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[Relation between mismatch repair genes and colon cancer.]

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Junli Ma
Junli Ma
Zeng Shan at Xiangya Hospital of Central South University
Zeng Shan
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Abstract

Mismatch repair (MMR) system is one form of DNA repair mechanisms, which plays an important role in rectifying the mismatch of base pairs, reducing gene mutations and keeping genome stability. Abnormal expression of MMR regulated by miRNA is closely related to the development of colon cancer. Functional defects of MMR (dMMR) with particular clinical characteristics can be used as a potential prognostic and predictive biomarker. This article reviews the relation between MMR system, miRNA and colon cancer.
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中南大学学报（医学版）
J Cent South Univ (Med Sci) 2014, 39(2) hp://www.csumed.org; hp://xbyx. xysm.net
190
错配修复基因和结肠癌的关系
马俊丽，曾珊
(中南大学湘雅医院肿瘤科，长沙 410008)
[摘要] 错配修复系统(mismatch repair，MMR)是机体DNA修复机制的一种形式，主要纠正碱基错配，防止基因
突变和维持基因组稳定性。miRNA介导的MMR表达异常和结肠癌的发生发展关系密切，MMR表达缺陷(dMMR)的结
肠癌有独特的临床特征，可以作为结肠癌的潜在预后及疗效预测因子。研究MMR与结肠癌的发生、MMR与miRNA的
相互关系及MMR疗效预测等具有非常重要的意义。
[关键词] 错配修复基因；微卫星不稳定性；miRNA；结肠癌
Relation between mismatch repair genes and colon cancer
MA Junli, ZENG Shan
(Department of Oncology, Xiangya Hospital, Center South University, Changsha 410008, China)
ABSTRACT Mismatch repair (MMR) system is one form of DNA repair mechanisms, which plays an important
role in rectifying the mismatch of base pairs, reducing gene mutations and keeping genome stability.
Abnormal expression of MMR regulated by miRNA is closely related to the development of colon
cancer. Functional defects of MMR (dMMR) with particular clinical characteristics can be used as
a potential prognostic and predictive biomarker. is article reviews the relation between MMR
system, miRNA and colon cancer.
KEY WORDS mismatch repair gene; microsatellite instability; miRNA; colon cancer
收稿日期(Date of reception)：2013-05-28
作者简介(Biography)： 马俊丽，硕士研究生，医师，主要从事消化道肿瘤方面的研究。
通信作者(Corresponding author)：曾珊，Email: zhengshan2000@yahoo.com
基金项目(Foundation item)：国家自然科学基金(30770971，30800518，81102046，81070362，81172470)；湖南省自然科学基金(11JJ2049，
12JJ3118)。is work was supported by the National Natural Science Foundation (30770971, 30800518, 81102046, 81070362, 81172470) and the Natural Science
Fund of Hunan Province, P. R. China (11JJ2049, 12JJ3118).
·
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· ·综 述·
DOI:10.11817/j.issn.1672-7347.2014.02.014
hp://xbyx.xysm.net/xbwk/leup/PDF/201402190.pdf
错配修复基因和结肠癌的关系 马俊丽，等 191
错配修复(mismatch repair，MMR)系统由一
系列特异性修复DNA错配的修复蛋白组成，包
括hMLH1，hMSH2，hPMS2，hMSH6等家族成
员，以上基因主要通过形成以下异源二聚体执
行相应功能：MutSα，MutSβ，MutLα，MutLβ和
MutLγ。hMSH2与hMSH6构成MutSα，识别单一
的碱基错配和插入/缺失环；MutSβ由hMSH2和
hMSH3构成，负责识别2~8个核苷酸的插入/缺失
环；hMLH1分别与hPMS2，PMS1构成MutLα，
MutLβ，定位于错配部位，并能协同Exo I，增
殖细胞核抗原(proliferating cell nuclear antigen，
PCNA)，DNA聚合酶消除错配，进行DNA再合
成。hMLH1与hMSH2是MMR的核心基因和蛋白，
它们之一异常则可能导致MMR系统功能受损或丧
失，不能进行复合物的组装，引起相应其他蛋白
如hMSH6，hPMS2等丢失；而MMR家族中其他基
因异常仅导致其编码蛋白的丢失。
MMR功能缺失(dMMR)减弱甚至丧失了本身
的校对功能，致使DNA错配积聚，广泛分布在生
物基因组中的短串联重复序列即微卫星重复序列
的拷贝数目发生变化，产生遗传不稳定性，称为
微卫星不稳定性(microsatellite instability，MSI)。
MSI分为高度微卫星不稳定性(MSI-H)和低度微卫
星不稳定性/微卫星稳定性(MSI-L/MSS)。MMR
基因中任何一个或几个基因突变均可产生MSI表
型。MMR基因正常(pMMR)则对应MSS。但是除
了MMR，还有其他途径维持微卫星的稳定性。
最近研究[1]发现组蛋白H3K36me3通过与MSH6的
proline-tryptophan-tryptophan-proline(PWWP)域直
接作用招募hMutSα，调控体内MMR；而细胞缺少
H3K36三甲基转移酶SETD2会表现为MSI，增加自
发突变率，该研究也解释了部分无明确MMR基因
突变而表型为MSI的原因。
免疫组织化学(immunohistochemistry，IHC)
检测hMLH1、hMSH2等蛋白表达情况和PCR进行
MSI状态分析是常用的dMMR筛查方法。IHC简单
实用、花费少，是较常用方法；但PCR能够鉴别
出被IHC忽略的dMMR，被认为是dMMR筛查的标
准方法。两种检测方法一致性达90%以上，联合应
用敏感性更高[2]。
1 MMR与结肠癌
MMR扮演管家基因角色，其功能异常导致
基因组稳定性降低，随机突变率增高。与细胞生
长、分化、凋亡或肿瘤转移有关的基因发生突
变，在DNA合成过程中丢失保真度，与肿瘤发生
发展密切相关。MMR改变普遍见于肠癌、胃癌、
子宫内膜癌等恶性肿瘤。
结肠癌发生过程复杂，主要涉及两种机制，
染色体不稳定性(chromosome instability，CIN)和
MSI。约85%结直肠癌表型为CIN，伴随高频率非
整倍体等位基因不平衡；其余发生和MSI相关。两
种机制的好发部位不同，CIN主要与远端结肠癌
有关，MSI则与近端相关，其发生率约是远端的10
倍，如遗传性非息肉病性结直肠癌(hereditary non-
polyposis colorectal cancer，HNPCC)，即Lynch综合征
(Lynch syndrome，LS)。散发性结肠癌可涉及上述两
种机制，但其MSI途径有别于LS。
1.1 LS
LS是常染色体显性遗传性疾病，发病率约为
3%~5%。患者发病年龄早，常伴有子宫内膜癌、
卵巢癌、胃癌等肠外肿瘤。LS发病主要和MMR基
因突变有关，hMLH1和hMSH2为最常见的突变基
因，约占90%。MMR无突变热点，突变位点分布
于整个编码区，多为点突变、小的插入或缺失，
广泛的基因重排发生率较低，基于不同的人群报
道，约占5%~20%，这种基因重排，多为丙氨酸重
复序列之间的同源性重组，难以通过多重连接依
赖探针扩增技术等常规方法检测发现，或许可以
部分解释基因内重排发生率较低的原因[3]。
LS中也存在MMR基因启动子区甲基化现象，
可能是LS发生的新机制。Tomita等[4]认为LS发生和
hMSH2启动子区高甲基化有关，由处于hMSH2上
游的上皮细胞黏附分子基因3'端缺失所致。
MMR表观突变与很多肿瘤的发生相关，这
种机制日益受到重视。部分LS无MMR的病理性
突变，但伴随hMLH1或hMSH2启动子区的表观
突变，说明主要基因的表观突变增加了遗传易感
性，与LS发生关系密切。
1.2 散发性结肠癌
基因尤其是抑癌基因的甲基化致使转录抑
制，与很多肿瘤的发生密切相关。经由MSI途径
发生的散发性结肠癌约占10%~15%，其中95%与
MLH1基因启动子区高甲基化有关，通常指启动子
区CpG岛甲基化(CpG island methylator phenotype，
CIMP)。
MSI散发性结肠癌发病年龄较高，常以低分
化、近端高发生率、肿瘤细胞淋巴浸润、女性好发
为特征，多数研究[5-7]表明这种表型有相对较好的预
后。并且常伴有BRAF V600E突变，而这种突变几乎
可以排除遗传性因素，是结肠癌预后不良指标，或
中南大学学报 (医学版 ), 2014, 39(2) hp://www.csumed.org; hp://xbyx.xysm.net
192
许可以联合MMR和BRAF基因检测进行临床预后分
析和指导用药[8-9]。CIMP能否作为预后因子正在探
索中，其中某些甲基化位点也许可以作为预后参考
指标。研究[10]提示结肠癌根治术后给予FOLFOX方
案化疗，3年无病生存期(disease-free survival，DFS)与
甲基化与否及MSI状态均无相关性，但是亚组分析
显示甲基化位点NEUROG1(+)和CDKN2A预示术后
复发时间提前，3年DFS较短。
2 MMR，miRNA与结肠癌
MiRNA是小的非编码RNA，约18~25个核苷酸
长度，在转录后水平起重要作用。通过与目的基
因mRNA的3'非编码区(3'UTR)结合发挥生物学作
用，调控mRNA表达。miRNA尽管占人类基因组的
1%~3%，却调控30%的基因表达。MiRNA广泛参与
细胞的增殖、分化、凋亡等过程，与恶性肿瘤的发
生发展密切相关。MiRNA介导MMR表达与结肠癌
的发生发展密切相关，并可能影响其化疗敏感性。
2.1 miRNA与结肠癌
已经证明miRNA在大多数肿瘤包括结肠癌
中异常表达，功能上可以作为致癌基因或抑癌基
因。在肿瘤中高表达的miRNA作用于抑癌基因，
抑制其表达，如miR-21在很多实体瘤中过表达，靶
向作用于同源性磷酸酶-张力蛋白(phosphatase and
tensin homolog，PTEN)和原肌球蛋白1(tropomyosin
1，TPM1)，在成瘤过程中发挥一定的作用[11]。
miR-372和miR-373作为致癌基因，作用于肿瘤抑
制基因LATS2[12]。部分miRNA在肿瘤中低表达或
不表达，如let-7，mi18a*，功能上类似于抑癌基
因，抑制原癌基因转录。
研究发现结肠癌中一系列miRNA表达异常，
它们作用的靶基因不同，但可能均参与了结肠癌
的发生发展。与正常组织相比，miR-135在腺瘤组
织中高表达，靶向作用于抑癌基因APC的3'UTR，
抑制其表达，持续激活下游的Wnt信号转导通路，
在结肠癌的发生发展中发挥重要作用[13-14]。相同
miRNA在正常组织、腺瘤和结肠癌中表达水平不
同，在疾病早期阶段可检测到的miRNA变化或许
正在变得越来越重要[15]。
2.2 MMR，miRNA和结肠癌
mRNA的3'UTR包含多个miRNA作用位点，
一种miRNA可以与多种mRNAs靶点结合，多种
miRNA也可以与一种mRNA相互作用，二者形成复
杂的调控系统。越来越多研究表明miRNA与MMR
相互作用在结肠癌的发病中发挥着重要作用。
miRNA的表达特点或许可以区分dMMR结肠癌和
错配修复正常(pMMR)结肠癌。
对比结肠癌和正常肠黏膜组织中735种
miRNA表达情况，发现其中39种差异明显，依
据MMR状态不同，miRNA差异情况有所不同：
miR-552，-592，-181c，-196b表达水平在dMMR
结肠癌中降低，pMMR结肠癌中升高；而miR-
625和-31表达水平与之相反[16]。Oberg等[15]证明
在dMMR和pMMR结肠癌中miR-31，miR-552，
miR-592，miR-224等表达差异有统计学意义，所
有的dMMR结肠癌，无论突变或甲基化来源，均呈
现相似的miRNA表达模式。但是有研究[17]表明：
miRNA和结肠癌发生类型有关，在和高度微卫星
不稳定性(MSI-H)相关的基因中(miR-155，-31，
-223，-26b，-92)，miR-31和-223在LS患者中过表
达。Lanza等[18]通过分析结肠癌中miRNA和mRNA
的表达，认为综合二者较单独RNA分析更能区分
MSI-H和MSS。miR-17~92家族中部分miRNA在
MSS结肠癌中表达上调，它们作为致癌基因，可能
部分解释MSS肿瘤有更激进的生物学行为的原因。
研究证明miRNA直接作用于MMR核心蛋白，
通过调控MMR的表达对结肠癌的发展产生影响。
miR31-5p直接作用于MLH1，沉默miR31-5p增加
MLH1的表达，并通过作用于MLH1蛋白调控细胞周
期，使其阻滞在S期[19]。原发性结肠癌中miR-155过
表达，似乎在MSI结肠癌中表达水平更高[20-21]。Valeri
等[21]认为过表达的miR-155下调结肠癌细胞和组织
中hMLH1，hMSH2，hMSH6蛋白的表达，可能是结
肠癌发生的另一种机制，尤其强调表型为MSI，而
MMR失活机制不明的结肠癌可能是因为miR-155过表
达造成的。多数MSI结肠癌伴随miR-155过表达，但
不是所有miR-155过表达结肠癌均表现为MSI，miR-
155可能影响其他DNA修复蛋白，二者之间的关系需
要进一步探讨验证。Valeri等[22]转染结肠癌细胞miR-
21后检测hMSH2，hMSH6蛋白的表达。结果证明
miR-21直接作用于hMSH2，hMSH6 mRNA的3'UTR，
下调MMR系统的重要组件hMSH2，hMSH6蛋白的表
达，增加MMR突变率和功能缺陷，导致MSI。进一
步通过细胞和动物试验表明，miR-21在体内外均可
因为下调hMSH2和hMSH6，减少G2/M阻滞和凋亡，
引起氟尿嘧啶(FU)抵抗。
3 MMR和结肠癌化疗
MMR作为结肠癌的生物标志物，在临床研究
中可进行预后和疗效预测。dMMR是早期结肠癌的
错配修复基因和结肠癌的关系 马俊丽，等 193
较好预后因子，II期结肠癌术后予以FU类单药辅
助治疗时要行MMR基因检测，dMMR结肠癌不会
从FU治疗中获益，pMMR患者才考虑辅助化疗。
MMR在晚期结肠癌预后作用尚未确定。
3.1 dMMR与FU
Tajima等[23]认为FU对结肠癌的细胞毒性依赖
于MMR基因完整性，并且和细胞内MutSα，MutSβ
水平有关，当二者并存时FU的细胞毒性最大。MMR
复合物状态或许可以为FU治疗提供敏感性分层。
由于体内FU代谢的复杂性，dMMR单因素
进行疗效预测需要进一步思考。研究[24]指出在
MMR基础上联合检测其他指标如胸苷酸合酶
(thymidylate synthase，TS)，TP53，CD8可进一步
筛选FU化疗的获益人群，TS高表达者给予FU治疗
效果好。和前述一致，dMMR者FU化疗降低了总
体生存期，但是CIMP患者可以从中获益。关于该
研究的意义需要进一步探索，以期筛选出最佳适
应患者。
3.2 dMMR与结肠癌其他化疗药物
dMMR结肠癌中，较多研究集中在FU，至于
加入奥沙利铂后，dMMR患者的DFS与pMMR患者
相似或稍高，差异无统计学意义[25]。Zaanan等[26]
则证明III期结肠癌中，MMR状态是FOLFOX辅助
化疗的独立预后因子。与pMMR患者相比，dMMR
患者的3年DFS延长有统计学意义。
dMMR结肠癌对伊立替康的敏感性尚无定
论，并且临床回顾性分析或前瞻性研究资料有
限。一项回顾性研究[27]表明晚期结肠癌中，
dMMR对伊立替康的反应率更高。CALGB 89803试
验[28]比较IFL方案(伊立替康、FU和亚叶酸钙)和LV
方案(FU和亚叶酸钙)治疗III期结肠癌的疗效，两
组患者总生存期(overall survival，OS)和DFS无明显
差别，但亚组分析显示IFL组能提高dMMR患者的5
年DFS，而pMMR患者未从伊立替康中获益。dMMR
结肠癌可能对伊立替康潜在获益，尚需大规模的临
床研究。
4 展 望
错配修复基因在结肠癌发病中的作用逐渐被
认识，但是存在许多问题仍需进一步阐述。MMR
系统成员庞杂，除已经熟知的十余种，其他MMR
基因有待发现，miRNA和MMR之间具体作用形式
需要进一步研究，它们之间相互作用模式可能更
复杂。肠癌发生不是由独立的基因改变引起，可
能涉及MMR和其他基因的相互作用，而这种相互
关系仍不太明确。在结肠癌治疗方面，MMR有一
定的指导作用，但是针对某一患者的个体化用药需
要进一步探讨，携带相同基因不同甲基化位点的患
者，其预后和用药是否也不同。分子基因研究仍
是我们努力的方向，为个体化治疗提供依据。
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(本文编辑 郭征)
本文引用：马俊丽, 曾珊. 错配修复基因和结肠癌的关系 [ J].
中南大学学报:医学版, 2014, 39(2): 190-194. DOI:10.11817/
j.issn.1672-7347.2014.02.014
Cite this article as: MA Junli, ZENG Shan. Relation between
mismatch repair genes and colon cancer[J]. Journal of Central South
University. Medical Science, 2014, 39(2): 190-194. DOI:10.11817/
j.issn.1672-7347.2014.02.014
... Several reports have suggested that MMR cellular levels are tightly regulated [21,22,24]. In addition to the well-characterized oncogenic events such as genetic amplification, mutation, and translocation, MMR dysregulation also leads to loss of the MMR system [34][35][36]. Bioinformatics analyses predicted the presence of a miR-137 seed region in the MSH2 3′UTR-with our in vitro results confirming MSH2 is targeted by miR-137. ...
... Several reports have suggested that MMR cellular levels are tightly regulated [21,22,24]. In addition to the well-characterized oncogenic events such as genetic amplification, mutation, and translocation, MMR dysregulation also leads to loss of the MMR system [34][35][36]. Bioinformatics analyses predicted the presence of a miR-137 seed region in the MSH2 3 UTR-with our in vitro results confirming MSH2 is targeted by miR-137. ...
MiR-137 Targets the 3′ Untranslated Region of MSH2: Potential Implications in Lynch Syndrome-Related Colorectal Cancer
Article
Full-text available
  • Sep 2021
Mismatch Repair (MMR) gene dysregulation plays a fundamental role in Lynch Syndrome (LS) pathogenesis, a form of hereditary colorectal cancer. Loss or overexpression of key MMR genes leads to genome instability and tumorigenesis; however, the mechanisms controlling MMR gene expression are unknown. One such gene, MSH2, exerts an important role, not only in MMR, but also in cell proliferation, apoptosis, and cell cycle control. In this study, we explored the functions and underlying molecular mechanisms of increased MSH2 expression related to a c.*226A>G variant in the 3′untranslated (UTR) region of MSH2 that had been previously identified in a subject clinically suspected of LS. Bioinformatics identified a putative binding site for miR-137 in this region. To verify miRNA targeting specificity, we performed luciferase gene reporter assays using a MSH2 3′UTR psiCHECK-2 vector in human SW480 cells over-expressing miR-137, which showed a drastic reduction in luciferase activity (p > 0.0001). This effect was abolished by site-directed mutagenesis of the putative miR-137 seed site. Moreover, in these cells we observed that miR-137 levels were inversely correlated with MSH2 expression levels. These results were confirmed by results in normal and tumoral tissues from the patient carrying the 3′UTR c.*226A>G variant in MSH2. Finally, miR-137 overexpression in SW480 cells significantly suppressed cell proliferation in a time- and dose-dependent manner (p < 0.0001), supporting a role for MSH2 in apoptosis and cell proliferation processes. Our findings suggest miR-137 helps control MSH2 expression via its 3′UTR and that dysregulation of this mechanism appears to promote tumorigenesis in colon cells.
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... The identification of suspected Lynch syndrome-related CRC is important, as patients with this subtype of Lynch syndrome could benefit from genetic counselling, surveillance, and specific treatments, such as subtotal colectomy, prophylactic hysterectomy and immune checkpoint inhibitors (Ma and Zeng, 2014;Boland et al., 2018). Moreover, the identification of this subtype of Lynch syndrome can result in early cancer detection and decreased cancer-related mortality of relatives with Lynch syndrome. ...
Characteristics of colorectal carcinoma patients with PMS2 defects detected by immunohistochemistry
Article
Full-text available
  • Aug 2020
  • EUR J CANCER PREV
Lynch syndrome is the most prevalent form of familial colorectal cancer (CRC) and is caused by pathogenic germline mismatch repair (MMR) gene mutations. MLH1, MSH2 and MSH6 mutations have been well studied, but the rate and characteristics of PMS2 mutations are rare, especially in China. This study enrolled 1706 unselected patients with CRC who underwent colorectal resection from June 2016 to November 2018, the MMR status and clinicopathological features were analysed. A total of 11.8% of patients with CRC had defects in at least one MMR-related protein. Among them, 8.3% were identified with PMS2 defects, and 3.1% of patients had isolated PMS2 defects. Compared with MMR-proficient CRC, PMS2-defect CRC occurred more frequently in the right colon and less frequently in the rectum, had more poorly differentiated and mucinous carcinoma cases, and had fewer perineural invasions and a lower pN stage but a more advanced pT stage and a larger tumour size. In the cases with PMS2 defect, there were fewer tumours in the right colon, fewer poorly differentiated cases and smaller tumour sizes than in the cases with both MLH1 and PMS2 defects. In addition, in cases with isolated PMS2 defects, there were more tumours in the right colon and, more mucinous carcinoma cases than in cases with MMR-proficient CRCs, but had a similar cancer onset age. This study identified the rate, clinicopathological and age characteristics of PMS2 defects in CRCs in China and highlighted the importance of universal screening and germline detection of PMS2 in CRC.
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Mutation analysis of related genes in hamartoma polyp tissue of Peutz-Jeghers syndrome
Article
Full-text available
  • Apr 2020
  • WORLD J GASTROENTERO
Background: Peutz-Jeghers syndrome (PJS) is a rare disease with clinical manifestations of pigmented spots on the lips, mucous membranes and extremities, scattered gastrointestinal polyps, and susceptibility to tumors. The clinical heterogeneity of PJS is obvious, and the relationship between clinical phenotype and genotype is still unclear. Aim: To investigate the mutation status of hereditary colorectal tumor-associated genes in hamartoma polyp tissue of PJS patients and discuss its relationship with the clinicopathological data of PJS. Methods: Twenty patients with PJS were randomly selected for this study and were treated in the Air Force Medical Center (former Air Force General Hospital) PLA between 2008 and 2017. Their hamartoma polyp tissues were used for APC, AXIN2, BMPR1A, EPCAM, MLH1, MLH3, MSH2, MSH6, MUTYH, PMS1, PMS2, PTEN, SMAD4, and LKB1/STK11 gene sequencing using next-generation sequencing technology. The correlations between the sequencing results and clinical pathological data of PJS were analyzed. Results: Fourteen types of LKB1/STK11 mutations were detected in 16 cases (80.0%), of which 8 new mutations were found (3 types of frameshift deletion mutations: c.243delG, c.363_364delGA, and c.722delC; 2 types of frameshift insertions: c. 144_145insGCAAG, and c.454_455insC; 3 types of splice site mutations: c.464+1G>T, c.464+1G>A, and c.598-1G>A); 9 cases (45.0%) were found to have 18 types of heterozygous mutations in the remaining 13 genes except LKB1/STK11. Of these, MSH2: c.792+1G>A, MSH6: c.3689C>G, c.4001+13C>CTTAC, PMS1: c.46C>t, and c.922G>A were new mutations. Conclusion: The genetic mutations in hamartoma polyp tissue of PJS are complex and diverse. Moreover, other gene mutations in PJS hamartoma polyp tissue were observed, with the exception of LKB1/STK11 gene, especially the DNA mismatch repair gene (MMR). Colorectal hamartoma polyps with LKB1/STK11 mutations were larger in diameter than those with other gene mutations.
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The Histone Mark H3K36me3 Regulates Human DNA Mismatch Repair through Its Interaction with MutS alpha
Article
Full-text available
  • Apr 2013
DNA mismatch repair (MMR) ensures replication fidelity by correcting mismatches generated during DNA replication. Although human MMR has been reconstituted in vitro, how MMR occurs in vivo is unknown. Here, we show that an epigenetic histone mark, H3K36me3, is required in vivo to recruit the mismatch recognition protein hMutSα (hMSH2-hMSH6) onto chromatin through direct interactions with the hMSH6 PWWP domain. The abundance of H3K36me3 in G1 and early S phases ensures that hMutSα is enriched on chromatin before mispairs are introduced during DNA replication. Cells lacking the H3K36 trimethyltransferase SETD2 display microsatellite instability (MSI) and an elevated spontaneous mutation frequency, characteristic of MMR-deficient cells. This work reveals that a histone mark regulates MMR in human cells and explains the long-standing puzzle of MSI-positive cancer cells that lack detectable mutations in known MMR genes.
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Contribution of Large Genomic Rearrangements in Italian Lynch Syndrome Patients: Characterization of a Novel Alu-Mediated Deletion
Article
Full-text available
Lynch syndrome is associated with germ-line mutations in the DNA mismatch repair (MMR) genes, mainly MLH1 and MSH2. Most of the mutations reported in these genes to date are point mutations, small deletions, and insertions. Large genomic rearrangements in the MMR genes predisposing to Lynch syndrome also occur, but the frequency varies depending on the population studied on average from 5 to 20%. The aim of this study was to examine the contribution of large rearrangements in the MLH1 and MSH2 genes in a well-characterised series of 63 unrelated Southern Italian Lynch syndrome patients who were negative for pathogenic point mutations in the MLH1, MSH2, and MSH6 genes. We identified a large novel deletion in the MSH2 gene, including exon 6 in one of the patients analysed (1.6% frequency). This deletion was confirmed and localised by long-range PCR. The breakpoints of this rearrangement were characterised by sequencing. Further analysis of the breakpoints revealed that this rearrangement was a product of Alu-mediated recombination. Our findings identified a novel Alu-mediated rearrangement within MSH2 gene and showed that large deletions or duplications in MLH1 and MSH2 genes are low-frequency mutational events in Southern Italian patients with an inherited predisposition to colon cancer.
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Management of stage II colon cancer - the use of molecular biomarkers for adjuvant therapy decision
Article
Full-text available
  • Feb 2013
  • BMC GASTROENTEROL
Background There is uncertainty on the benefit of adjuvant chemotherapy in patients with stage II colorectal cancers. The aim of this study is to investigate the combined role of clinical, pathological and molecular parameters to identify those stage II patients who better benefit from adjuvant therapy. Methods We examined 120 stage II colon cancer patients. Of these, 60 patients received adjuvant 5-FU chemotherapy after surgery and the other 60 did not receive therapy. Immunohistochemical (IHC) analyses were performed to evaluate the expressions of Thymidylate synthetase (TYMS), TP53 (p53), β-catenin (CTNNB1) and CD8. For TYMS, its mRNA expression levels were also investigated by real time qRT-PCR. The entire case study was characterized by the presence of a defect in the MMR (mismatch repair) system, the presence of the CpG island methylator phenotype (CIMP or CIMP-High) and for the V600E mutation in the BRAF gene. At the histo-pathological level, the depth of tumour invasion, lymphovascular invasion, invasion of large veins, host lymphocytic response and tumour border configuration were recorded. Results The presence of the V600E mutation in the BRAF gene was a poor prognostic factor for disease free and overall survival (DFS; hazard ratio [HR], 2.57; 95% CI: 1.03 -6.37; p = 0.04 and OS; HR, 3.68; 95% CI: 1.43-9.47; p < 0.01 respectively), independently of 5-FU treatment. Adjuvant therapy significantly improved survival in patients with high TYMS levels (p = 0.04), while patients with low TYMS had a better outcome if treated by surgery alone (DFS; HR, 6.07; 95% CI, 0.82 to 44.89; p = 0.04). In patients with a defect in the MMR system (dMMR), 5-FU therapy was associated to reduced survival (DFS; HR, 37.98; 95% CI, 1.04 to 1381.31; p = 0.04), while it was beneficial for CIMP-High associated tumours (DFS; HR, 0.17; 95% CI, 0.02 to 1.13; p = 0.05). Conclusions Patients’ characterization according to MMR status, CIMP phenotype and TYMS mRNA expression may provide a more tailored approach for adjuvant therapy in stage II colon cancer.
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Both hMutSα and hMutSß DNA Mismatch Repair Complexes Participate in 5-Fluorouracil Cytotoxicity
Article
Full-text available
Patients with advanced microsatellite unstable colorectal cancers do not show a survival benefit from 5-fluorouracil (5-FU)-based chemotherapy. We and others have shown that the DNA mismatch repair (MMR) complex hMutSα binds 5-FU incorporated into DNA. Although hMutSß is known to interact with interstrand crosslinks (ICLs) induced by drugs such as cisplatin and psoralen, it has not been demonstrated to interact with 5-FU incorporated into DNA. Our aim was to examine if hMutSß plays a role in 5-FU recognition. We compared the normalized growth of 5-FU treated cells containing either or both mismatch repair complexes using MTT and clonogenic assays. We utilized oligonucleotides containing 5-FU and purified baculovirus-synthesized hMutSα and hMutSß in electromobility shift assays (EMSA) and further analyzed binding using surface plasmon resonance. MTT and clonogenic assays after 5-FU treatment demonstrated the most cytotoxicity in cells with both hMutSα and hMutSß, intermediate cytotoxicity in cells with hMutSα alone, and the least cytotoxicity in cells with hMutSß alone, hMutSß binds 5-FU-modified DNA, but its relative binding is less than the binding of 5-FU-modified DNA by hMutSα. Cytotoxicity induced by 5-FU is dependent on intact DNA MMR, with relative cell death correlating directly with hMutSα and/or hMutSß 5-FU binding ability (hMutSα>hMutSß). The MMR complexes provide a hierarchical chemosensitivity for 5-FU cell death, and may have implications for treatment of patients with certain MMR-deficient tumors.
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MicroRNA-31-5p modulates cell cycle by targeting human mutL homolog 1 in human cancer cells
Article
  • Mar 2013
  • TUMOR BIOL
MicroRNAs (miRNAs) and DNA mismatch repair (MMR) have been linked to human cancer progression. Human mutL homolog 1 (hMLH1), one of the core MMR genes, defects in lung cancer development. However, the interaction between miRNAs and MMR genes and their regulatory effect on cell cycle remain poorly understood. In this study, we investigated the role of miR-31-5p in hMLH1 gene expression and the effect of miR-31-5p on cell cycle in non-small cell lung cancer (NSCLC). We found that miR-31-5p was inversely correlated with hMLH1 expression in NSCLC cell lines and hMLH1 was a direct target of miR-31-5p. Knockdown of miR-31-5p induced a cell cycle arrest at G2/M phase and increased hMLH1 protein expression in NSCLC cells. Conversely, overexpression of miR-31-5p significantly induced cell cycle arrest at S phase and decreased hMLH1 protein expression. Furthermore, knockdown of hMLH1 upregulated miR-31-5p expression and caused cell cycle arrest at S phase. Data from this study revealed that miR-31-5p modulates cell cycle by targeting hMLH1 protein at the posttranscriptional level in NSCLC, which may represent a novel therapy strategy for lung cancer by targeting miR-31-5p.
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[A novel genetic disorder of Lynch syndrome - EPCAM gene deletion]
Article
  • Feb 2013
Lynch syndrome, also known as hereditary non-polyposis colorectal cancer, HNPCC, is a cancer-predisposing autosomal-dominant hereditary disorder caused by defects of the mismatch repair(MMR)system during DNA replication. Not only colorectal cancer, but malignancies in various organs, e.g., endometrium, stomach, small intestine, and urinary tract, occur in people of a relatively young age and accumulate in the family; therefore, this syndrome is considered to be a very important clinical entity with regard to the cancer treatment strategy. Germline mutations of 4 MMR genes, e.g., MLH1, MSH2, MSH6 and PMS2, had been identified as the cause of this disease, however, a novel mechanism, epigenetic inactivation of MSH2 gene due to hypermethylation of promotor region by the deletion of 3'part of epithelial cell adhesion molecule(EPCAM) gene which is located upstream of the MSH2 gene, has been reported in recent years. Therefore, it should be kept in mind in genetic testing and/or counseling for Lynch syndrome case with MSH2 defect that there might be a deletion of the EPCAM gene. In this review, the significance of the EPCAM gene defect in the management for Lynch syndrome is briefly introduced.
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Overexpression of microRNAs-155 and 21 targeting mismatch repair proteins in inflammatory bowel diseases
Article
  • Jan 2013
  • CARCINOGENESIS
Microsatellite instability (MSI) due to mismatch repair (MMR) deficiency is reported in 5-10% of colorectal cancers complicating inflammatory bowel diseases (IBD-CRCs). The molecular mechanisms underlying MMR-deficiency may be different in IBD-CRCs, and in sporadic and hereditary MSI tumours. Here we hypothesize that over-expression of miR-155 and miR-21, two inflammation-related miRNAs that target core MMR proteins, may constitute a pre-neoplastic event for the development of MSI IBD-CRCs. We studied miR-155 and miR-21 expression using Real-Time Quantitative PCR in MSI (n=10) and MSS (microsatellite stable, n=10) IBD-CRCs, and in MSI (n=32) and MSS (n=30) non-IBD CRCs. We also screened colonic samples from IBD patients without cancer (n=18) and used healthy colonic mucosa as controls (n=20). MiR-155 and miR-21 appeared significantly over-expressed in the colonic mucosa of IBD subjects without CRC, but also in neoplastic tissues of IBD patients compared to non-IBD controls (p<0.001). Importantly, in patients with IBD-CRCs, miR-155 and miR-21 over-expression extended to the distant non-neoplastic mucosa (p<0.001). Ratios of expressions in tumours versus matched distant mucosa revealed a nearly significant association between miR-155 over-expression and MSI in IBDs (p=0.057). These results show a strong deregulation of both MMR-targeting miRNAs in IBD subjects with or without cancer. MiR-155 over-expression being particularly associated to MSI IBD-CRCs and extending to distant non-neoplastic mucosa, strongly suggests that a pre-neoplastic miR-155 field defect may promote MSI-driven transformation of the colonic mucosa. The detection and monitoring of miR-155 field defect may therefore have implications for the prevention and treatment of MSI IBD-CRCs.
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Methylation and microsatellite status and recurrence following adjuvant FOLFOX in colorectal cancer
Article
The prognostic impact of CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) on the treatment outcome of colon cancer patients receiving adjuvant 5-fluorouracil/ leucovorin/oxaliplatin (FOLFOX) is unclear. We investigated CIMP and MSI status in colorectal cancer patients treated with adjuvant FOLFOX. Stage II and III sporadic colorectal cancer patients who underwent curative resection followed by adjuvant FOLFOX were included. Eight CpG island loci (CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, CDKN2A (p16), RUNX3 and SOCS1) and 5 microsatellite markers were examined. Disease-free survival (DFS) was analyzed according to CIMP and MSI status. A total of 322 patients were included: male/female 192/130, median age 61 years (range 30-78), proximal/distal location 118/204, and stage II/III 43/279. CIMP status was high in 25 patients (7.8%) and 21 patients (6.5%) had MSI-high tumor. CIMP/MSI status was not significantly associated with DFS: 3-year DFS 100% in CIMP(-)/MSI(+), 84% in CIMP(-)/MSI(-), 82% in CIMP(+)/MSI(-), and 75% in CIMP(+)/MSI(+) (p = 0.33). Results of exploratory analysis showed that concurrent methylation at NEUROG1 and CDKN2A (p16) was associated with shorter DFS: 3-year DFS 69% in NEUROG1(+)/CDKN2A (p16)(+) vs. 87% in NEUROG1(-)/CDKN2A (p16)(-) (p = 0.006). In conclusion, concurrent methylation of NEUROG1 and CDKN2A (p16) is associated with recurrence following adjuvant FOLFOX in stage II/III colorectal cancer.
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Clinicopathologic features and prognostic analysis of MSI-high colon cancer
Article
  • Nov 2011
  • INT J COLORECTAL DIS
The objectives of the study were to estimate the incidence and clarify the clinicopathologic feature of sporadic microsatellite instability (MSI)-high (MSI-H) colon cancer. Furthermore, the role of MSI in colon cancer prognosis was also investigated. Microsatellite status was identified by genotyping. The clinicopathologic differences between two groups (MSI-H vs. MSI-L/S) and the prognostic value of MSI were analyzed. From 1993 to 2006, 709 sporadic colon cancer patients were enrolled. MSI-H colon cancers showed significant association with poorly differentiated (28.3% vs. 7.2%, p = 0.001), proximally located (76.7% vs. 34.5%, p = 0.001), more high mucin-containing tumor (10.0% vs. 5.1%, p = 0.001) and female predominance (56.7% vs. 30.2%, p = 0.001). In multivariate analysis, MSI-H is an independent factor for better overall survival (HR, 0.459; 95% CI, 0.241-0.872, p = 0.017). Based on the hospital-based study, MSI-H colon cancers demonstrated distinguished clinicopathologic features from MSI-L/S colon cancers. MSI-H is an independent favorable prognostic factor for overall survival in colon cancer.
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