肝细胞癌WNT/β-catenin信号通路相关基因异常甲基化的研究
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摘要
肝细胞癌（Hepatocellular carcinoma，HCC）是世界范围内最常见且恶性程度最高的肿瘤之一，根据世界卫生组织GLOBOCAN2008数据统计，位居男性恶性肿瘤发病率的第五位，死亡顺位的第二位，女性恶性肿瘤发病率的第七位，死亡顺位的第六位。由于原发性肝癌不易早期发现，每年的死亡人数和确诊人数（626000）基本相当。由于目前缺乏有效的治疗方法，临床学者们致力于肝癌形成过程中可能涉及的基因组学、蛋白组学以及信号传导等方面的研究，进一步了解其发生发展的分子生物学机制，寻找新的治疗途径。
从遗传学角度，肝细胞癌同其他肿瘤一样，存在基因组不稳定性或遗传学不稳定性，随着人类基因组序列的完成，人们开发了全基因组范围检测肿瘤细胞染色体异常的基于基因芯片的比较基因组杂交(microarray comparative genomic hybridization, arrayCGH)技术能在更高的解析度发现肿瘤基因组的异常。绘制出了较为精细的肝癌全基因组异常图谱。除了染色体存在不稳定外，肝癌样本也存在一些基因的突变，导致抑癌基因失活或者癌基因活化。已证实抑癌基因p53基因的失活与HCC的发生发展密切相关。众多研究表明HCC常发生高频率p53基因的杂合性缺失LOH（loss of heterozygosity）（25%-80%），p53第249位密码子突变外及其他密码子突变也被证实与肝癌的发生发展有密切的关系。最近，其他抑癌基因如PTEN等也受到重视，研究者发现一些(9.5%)肝癌患者PTEN存在第5和8外显子的突变。与机体发育和细胞分化功能相关的Wnt/β-catenin也被累及，Terris等人发现β-catenin基因第3外显子在肝癌中的突变率是19%，突变导致β-catenin蛋白稳定性增加，导致β-catenin蛋白聚集、活性增加引发肝癌。肿瘤基因组不稳定性和基因突变等异常会影响基因的转录表达。
肿瘤表观遗传改变在细胞的基因表达上发挥举足轻重的作用，表观遗传学机制中，DNA甲基化和microRNA与肝癌发生的关系是得到最为深入研究的表观遗传学机制。近年来积累的资料表明，CpG岛高甲基化导致的抑癌基因和错配修复基因等重要肝癌相关基因的表达沉默，是肿瘤的发生和发展过程中的重要分子事件之一。这些基因参与肝癌发生的多阶段过程。WNT/β-catenin信号途径异常激活可以启动下游多种癌基因的转录并导致细胞癌变，是肝癌发生的主要分子机制之一。Wnt拮抗剂基因表达失调与肿瘤形成之间有着密切的关系，目前这一家族中的多个基因已被确立为候选的肿瘤抑制基因。然而这类基因有一个显著特点是：基因突变并不是导致其失活的主要遗传学机制。相比之下，由启动子区CpG岛发生异常甲基化导致的基因表观遗传学失活在肿瘤中则更为普遍。目前很多研究表明Wnt拮抗剂基因的表观遗传学失活与肝癌的发生有着密切关系。已有多项研究报道了多种细胞外Wnt拮抗基因，如SFRP1，SFRP2，SFRP4，SFRP5，WIF1，DKK1，DKK2，DKK3基因启动子区高甲基化造成的拮抗基因表达下调，在肝癌中持续激活WNT/β-catenin信号通路。目的：检测肝细胞癌中WNT/β-catenin信号通路相关基因甲基化状态，探索具有临床诊断治疗意义的分子标记物。
方法：
1、本实验室前期工作中对中国东北地区45例肝细胞癌组织应用高通量寡核苷酸arrayCGH进行了基因组DNA拷贝数的分析，此次通过总结绘制基因组失平衡染色体图谱，得到了WNT/β-catenin信号途径相关基因在人肝细胞癌中DNA拷贝数变化的数据。
2、收集新鲜肝癌组织20例，与其配对的癌旁组织20例，配对的外周血20例。与肝癌无关疾病的肝脏组织10例作为正常对照。
3、提取肝组织和外周血全基因组DNA后，应用Sanger双脱氧链终止法对Wnt拮抗基因APC和抑癌基因PTEN、TP53进行全部编码区测序。
4、亚硫酸盐处理基因组DNA。
5、应用甲基化特异性PCR（methylation-specific PCR, MSP）作为定性的甲基化检测方法对APC、AXIN2、FZD9、SFRP1、SOX17、WIF1和WNT2共7个Wnt途径相关基因进行甲基化检测初筛。
6、应用硫化测序（Bisulfite sequencing, BS）验证MSP结果，并得到甲基化位点更详细的定点定量数据。
7、统计学分析不同组织类型之间甲基化程度的差异是否具有统计学意义。
结果：
1、肝癌细胞基因组存在广泛的基因组不稳定性，多个染色体区段发生异常改变。其中扩增区常见于染色体1q，5p，5q，6p，7q，8q，17q，20p和20q。而LOH区常见于1p，4q，6q，8p，9p，13q，14q，16p，16q，17p，18q和21q。
2、在20例肝癌样本，我们分别在1例病例中检测到抑癌基因APC、PTEN、TP53体细胞突变，其中APC、TP53突变阳性病例中，同时合并有LOH，提示APC、TP53的异常对肝癌发生发展的作用。
3、WNT/β-catenin信号途径中的6个相关基因（APC、FZD9、SFRP1、SOX17、WIF1和WNT2）在肝癌中呈异常甲基化。其中FZD9、SFRP1、SOX17、WIF1和WNT2高甲基化在肝癌组织和正常组织差异有统计学意义，有潜力作为肝癌早期诊断指标之一。
4、不同基因在肝癌组织中甲基化模式不同，有的呈全或无，有的呈散在、点状分布。
5、肝癌患者外周血甲基化模式同肝组织不同。
6、AXIN2在肝癌组织、癌旁组织、正常肝组织和外周血中均未发现异常甲基化，而许多文献曾报道在直肠癌、肺癌等肿瘤中高甲基化现象常见。
结论：
发现肝癌细胞基因组存在广泛的基因组不稳定性，多个染色体区段发生异常改变。发现的扩增区和LOH区与既往文献报道相符。除了染色体存在不稳定外，肝癌样本也存在一些基因的突变，肝癌样本也存在一些基因的突变，我们检测到了抑癌基因APC、PTEN、TP53体细胞突变，其中APC、TP53突变阳性病例中，同时合并有LOH，提示APC、TP53的异常对肝癌发生发展的作用。WNT/β-catenin信号途径中的6个相关基因（APC、FZD9、SFRP1、SOX17、WIF1和WNT2）在肝癌中呈异常甲基化。我们前期实验工作发现，这6个基因的DNA拷贝数在肝癌中呈明显扩增，但根据文献报道，这些基因在肝癌中均表达下调。提示基因异常甲基化是基因沉默的机制之一。FZD9、SFRP1、SOX17、WIF1和WNT2异常甲基化有潜力作为肝癌早期诊断指标之一。基因突变、杂合性缺失（LOH）等在肿瘤演进中存在随机的、个体化的特点，尚难以在其中找到共性的、适用于所有肿瘤或同类肿瘤的标志物，有其局限性的一面，相对而言，异常甲基化在肿瘤中属于普遍现象。可开发更为广泛适用的肿瘤生物学标志。
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.8376_8377delAT，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.
从遗传学角度，肝细胞癌同其他肿瘤一样，存在基因组不稳定性或遗传学不稳定性，随着人类基因组序列的完成，人们开发了全基因组范围检测肿瘤细胞染色体异常的基于基因芯片的比较基因组杂交(microarray comparative genomic hybridization, arrayCGH)技术能在更高的解析度发现肿瘤基因组的异常。绘制出了较为精细的肝癌全基因组异常图谱。除了染色体存在不稳定外，肝癌样本也存在一些基因的突变，导致抑癌基因失活或者癌基因活化。已证实抑癌基因p53基因的失活与HCC的发生发展密切相关。众多研究表明HCC常发生高频率p53基因的杂合性缺失LOH（loss of heterozygosity）（25%-80%），p53第249位密码子突变外及其他密码子突变也被证实与肝癌的发生发展有密切的关系。最近，其他抑癌基因如PTEN等也受到重视，研究者发现一些(9.5%)肝癌患者PTEN存在第5和8外显子的突变。与机体发育和细胞分化功能相关的Wnt/β-catenin也被累及，Terris等人发现β-catenin基因第3外显子在肝癌中的突变率是19%，突变导致β-catenin蛋白稳定性增加，导致β-catenin蛋白聚集、活性增加引发肝癌。肿瘤基因组不稳定性和基因突变等异常会影响基因的转录表达。
肿瘤表观遗传改变在细胞的基因表达上发挥举足轻重的作用，表观遗传学机制中，DNA甲基化和microRNA与肝癌发生的关系是得到最为深入研究的表观遗传学机制。近年来积累的资料表明，CpG岛高甲基化导致的抑癌基因和错配修复基因等重要肝癌相关基因的表达沉默，是肿瘤的发生和发展过程中的重要分子事件之一。这些基因参与肝癌发生的多阶段过程。WNT/β-catenin信号途径异常激活可以启动下游多种癌基因的转录并导致细胞癌变，是肝癌发生的主要分子机制之一。Wnt拮抗剂基因表达失调与肿瘤形成之间有着密切的关系，目前这一家族中的多个基因已被确立为候选的肿瘤抑制基因。然而这类基因有一个显著特点是：基因突变并不是导致其失活的主要遗传学机制。相比之下，由启动子区CpG岛发生异常甲基化导致的基因表观遗传学失活在肿瘤中则更为普遍。目前很多研究表明Wnt拮抗剂基因的表观遗传学失活与肝癌的发生有着密切关系。已有多项研究报道了多种细胞外Wnt拮抗基因，如SFRP1，SFRP2，SFRP4，SFRP5，WIF1，DKK1，DKK2，DKK3基因启动子区高甲基化造成的拮抗基因表达下调，在肝癌中持续激活WNT/β-catenin信号通路。目的：检测肝细胞癌中WNT/β-catenin信号通路相关基因甲基化状态，探索具有临床诊断治疗意义的分子标记物。
方法：
1、本实验室前期工作中对中国东北地区45例肝细胞癌组织应用高通量寡核苷酸arrayCGH进行了基因组DNA拷贝数的分析，此次通过总结绘制基因组失平衡染色体图谱，得到了WNT/β-catenin信号途径相关基因在人肝细胞癌中DNA拷贝数变化的数据。
2、收集新鲜肝癌组织20例，与其配对的癌旁组织20例，配对的外周血20例。与肝癌无关疾病的肝脏组织10例作为正常对照。
3、提取肝组织和外周血全基因组DNA后，应用Sanger双脱氧链终止法对Wnt拮抗基因APC和抑癌基因PTEN、TP53进行全部编码区测序。
4、亚硫酸盐处理基因组DNA。
5、应用甲基化特异性PCR（methylation-specific PCR, MSP）作为定性的甲基化检测方法对APC、AXIN2、FZD9、SFRP1、SOX17、WIF1和WNT2共7个Wnt途径相关基因进行甲基化检测初筛。
6、应用硫化测序（Bisulfite sequencing, BS）验证MSP结果，并得到甲基化位点更详细的定点定量数据。
7、统计学分析不同组织类型之间甲基化程度的差异是否具有统计学意义。
结果：
1、肝癌细胞基因组存在广泛的基因组不稳定性，多个染色体区段发生异常改变。其中扩增区常见于染色体1q，5p，5q，6p，7q，8q，17q，20p和20q。而LOH区常见于1p，4q，6q，8p，9p，13q，14q，16p，16q，17p，18q和21q。
2、在20例肝癌样本，我们分别在1例病例中检测到抑癌基因APC、PTEN、TP53体细胞突变，其中APC、TP53突变阳性病例中，同时合并有LOH，提示APC、TP53的异常对肝癌发生发展的作用。
3、WNT/β-catenin信号途径中的6个相关基因（APC、FZD9、SFRP1、SOX17、WIF1和WNT2）在肝癌中呈异常甲基化。其中FZD9、SFRP1、SOX17、WIF1和WNT2高甲基化在肝癌组织和正常组织差异有统计学意义，有潜力作为肝癌早期诊断指标之一。
4、不同基因在肝癌组织中甲基化模式不同，有的呈全或无，有的呈散在、点状分布。
5、肝癌患者外周血甲基化模式同肝组织不同。
6、AXIN2在肝癌组织、癌旁组织、正常肝组织和外周血中均未发现异常甲基化，而许多文献曾报道在直肠癌、肺癌等肿瘤中高甲基化现象常见。
结论：
发现肝癌细胞基因组存在广泛的基因组不稳定性，多个染色体区段发生异常改变。发现的扩增区和LOH区与既往文献报道相符。除了染色体存在不稳定外，肝癌样本也存在一些基因的突变，肝癌样本也存在一些基因的突变，我们检测到了抑癌基因APC、PTEN、TP53体细胞突变，其中APC、TP53突变阳性病例中，同时合并有LOH，提示APC、TP53的异常对肝癌发生发展的作用。WNT/β-catenin信号途径中的6个相关基因（APC、FZD9、SFRP1、SOX17、WIF1和WNT2）在肝癌中呈异常甲基化。我们前期实验工作发现，这6个基因的DNA拷贝数在肝癌中呈明显扩增，但根据文献报道，这些基因在肝癌中均表达下调。提示基因异常甲基化是基因沉默的机制之一。FZD9、SFRP1、SOX17、WIF1和WNT2异常甲基化有潜力作为肝癌早期诊断指标之一。基因突变、杂合性缺失（LOH）等在肿瘤演进中存在随机的、个体化的特点，尚难以在其中找到共性的、适用于所有肿瘤或同类肿瘤的标志物，有其局限性的一面，相对而言，异常甲基化在肿瘤中属于普遍现象。可开发更为广泛适用的肿瘤生物学标志。
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.8376_8377delAT，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.
引文
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