Cytogenetic and molecular genetic alterations in hepatocellular carcinoma
Invited Review

Cytogenetic and molecular genetic alterations in hepatocellular carcinoma

Sze-hang Lau, Xin-yuan Guan1

Department of Clinical Oncology, Faculty of Medicine, The University of Hong Kong, Hong Kong, China

1Correspondence to Dr Xin-yuan GUAN.

Fax 852-2816-9126.
E-mail xyguan@hkucc.hku.hk

Abstract: Specific chromosome aberrations are frequently detected during the development of hepatocellular carcinoma. Molecular cytogenetic approaches such as comparative genomic hybridization and loss of heterozygosity analyses have provided fruitful information on changes in HCC cases at the genomic level. Mapping of chromosome gains and losses have frequently resulted in the identification of oncogenes and tumor suppressors, respectively. In this review, we summarize some frequently detected chromosomal aberrations reported for hepatocellular carcinoma cases using comparative genomic hybridization and loss of heterozygosity studies. Focus will be on gains of 1q, 8q, and 20q, and losses of 4q, 8p, 13q, 16q, and 17p. We then examine the candidate oncogenes and tumor suppressors located within these regions, and explore their possible functions in hepatocarcinogenesis. Finally, the impact of microarray-based screening platforms will be discussed.

Keywords: hepatocellular carcinoma; chromosome aberrations; oncogenes; tumor suppressor genes

Submitted Feb 17, 2005. Accepted for publication Mar 25, 2005.

doi: 10.1111/j.1745-7254.2005.00126.x

Introduction

Hepatocellular carcinoma (HCC) is one of the most common human malignant neoplasms, with a particularly high incidence in Chinese and African populations[1]. In 2000, HCC ranked as the fifth most frequent cancer, but the third leading cause of cancer death worldwide[2]. Unlike other cancers, the main causative agents for HCC, hepatitis B virus (HBV), hepatitis C virus (HCV) and aflatoxin (AFB), are well studied. Yet little is known about the molecular pathogenesis of HCC[3]. In fact, the majority of HCC are associated with a background of chronic liver disease. Therefore, hepatocarcinogenesis is believed to be a long-term process that involves multiple genetic alterations.

Recurrent chromosome alterations

Chromosome aberrations are a hallmark of solid tumors and it has been known for decades that chromosome rearrangements exist in most if not all human tumors[4]. Addi-tionally, cytogenetic study followed by molecular analysis of recurring chromosome changes has greatly facilitated the identification of crucial oncogenes and tumor suppressors[5]. For example, the tumor suppresor gene Rb was identified based on the observation of chromosome deletion del(13)(q14) in retinoblastoma[6] and the proto-oncogene myc was shown to be involved in the chromosome translocation t(8;14) in human Burkett’s lymphoma[7]. The conventional comparative genomic hybridization (CGH) method provides a powerful means of global analysis of segmental chromosome gains or losses[8]. Since it was developed, CGH has been widely applied to the detection of recurrent chromosomal alterations in various cancers, including HCC[9–13]. Frequent non-random chromosomal gains and losses detected by CGH are summarized in Table 1, including gains of 1q, 6p, 8q, 17q, and 20q, and losses of 1p, 4q, 5q, 6q, 8p, 9p, 10q, 13q, 16q, 17p, 19p, and 22q[9–13]. In addition, the loss of heterozygosity (LOH) assay is used to define chromosomal regions with allelic deletions, and results revealed that LOH was frequently detected in 1p, 4q, 6q, 8p, 13q, 16q, and 17p[14–16]. These studies suggest the presence of multiple oncogenes or tumor suppressor genes in regions of recurrent gain or loss, respectively.

Table 1
Table 1 Summary of chromosomal gains and losses revealed by comparative genomic hybridization analyses
Full table

To avoid overlap with comprehensive reviews that have been published recently[3,17], this review will mainly focus on chromosomal gains on 1q, 8q, and 20q, and losses on 4q, 8p, 13q, 16q, and 17p in HCC. For simplicity, gene symbols will be used for all genes described throughout the review (details for all genes are summarized in Table 2).

Table 2
Table 2 Summary of candidate cancer-related genes within abnormal chromosomal regions
Full table

Chromosome gains

Gain of 1q Gain of 1q is one of the most frequently detected alterations in HCC and has been suggested as an early genomic lesion in the process of HCC development[18], although the target oncogene within the amplified region has not been identified. CGH studies showed that the gain of 1q was detected in 58%–86% of HCC cases[9–13], with about 10% high-copy number amplification[12]. Several minimal amplifying regions (MAR) were mapped including 1q12-q22[12,19], 1q23.3-q25.3[20] and 1q24.2-1q43[21]. In an attempt to identify candidate oncogene(s) at 1q12-q22, Wong et al examined the expression level of several candidate onco-genes in this region using reverse transcription-polymerase chain reaction (RT-PCR). Significant overexpression of 4 genes known as JTB, SHC1, CCT3, and COPA were observed in tumors as compared with paired adjacent liver tissues[22].

Gain of 8q Gain of 8q is frequently observed in HCC (48%–77%)[9–13]. The gain of 8q is also frequently detected in many other solid tumors including those of the prostate[23], lung[24], esophagus[25], nasopharynx[26], ovary[27] and breast[28]. Amplification and overexpression of the c-myc oncogene has been reported in various solid tumors, including HCC. In our recent study, amplification of c-myc was correlated with HCC tumor size, tumor metastasis and recurrence. Amplification of c-myc was found in 11/13 (85%), 9/25 (36%) and 4/12 (33%) cases of large HCC (>9 cm), medium HCC (4–9 cm) and small HCC (<4 cm), respectively. In addition, a higher frequency of c-myc amplification was detected in metastatic tumors (45%) than in their primary HCC (29%), and in recurrent tumors (60%) than in their primary HCC (38%)[18]. Because the gain of 8q involved the entire long arm in this study, it is highly likely that one or more novel oncogenes are involved in HCC development apart from c-myc. In a recent report, Okamoto et al suggested 2 candidate oncogenes as amplification targets at 8q23-q24 known as PTK2 and EIF3S3. PTK2 encodes focal adhesion kinase, while EIF3S3 encodes the p40 subunit of eukaryotic translation initiation factor 3. Overexpression of these genes may be involved in HCC progression[29].

Gain of 20q Gain of 20q was detected in 3/5 CGH studies with a frequency of 20%–37% [9,10,12]. Gain of 20q was also frequently detected in breast cancer, and a steroid receptor coactivator, AIB1, has been identified within the commonly amplified region at 20q12[30,31]. Interestingly, gain of 20q is significantly associated with clinical stages and tumor size of HCC. Guan et al reported that gain of 20q occurred in 10/40 (25%) of stage II and III HCC but in 0/10 stage I HCC[12]. In addition, Wong et al reported that significantly more 20q gain was found in non-cirrhotic HCC. Strikingly, gain of 20q was observed in 9/12 (75%) non-cirrhotic, while only in 16/55 (29%) cirrhotic HCC (P=0.003)[10]. In our recent study, amplification of the AIB1 gene was correlated with HCC metastasis and recurrence. The amplification frequency of AIB1 was significantly higher in metastatic HCC (41%) than in matched primary HCC (23%), as well as being higher in recurrent HCC (60%) than in matched primary HCC (29%) (P < 0.05)[18].

Chromosome losses

Loss of 4q Loss of chromosome 4q was detected by CGH in 32%–70% of HCC cases[9–13]. Deletion of 4q has been well described because of its specificity for HCC. Several minimal deletion regions (MDR) on 4q have been detected by LOH including 4q12-q23, 4q13.1-q21.23, 4q13-q34, 4q21-q25, 4q24-q28, 4q28.2-q34.3, and 4q35[32–34]. In addition, Yeh et al reported recently the mapping of a 17.5 cM MDR between D4S1534 and D5S1572 by screening 149 HCC: a few candidate genes were found to be downregulated[35]. Biological evidence for tumor suppressor genes on 4q comes from a study that reversed the tumorigenic phenotype of a teratoma cell line by introducing a normal chromosome 4 into malignant teratoma cells using microcell-mediated chromosome transfer[36]. To date, candidate tumor suppressor genes correlated with 4q loss have remained unidentified. A recent report by Deng et al described the role of PRDM5 at 4q26 as a suppressor in various tumors including HCC. PRDM5 encodes a protein belonging to the PR-domain protein family. Epigenetic silencing was evident for this gene and its overexpression resulted in proapoptotic and growth suppressive functions[37].

Loss of 8p Loss of 8p is well documented in HCC with a frequency of 29%–77%[9–13]. By analyzing 50 HCC cases, we observed a correlation of 8p loss with advanced stage of disease and tumor size; most strikingly, 8p loss was present in 8/13 (62%) of large, 7/25 (28%) of medium, but only 1/12 (8%) of small HCC cases (P=0.014)[12]. Furthermore, 8p deletion was associated with HCC metastasis, suggesting the presence of one or more candidate gene(s) are involved in HCC progression[19]. Using the high-resolution LOH strategy, Jou et al reported that 50% of HCC showed LOH in chromosome 8p and the percentages of 3 MDR at 8p23.3-p23.1 (D8S504–D8S277), 8p22-p21.3 (D8S1106–D8S258) and 8p21.3-p12 (D8S258–D8S283) were 71%, 69%, and 63%, respectively[34]. Several candidate tumor suppressors have been isolated within these regions, including PINX1 at 8p23[38], FGL1 at 8p22[39] and DLC1[40] at 8p22-p21.3 regions. In the 8p23 region, Liao et al observed the under-expression of PINX1 (LPTS) in HCC cells and tissues, while ectopic expression of this gene in liver cancer cells SMMC-7721 resulted in growth suppression[38]. At 8p22, Yan et al reported reduced or undetectable expression of FGL1 in HCC speci-mens, and found a significant correlation with tumor differentiation[39]. Interestingly, Wong et al reported the significant under-expression of DLC1 mRNA and promoter hyper-methylation was found in 24% of primary HCC cases, suggesting a role of epigenetic silencing in DLC1 downregula-tion[41]. DLC-1 belongs to the rho family GTPase-activating proteins (RhoGAP) specific to RhoA and cdc42, which are implicated in cell migration control. Re-expression of DLC1 in HCC cells resulted in caspase-3-dependent apoptosis, inhibition of cell growth and invasiveness in vitro, and reduction of tumor formation in nude mice[42]. Furthermore, candidate tumor suppressors known as PDGFRL[43] and TUSC3[44] were identified in the 8p22-p21.3 region.

Loss of 13q Loss of 13q was detected in 16%–37% of HCC cases[9–13]. LOH analyses also showed a high percentage of allelic deletion on 13q (40%–55%)[14–16]. Jou et al identified 5 MDR with high frequencies of LOH including 13q12.11-q12.3 (71%), 13q13.1-q22.1 (79%), 13q31.3-q32.2 (67%), 13q32.2-q33.3 (73%) and 13q33.3-q34 (75%)[34]. MDR in 13q13.1-q22.1 (D13S171–D13S156) showed the highest frequency of LOH, and well-documented tumor suppressors including BRCA2 and RB are located in this region. In 13q33.3-q34 MDR, one candidate tumor suppressor known as P33ING1 has been isolated[45]. Overexpression of p33ING1 inhibited cell cycle progression and the repression function of p33ING1 was enhanced by the Ras/Raf pathway[46]. Recently, another candidate tumor suppressor known as deleted in liver cancer 2 (DLC2) was identified on 13q12.3. It shows homology to tumor suppressor gene DLC1 located at 8p22-p21.3. DLC2 encodes a protein with RhoGAP, SAM and START domains and is significantly underexpressed in only 8 of 45 (18%) HCC, as compared with its ubiquitous expression in normal tissues[47].

Loss of 16q One of the most frequent losses in HCC cases is located in the long arm of chromosome 16. Loss of 16q was detected in 30%–70% of HCC cases by CGH studies[9–13]. LOH also revealed allelic deletions in 55%–59% of HCC[14–16]. Two MDR at 16q in HCC have been reported to include 16q12.1-q23.1 (71%) and 16q23.1-q24.1 (71%)[33]. Several candidate tumor suppressors have been reported in recent publications. The loss of 16q has also been reported as a frequent genomic alteration in ovarian[27] and prostate cancer[48]. The loss of 16q24.1-q24.2 in prostate cancer has been associated with aggressive behavior of the disease, recurrent growth, poor differentiation of the tumor, and a poor prognosis for the patient[48]. The best-studied tumor suppressor gene is E-cadherin (CDH1) at 16q22.1. Inter-estingly, expression of the E-cadherin gene is reduced by a hypermethylation mechanism although no somatic mutations of the E-cadherin gene in HCC have yet been described[49,50]. Bando et al identified a distinct commonly deleted region at 16q24.1-24.2 (D16S534 and D16S3091) near novel sequence HSRG1[51]. Another candidate tumor suppressor known as WWOX was identified at 16q23.3-q24.1: absence or reduced expression of WWOX was observed in 60% of HCC cell lines compared with normal liver[52].

Loss of 17p Loss of 17p is one of the most frequent chromosomal alterations in HCC as determined by both CGH[2,11–13] and LOH[14–16]. Deletion of 17p is common in various cancers including breast[53] and colon cancer[54]. The frequent deletion of 17p may affect the tumor suppressor gene TP53 on 17p13.1. TP53 is frequently inactivated in various types of malignant tumors, including HCC[55]. However, Yumoto et al showed that loss of 17p occurred in 18/31 HCC cases (58%) by LOH, whereas TP53 mutation was only observed in 8/31 HCC cases (26%)[56]. Our recent study revealed that loss of one allele at 17p13.3 distal to the TP53 gene was observed in 48/94 HCC (51%), whereas LOH at 17p13.1, near the TP53 gene, was detected in 30/94 HCC (32%) and p53 mutation was only detected in 22/94 HCC (23%)[57]. These results suggest that another tumor suppressor gene at 17p13.3 may be involved in the pathogenesis of HCC. One candidate suppressor, C17orf25, has been isolated from this region[58]. Downregulated expression of C17orf25 was found in HCC samples matched to adjacent non-tumor liver tissues. Furthermore, transfection of C17orf25 into the hepatocellular carcinoma cell SMMC-7721 inhibited cell growth[58].

Conclusion

Molecular cytogenetic approaches such as CGH and LOH provide important clues to the identification of novel genes involved in the multistage development of HCC. In this review, we discussed some of the most common chromosome aberrations, including gains of 1q, 8q, and 20q, and loss of 4q, 8p, 13q, 16q, and 17p, followed by candidate oncogenes and tumor suppressors that may be involved within these regions. Due to the heterogeneity of HCC, it has been suggested that gains or losses of specific chromosomal loci rarely affect more than half of them, and specific HCC-related genes have not been identified in most of the above-mentioned chromosomal regions[3]. Results obtained with traditional approaches such as cytogenetic CGH and LOH studies are relatively low in resolution and are labor intensive. Advances in microarray-based approaches are beginning to reverse this situation by providing an integrated view of genetic alterations during tumorigenesis and progression[59].

Different microarray-based CGH (aCGH) platforms are emerging that allow high-resolution genomic profiling[60–62]. For instance, Hashimoto et al reported the use of cytogenetic CGH and aCGH in the identification of genes involved in HCV-related HCC[63]. In a recent article, Ishkanian et al reported the construction of submegabase resolution tiling genomic arrays (SMRT arrays) containing 32 433 overlapping BAC clones. This allows the detection of cryptic chromosomal changes down to microgain and microloss resolution[64]. In addition, some groups have used oligonucleotide arrays for high-resolution CGH and LOH studies. Single nucleotide polymorphism (SNP) arrays have been employed for high-resolution LOH studies in small cell lung cancer samples[65] and have permitted the simultaneous detection of copy number changes and LOH in a single experiment[66].

In another approach, chromosomal bias of gene expression signature was studied in HCC cases[67,68]. Using a recently developed technique known as expression balance map analysis, Midorikawa et al identified regions with frequent chromosomal aberrations in HCC based solely on expression data. Common alterations, including gain of 1q21-q23 and loss of 4q13, were found in 74% and 48% of HCC cases, respectively[67]. These matched well with previous CGH analyses. In conclusion, a combination of these powerful technologies provides a more comprehensive view of the development of HCC, and identification of novel gene candidates may provide better diagnosis and prognostication of HCC.

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Cite this article as: Lau Sh, Guan Xy. Cytogenetic and molecular genetic alterations in hepatocellular carcinoma. Acta Pharmacologica Sinica 2005;26(6):659-665. doi: 10.1111/j.1745-7254.2005.00126.x