Methylation Of Multiple Genes Involved In WNT/β-catenin Pathway In Hepatocellular Carcinoma

Hepatocelluar carcinoma （HCC） is one of the most common malignancies throughout theworld，ranking sixth in incidence and third in mortality among tumors of all sites. It is usuallyan aggressive malignancy with a5-year survival rate of as low as14%. The5-year survivalrate of patients with early-stage HCC is26%but only2%when it is found after metastasis todistant organs. Therefore, early detection is critical for effective treatment of HCC. Thecurrent circulating marker, alpha-fetoprotein （AFP）, and its fucosylated glycoform L3, are oflimited value, with a sensitivity of only40%to60%. Thus, there is an urgent need for a bettermarker or panel of markers for the early detection of HCC. However, well understanding ofthe molecular pathways of hepatocarcinogenesis is vital for identifying genetic markers anddeveloping targeted treatments.Genomic study on HCC focuses on DNA sequence changes, chromosomal aberrationsand epigenetic abnormalities. According to the current understanding, most HCC patientscontracted the disease from the accumulation of genetic abnormalities, probably induced byexterior etiological factors especially HBV and HCV infections. Chromosomal instability,including gain or loss of the genomic DNA copy number, is commonly seen in HCC.Chromosomal amplification regions often harbor oncogenes, whereas the chromosomaldeletion regions often include tumor suppressor genes, both conferring a growth advantagefor tumorigenesis in HCC. To identify these crucial regions in hepatocarcinogenesis, anumber of approaches have been employed to detect the genomic alterations in HCC samples.The widespread use of aCGH （microarray comparative genomic hybridization, arrayCGH）has prompted the development of computational analyses to identify the chromosomeaberrations with much higher resolution. Mutation of tumor suppressor genes also involved inhepatocarcinogenesis. A variety of studies in recent years provided evidence that the p53tumor suppressor gene plays a major role in hepatocarcinogenesis. A number of studiesclearly support the findings of a positive correlation between the249serp53gene mutation andthe AFB1exposure. In contrast, p53mutation may occur as a late event in carcinogenesis without a typical mutational pattern in areas with low AFB1intake. A series of studiessupport this hypothesis: dedifferentiated cellular subpopulations developed after p53mutations occurred within HCC, more severe cellular atypia exists in areas with loss ofheterozygosity （LOH） of p53within HCC, and finally, p53mutations preferentially occur inmoderately to poorly differentiated HCC along with or after p53LOH.A number of studies have indicated that promoter hypermethylation may be a keymechanism involved in the inactivation of some tumor suppressor genes in HCC. Promotermethylation and subsequent loss of protein expression have been demonstrated in manyoncogenes and tumor suppressor genes in HCC. WNT/β-catenin pathway is the bestcharacterized WNT signaling pathway, regulates the stability of transcription co-activatorβ-catenin and thus activates the expression of a set of target genes, which in turn regulates cellproliferation, behavior and survival. Aberrant activation of the WNT/β-catenin signalingpathway is frequently involved in a broad spectrum of human malignancies. Alternative togenetic deletions and point mutations, epigenetic inactivation of negative WNT regulators,through DNA methylation of promoter CpG islands and/or histone modification, leads to theactivation or amplification of aberrant WNT/β-catenin signaling. Downregulation ofextracellular WNT antagonists such as SFRP1, SFRP2, SFRP4, SFRP5, WIF1, DKK1, DKK2and DKK3due to abnormal promoter methylation correlates with constitutive activation ofcanonical WNT/β-catenin signaling in HCC. The growing list of epigenetically silencedWNT antagonists involved in HCC indicates an important role for epigenetic inactivationevents in tumor initiation and progression. Epigenetic silencing of negative WNT regulators isbelieved to induce abnormal WNT/β-catenin activation in the earliest stage of tumorigenesis,even before the appearance of key pathway mutations. Epigenetic changes （promotermethylation or histone methylation/deacetylation） are pharmacologically reversible, usingepigenetic agents including DNA methylatransferase inhibitors （5-aza-2’-deoxycytidine,Zebularine） and histone deacetylase inhibitors （TSA, SAHA and PXD101）. Restoration of theexpression of negative WNT regulators in silenced tumor cells, through either DNAdemethylation/histone remodeling or ectopic expression, results in the blockade of β-catenin/TCF-dependent transcription, inhibition of tumor cell proliferation, and induction of tumor cell apoptosis in multiple cancers. Thus, epigenetic modulation of WNT/β-cateninsignaling could be an attractive therapeutic strategy for WNT-addicted cancers.Purpose:We investigated promoter methylation status of seven genes related with WNT/β-catenin signaling pathway in patients with hepatocellular carcinoma （HCC）.Methods：1. Our previous study identified genomic imbalances using whole genomic aCGH（microarray comparative genomic hybridization, arrayCGH）. We drew an overview idiogrammap defined the regions of gains and losses according to the result in HCC. In this study, wemapped the WNT pathway related genes into the HCC ideogram.2. A total of20HCC tissue specimens paired with their corresponding adjacentnon-cancerous tissues, and their peripheral blood were obtained. In addition,10non-HCC-related normal liver tissues from patients with hepatolithiasis or hepatichemangioma were collected as normal control.3. Mutation detection of WNT antagonist APC and tumor suppressor PTEN, TP53usingSanger sequencing.4. Bisulfite treatment of genomic DNA.5. Methylation status of APC, AXIN2, FZD9, SFRP1, SOX17, WIF1and WNT2wasanalyzed by MSP （methylation-specific PCR）.6. BS（Bisulfite sequencing, BS）as a golden standard to evaluate methylation pattern inspecific genes especially in tumor was employed to confirm MSP result, and obtain detailedmethylation pattern.7. The differences in methylation level between HCC, the adjacent non-cancerous tissuesand normal liver tissues were evaluated using Student’s t test. P <0.05was consideredstatistically significant.Results:1. The most frequent genomic imbalances detected in HCC samples were gains of1q,5p,5q,6p,7q,8q,17q,20p and20q, and losses of1p,4q,6q,8p,9p,13q,14q,16p,16q，17p,18q and21q. 2. APC mutation in exon15（c.8376₈377delAT，p.I2666X） was found in1HCC tissuesample, and LOH of APC was identified in this particular case as well. Mutation of PTEN andTP53was also found in1sample respectively.3. Comparing with HCC tumor adjacent tissue and normal liver tissue, FZD9, SFRP1,SOX17, WIF1and WNT2were hypermethylated in HCC tissue （P<0.05）. Hypermethylation ofAPC was found in HCC tissue, tumor adjacent tissue and normal liver tissue, but not inperipheral blood.4. Different methlytion pattern was observed in these6genes. In targeted promoterregion, APC hypermethylation was seen in all or none of the CG loci in case-specific pattern.In contrast, hypermethylation of other genes was scattered in the whole promoter region.5. Methylation pattern of liver tissue was not consistent with peripheral blood.6. Hypermethylation of AXIN2was not found in HCC.Conclusion:Chromosomal instability, including gain or loss of the genomic DNA copy number, isfrequently observed in HCC. Loss of function of tumor suppressor genes APC and TP53playsimportant role in hepatocarcinogenesis. In our pilot study, WNT/β-catenin signaling pathwayrelated genes APC, FZD9, SFRP1, SOX17, WIF1and WNT2showed DNA copy number gainin HCC. And up-regulation of these genes was documented in previous study. In this study,We investigated promoter methylation status and found hypermethylation in FZD9、SFRP1、SOX17、WIF1and WNT2. It suggested that hypermethylation of these genes is one of the themechanisms for gene inactivation in HCC. FZD9、SFRP1、SOX17、WIF1and WNT2couldbe used as biomarkers for early diagnosis of HCC. Our study suggested that promoterhypermethylation of APC is a common event in HCC. In addition to hypermethylation, LOHand gene mutation also may play a role in the regulation of APC.