the Johns Hopkins Institutional Review Board and found to be exempt. No chemotherapy or radiation therapy was instituted before tumor excision or FNA procedure. The case distribution in different clinicopathological categories is summarized in Table 1. The tissue was fixed in buffered formalin for surgical specimens or in ethanol-based fixative for FNA specimens. Consecutive 4-μm sections were cut from paraffin-embedded tissue blocks and mounted for histopathological evaluation using conventional hematoxylin and eosin (H PubMed Scopus Google Scholar of DNA was by with of for at of of and of were and the was at for DNA was by of the DNA with of with and in DNA was at DNA promoter regions of SOCS-1, APC, E-cadherin, GSTP, p15, p16, RAR-β, p14, and genes were in a with using as M M C Herman J of promoter methylation in of the PubMed Scopus Google Scholar DNA methylation of CpG was by using for both methylated and M M C Herman J of promoter methylation in of the PubMed Scopus Google Scholar of were to of one a in which CpG are to and the a in which CpG are methylated by DNA were for of were on of DNA methylation of all SOCS-1, was in all 51 cases of HCC. of the of the of the SOCS-1 the of SOCS-1 was in surgical resection specimens of HCC, but in specimens. only data of promoter methylation of SOCS-1 in 26 HCC cases of specimens was in the or exact on the in the were to the of TSG methylation in HCC, and nontumor liver. or exact were also to the TSG methylation and clinicopathological such as status and A analysis was to the TSG methylation and the of tumor The frequency of promoter methylation of nine genes in 51 cases of HCC, cirrhotic livers, and 14 nontumor liver tissues was using and is shown in Table cases of HCC had methylation of at least one The most frequently methylated TSG promoters in HCC were: SOCS-1 (65%), GSTP (54%), APC (53%), E-cadherin (49%), and p15 of the genes, APC, has been to be methylated frequently in IH Lo YM Yeo W Lau WY Johnson PJ Frequent p15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients.Clin Cancer Res. 2000; 6: 3516-3521PubMed Google Scholar, 19Yoshikawa H Matsubara K Qian GS Jackson P Groopman JD Manning JE Harris CC Herman JG SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity.Nat Genet. 2001; 28: 29-35PubMed Google Scholar, 20Tchou JC Lin X Freije D Isaacs WB Brooks JD Rashid A De Marzo AM Kanai Y Hirohashi S Nelson WG GSTP1 CpG island DNA hypermethylation in hepatocellular carcinomas.Int J Oncol. 2000; 16: 663-676PubMed Google Scholar, T R K Frequent of E-cadherin by genetic and epigenetic in the of hepatocellular Cancer Res. 2001; Google Scholar The genes studied demonstrated frequent methylation: and The frequency of methylation of E-cadherin, GSTP, APC, p15, SOCS-1, p16, and promoters was in both cirrhotic and nontumor was methylation of p14, p15, or in either nontumor or cirrhotic Methylation of SOCS-1, GSTP, APC, E-cadherin, and p15 was significantly more frequent in HCC than in nontumor (P = P = P = P = and P = Methylation of SOCS-1, GSTP, and p15 was also more frequently seen in HCC than cirrhosis (P = P = and P = Methylation was also more frequent in HCC than cirrhosis for APC and E-cadherin but these were not (P = and P = was in the methylation frequency of tested TSGs nontumor liver and cirrhosis (P of TSG Methylation in HCC and = = = of cases for SOCS-1 analysis is of cases for SOCS-1 analysis is in a Methylation status of TSGs APC, p15, p14, p16, and was in the analysis of methylation Methylation of SOCS-1 was not because of the in surgical specimens and Methylation of multiple TSGs was seen in HCC of the HCC cases had three or more TSG promoters methylated in comparison to 0% in nontumor and 13% in cirrhotic (P = 0.001). Methylation of or more TSG promoters was only observed in HCC cases and Table Methylation of two or three TSGs was not associated with either HCC or TSG methylation was more frequently seen in nontumor liver than in HCC (P = of HCCs methylation of TSGs in comparison to in cirrhosis and in nontumor Methylation of TSG in HCC and = = = in a on the methylation shown in 51 cases of HCC can be into three methylation and methylation cases of HCC methylation of nine TSGs the methylation cases (54%), which methylation of one to three TSG the methylation 14 cases which had methylation of more than TSG The most frequently methylated TSGs in the methylation were APC, E-cadherin, SOCS-1, p15, and cases of HCC TSG methylation of either APC or E-cadherin or of the methylated HCC cases a methylation APC and of HCC methylation to clinicopathological data revealed several TSG methylation was more common among or HCC than HCC Methylation of SOCS-1, APC, and p15 was associated with HCC to HCC (P = P = and P = Although not to be a methylation of p16, and in HCC was and HCC in the frequency of methylation of tested TSGs (P 0.05). was the frequency of TSG promoter methylation and or the histopathological of the tumor (P 0.05). HCC, the major type of liver is one of the most common and a of in many Although many of the major and for HCC have been the genetic and epigenetic pathways to of liver have The distribution of HCC further to the common genetic and epigenetic for hepatocarcinogenesis. study indicated that of two DNA and was significantly in HCC compared to nontumor Y Kanai Y M H H Hirohashi S of for DNA and and DNA methylation status on CpG and regions during human 2001; PubMed Scopus Google Scholar that epigenetic mechanisms may be It be important to the genes silenced through promoter CpG island hypermethylation during hepatocarcinogenesis. We have studied methylation of nine TSGs involved in several transduction pathways in 51 cases of Western HCC. We found that methylation of TSG promoters was a frequent event in HCC, as 82% cases of HCC had at least one TSG promoter nine TSGs the most frequently methylated were SOCS-1, GSTP, APC, E-cadherin, and comparison to nontumor liver methylation of these TSGs was associated with HCC. Methylation of SOCS-1, GSTP, and p15 was also significantly more frequent in HCC than in cirrhotic liver. studies have also shown frequent methylation of SOCS-1, p15, and E-cadherin genes in Chinese and Japanese IH Lo YM Yeo W Lau WY Johnson PJ Frequent p15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients.Clin Cancer Res. 2000; 6: 3516-3521PubMed Google Scholar, 19Yoshikawa H Matsubara K Qian GS Jackson P Groopman JD Manning JE Harris CC Herman JG SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity.Nat Genet. 2001; 28: 29-35PubMed Google Scholar, Y T J K H is by CpG methylation in human hepatocellular 1999; 116: Full Text Full Text PDF PubMed Scopus Google Scholar It that silencing of TSGs through promoter CpG island methylation may play an important role during hepatocarcinogenesis. It be that comparison tissues were not but cirrhotic liver from patients with HCC and nontumor liver from patients with liver such as studies have found methylation in liver from patients H Matsubara K Qian GS Jackson P Groopman JD Manning JE Harris CC Herman JG SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity.Nat Genet. 2001; 28: 29-35PubMed Google Scholar However, to the of methylation in it is important to study hepatic tissue Our data suggest that gene methylation may be in HCC from nonmalignant liver. The most methylation in HCC is the methylation of multiple Although or TSG methylation can be seen in nontumor and cirrhotic liver the majority of HCC cases three or more methylated methylation of or more TSG promoters was only seen in HCC. multiple TSG methylation patterns were also observed in HCC by using a of genes involved in M P B A S M A G Methylation of cell gene in cirrhosis and associated hepatocellular carcinoma.Hepatology. PubMed Scopus Google Scholar the of multiple TSGs than a this that of multiple transduction pathways is during hepatocarcinogenesis. studies have that the transduction may be important in hepatocarcinogenesis. this pathway, the with E-cadherin at the is by and the Nelson and APC and signaling 1997; PubMed Scopus Google Scholar, and in human Cancer. PubMed Scopus Google Scholar, M The of APC, E-cadherin and in tumour and 1997; PubMed Scopus Google Scholar, A S through and 1999; 56: PubMed Scopus Google Scholar, J of signaling in tumor 2000; PubMed Scopus Google Scholar many human neoplasms, this is in the and of an of on the which in the of a of genes involved in and cell This may result from mutations in APC, E-cadherin, or of HCCs of in many Y D of in hepatocellular carcinoma is associated with cell J 1999; Full Text Full Text PDF PubMed Google Scholar, T H Y M in hepatocellular a of in the 17: PubMed Scopus Google Scholar, mutation and in hepatocellular and 2001; 92: PubMed Scopus Google Scholar However, mutations in the APC, or genes to be in MF Absence of APC gene mutation in the mutation in hepatocellular 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A B P M J C A C mutations of the gene are frequent in and human hepatocellular Natl 1998; PubMed Scopus Google Scholar, H H A M G H mutations are frequent in human hepatocellular carcinomas associated with hepatitis C virus J 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, P J C C J D G P J mutations in hepatocellular carcinoma with a of of 1999; PubMed Scopus Google Scholar, YM mutations are associated with a of hepatocellular carcinoma negative for hepatitis B virus and with J 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J X Y and of the gene may play an important role in hepatocellular carcinoma among Chinese Cancer Oncol. 2001; PubMed Scopus Google Scholar, H K Y T T M S K Frequent in human hepatocellular Res. 2001; PubMed Scopus Google Scholar, H and hepatocellular Genet. 2000; PubMed Scopus Google Scholar that mechanisms may be We found methylation of APC, E-cadherin, or both in 82% of HCCs. of methylated HCC cases APC and E-cadherin methylation, that inactivation of either APC or E-cadherin may to the of in HCC. of the JAK/STAT has also been in hepatocarcinogenesis. of signaling (SOCS-1, also as and is a protein that the JAK/STAT by to study has indicated that the SOCS-1 gene was methylated in the majority of Chinese HCC H Matsubara K Qian GS Jackson P Groopman JD Manning JE Harris CC Herman JG SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity.Nat Genet. 2001; 28: 29-35PubMed Google Scholar The tumor suppressor function of SOCS-1 was further demonstrated by of cell and of SOCS-1 into HCC cell H Matsubara K Qian GS Jackson P Groopman JD Manning JE Harris CC Herman JG SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity.Nat Genet. 2001; 28: 29-35PubMed Google Scholar a multiplex we have the methylation status of SOCS-1 in 26 FNA specimens of Western HCC. with we have found that the SOCS-1 gene is frequently methylated in Western HCC. Methylation of SOCS-1 was more frequent in and HCC than HCC. It has been shown that of the JAK/STAT by of was observed both in HBx and in that SOCS-1 was YH Yun Y HBx protein of hepatitis B virus activates Jak1-STAT signaling.J Biol Chem. 1998; 273: 25510-25515Crossref PubMed Scopus (214) Google Scholar, 19Yoshikawa H Matsubara K Qian GS Jackson P Groopman JD Manning JE Harris CC Herman JG SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity.Nat Genet. 2001; 28: 29-35PubMed Google Scholar methylation of SOCS-1 of JAK/STAT signaling in HCC to be further Our study to the thought that an of by infection and silencing of SOCS-1 may play a role in hepatocarcinogenesis. study has indicated that the gene is frequently in HCC through or promoter M C H is a major inactivation in hepatocellular 2000; PubMed Scopus Google Scholar However, the frequency of promoter methylation in A frequency of methylation has been in Asian Y T J K H is by CpG methylation in human hepatocellular 1999; 116: Full Text Full Text PDF PubMed Scopus Google Scholar, Y S M Wang G F A in human liver J Cancer. PubMed Scopus Google Scholar associated with Recent studies from and a frequency of promoter methylation to M C H is a major inactivation in hepatocellular 2000; PubMed Scopus Google Scholar, CH Wang JC mutations and methylation of and in hepatocellular carcinoma in J Cancer. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar we found that only of Western HCC promoter methylation. The these studies on methylation may be because of it could also be because of the different for analysis. The p15 which encodes is methylated in several human neoplasms, JG Civin CI Issa JP Collector MI Sharkis SJ Baylin SB Distinct patterns of inactivation of p15INK4B and p16INK4A characterize the major types of hematological malignancies.Cancer Res. 1997; 57: 837-841PubMed Google Scholar It has been that p15 promoter methylation is in of Chinese HCC and of HCC and IH Lo YM Yeo W Lau WY Johnson PJ Frequent p15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients.Clin Cancer Res. 2000; 6: 3516-3521PubMed Google Scholar we found that of HCC p15 promoter methylation. methylation of p15 is not seen in we the of p15 methylation with infections in HCC. we found that p15 promoter methylation, with SOCS-1 and APC, is more frequently seen in and HCC than HCC. Our study that silencing of p15 through promoter methylation may be involved in hepatocarcinogenesis. However, the of p15 promoter methylation in cirrhosis in study the that of p15 may be an early event of M P B A S M A G Methylation of cell gene in cirrhosis and associated hepatocellular carcinoma.Hepatology. PubMed Scopus Google Scholar we have shown that silencing of TSGs through promoter methylation is a frequent event in HCC. abnormalities in HCC not only play important during hepatocarcinogenesis, but also be in the of HCC.