Muto Yuta, Maeda Takafumi, Suzuki Koichi, Kato Takaharu, Watanabe Fumiaki, Kamiyama Hidenori, Saito Masaaki, Koizumi Kei, Miyaki Yuichiro, Konishi Fumio, Alonso Sergio, Perucho Manuel, Rikiyama Toshiki
Department of Surgery, Saitama Medical Center, Jichi Medical University, 1-847, Amanuma-cho, Omiya-ku, Saitama 330-8503, Japan.
BMC Cancer. 2014 Jun 25;14:466. doi: 10.1186/1471-2407-14-466.
Recent work led to recognize sessile serrated adenomas (SSA) as precursor to many of the sporadic colorectal cancers with microsatellite instability (MSI). However, comprehensive analyses of DNA methylation in SSA and MSI cancer have not been conducted.
With an array-based methylation sensitive amplified fragment length polymorphism (MS-AFLP) method we analyzed 8 tubular (TA) and 19 serrated (SSA) adenomas, and 14 carcinomas with (MSI) and 12 without (MSS) microsatellite instability. MS-AFLP array can survey relative differences in methylation between normal and tumor tissues of 9,654 DNA fragments containing all NotI sequences in the human genome.
Unsupervised clustering analysis of the genome-wide hypermethylation alterations revealed no major differences between or within these groups of benign and malignant tumors regardless of their location in intergenic, intragenic, promoter, or 3' end regions. Hypomethylation was less frequent in SSAs compared with MSI or MSS carcinomas. Analysis of variance of DNA methylation between these four subgroups identified 56 probes differentially altered. The hierarchical tree of this subset of probes revealed two distinct clusters: Group 1, mostly composed by TAs and MSS cancers with KRAS mutations; and Group 2 with BRAF mutations, which consisted of cancers with MSI and MLH1 methylation (Group 2A), and SSAs without MLH1 methylation (Group 2B). AXIN2, which cooperates with APC and β-catenin in Wnt signaling, had more methylation alterations in Group 2, and its expression levels negatively correlated with methylation determined by bisulfite sequencing. Within group 2B, low and high AXIN2 expression levels correlated significantly with differences in size (P = 0.01) location (P = 0.05) and crypt architecture (P = 0.01).
Somatic methylation alterations of AXIN2, associated with changes in its expression, stratify SSAs according to some clinico-pathological differences. We conclude that hypermethylation of MLH1, when occurs in an adenoma cell with BRAF oncogenic mutational activation, drives the pathway for MSI cancer by providing the cells with a mutator phenotype. AXIN2 inactivation may contribute to this tumorigenic pathway either by mutator phenotype driven frameshift mutations or by epigenetic deregulation contemporary with the unfolding of the mutator phenotype.
最近的研究使人们认识到无蒂锯齿状腺瘤（SSA）是许多具有微卫星不稳定性（MSI）的散发性结直肠癌的前体。然而，尚未对SSA和MSI癌症中的DNA甲基化进行全面分析。
我们采用基于阵列的甲基化敏感扩增片段长度多态性（MS-AFLP）方法，分析了8例管状腺瘤（TA）、19例锯齿状腺瘤（SSA）、14例具有微卫星不稳定性（MSI）的癌和12例无微卫星不稳定性（MSS）的癌。MS-AFLP阵列可检测人类基因组中包含所有NotI序列的9654个DNA片段在正常组织和肿瘤组织之间的甲基化相对差异。
对全基因组高甲基化改变进行无监督聚类分析发现，这些良性和恶性肿瘤组之间或组内无论在基因间、基因内、启动子或3'端区域的位置如何，均无主要差异。与MSI或MSS癌相比，SSA中低甲基化的频率较低。对这四个亚组之间的DNA甲基化进行方差分析，确定了56个差异改变的探针。该探针子集的层次树显示出两个不同的簇：第1组，主要由具有KRAS突变的TA和MSS癌组成；第2组具有BRAF突变，由具有MSI和MLH1甲基化的癌（2A组）和无MLH1甲基化的SSA（2B组）组成。在Wnt信号通路中与APC和β-连环蛋白协同作用的AXIN2在第2组中有更多的甲基化改变，其表达水平与亚硫酸氢盐测序确定的甲基化呈负相关。在2B组内，AXIN2的低表达和高表达水平与大小差异（P = 0.01）、位置差异（P = 0.05）和隐窝结构差异（P = 0.01）显著相关。
AXIN2的体细胞甲基化改变及其表达变化，根据一些临床病理差异对SSA进行分层。我们得出结论，当MLH1高甲基化发生在具有BRAF致癌突变激活的腺癌细胞中时，通过为细胞提供突变体表型来驱动MSI癌症的发生途径。AXIN2失活可能通过突变体表型驱动的移码突变或与突变体表型展开同时发生的表观遗传失调，促成这一致癌途径。
