The genetic and epigenetic events of hepatocarcinogenesis are relatively poorly understood. However, it is likely there is geographical variation in the mechanisms involved. The aim of the current study was to investigate the role of chromosomal instability (CIN) and CpG island methylation, globally and in relation to specific candidate tumour suppressor genes (TSGs), in hepatocellular carcinomas (HCCs) from Australia and South Africa. The role of the putative TSG p14ARF was examined and the relationship between p14, p16 and p53 was assessed in Australian and South African HCCs. Additionally, the cell adhesion molecule E-cadherin and the cell signalling protein β-catenin were studied in the same series of HCCs. Genomic DNA was extracted from malignant and non-malignant liver tissue from 37 Australian and 24 South African cases. In addition, samples of histologically normal liver tissue from 20 transplant donor livers were also studied. CIN was examined at 8 chromosomal regions (1p, 4p, 4q, 8p, 9p, 13q, 16q and 17p), using 23 microsatellite markers. Methylation status of six genes implicated in malignant transformation (p14, p16, p15, RIZ1, E-cadherin and O6-MGMT) were examined using methylation specific PCR (MSP), a qualitative assay, while CpG island methylation of 5 methylated in tumour clones (MINTs 1, 2, 12, 25 and 31) was assessed using combined bisulfite restriction analysis (COBRA), a quantitative assay. The prevalence of CIN across all chromosomal regions was significantly higher in South African HCCs compared to Australian HCCs (53 Vs 33%, p<0.001, χ2). In contrast, the prevalence of promoter methylation as assessed by MSP was significantly higher in Australian compared with South African cases in both malignant and non-malignant liver tissue (p<0.01 and <0.001 respectively χ2)- Data from MINT assays revealed an increasing degree of CpG island methylation in the progression from histologically normal liver tissue, through non-malignant liver tissue to malignant liver. CpG island methylation was more frequent, and CIN less frequent in Australian HCCs, suggesting that there may be different pathways of hepatocarcinogenesis in Australia and South Africa. This study confirms that CpG island methylation is a common and early event in hepatocarcinogenesis, preceding malignant transformation.
Mutational inactivation of the p53 is one of the most common abnormalities in HCC, particularly in those from sub-Saharan Africa. However mutations of the p53 gene are relatively uncommon in Australian HCCs. Therefore, silencing of p14 may be an alternate mechanism of inactivating p53 as p14 binds to MDM2 resulting in a stabilisation of p53. Loss of heterozygosity (LOH), mutation, promoter methylation and protein expression analyses was examined for p14, p16 and p53. LOH at 9p21 was more prevalent in South African HCCs (p=0.04). Rates of p53 LOH were comparable between the two groups. Hypermethylation of the p14 and p16 promoters was prevalent in Australian HCCs No significant association between p14 and p53 was seen in this study suggesting that p14 inactivation does not substitute for p53 loss.
E cadherin and β-catenin forms the cadherin/catenin complex, required for strong cell adhesion. Inactivation of this complex facilitates invasion into surrounding tissues. Evidence of E-cadherin inactivation by CIN and promoter methylation were sought by LOH analysis at 16q22 and methylation-specific PCR respectively. β-catenin mutations were assessed by denaturing high performance liquid chromatography, while E-cadherin and β-catenin protein expression were studied by immunohistochemistry. LOH at 16q22 was more frequent in South African HCCs (50% Vs 11%, p<0.05, χ2). Methylation was more likely to be detected in patients over the age of 50 (p<0.001, χ2) and was prevalent in non-malignant liver. No Pcatenin mutations were identified. No abnormalities in protein expression were detected through β-catenin or E-cadherin immunohistochemistry. Contrary to previous observations in HCCs from other countries, neither E-cadherin nor β-catenin appears to play a role in hepatocarcinogenesis in Australian and South African HCCs. The higher prevalence of LOH, but lower prevalence of promoter methylation for E-cadherin in South African compared with Australian HCCs is consistent with previous results. The current study confirms and extends earlier observation that there are fundamental differences in the mechanisms of hepatocarcinogenesis in Australia and South Africa with CpG island methylation being more prevalent in Australian HCCs, while CIN plays a major role in South African HCCs.