DNA错配修复功能异常对乳房外Paget病5-FU化疗敏感性的影响与机制

Extramammary Paget's disease (EMPD) is a rare skin cancer. We have found that the germline mutations and reduced expression of DNA mismatch repair (MMR) genes were common in EMPD, and also found that there was a significantly discrepancy in the sensitivity of 5-FU chemotherapy in patients with EMPD. We propose that MMR abnormalities may play an important role in affecting the 5-FU chemotherapeutic sensitivity in EMPD. The aims of our study are firstly to analyze the correlation between MMR status and 5-FU sensitivity; to reveal the different mechanisms and effects between the MMR genes mutations and decreased proteins expression on EMPD cells in 5-FU induced cell apoptosis and autophagic cell death pathways via in vitro and in vivo (nude mice) tests; finally, to evaluate the predictive value of MMR analysis in the 5-FU sensitivity in EMPD. This study will explore the mechanisms and effects of MMR alterations in EMPD on the 5-FU chemotherapeutic sensitivity, and provide new ideas for the treatment of this cancer.

乳房外Paget病（EMPD）是一种罕见的皮肤肿瘤。我们发现DNA错配修复（MMR）基因胚系突变和蛋白表达下降在EMPD具有较高的检出率，并发现5-FU化疗敏感性在EMPD患者中存在明显差异。我们提出假说认为MMR异常可能是影响EMPD 5-FU敏感性的重要机制。本研究拟首先分析EMPD患者的MMR状态，明确5-FU敏感性与MMR状态的关联；通过体外和体内（裸鼠模型）实验，揭示MMR基因突变、MMR蛋白表达下降和MMR正常的EMPD细胞在5-FU诱导的细胞凋亡和自噬性死亡通路的差异；最后，评价MMR功能分析在预测EMPD对5-FU敏感性的价值。本研究将探索MMR功能异常在EMPD对5-FU敏感性的影响及机制，为EMPD治疗提供新思路。

本项目聚焦DNA错配修复（MMR）系统在一种罕见的皮肤肿瘤——乳房外Paget病（EMPD）中的状态变化及其影响EMPD 5-FU化疗敏感性的重要机制。研究通过免疫组化方法完成对57例EMPD肿瘤组织MMR蛋白表达的检测，明确我国EMPD样本的MMR蛋白表达水平。分析MMR关键蛋白MSH2基因甲基化程度，完成对我国EMPD样本的MSH2启动子甲基化水平的评估，确定CpG6（-87）和CpG3（-98）是EMPD患者中最常见的两个甲基化位点。.本项目还立足于临床实践，利用甲基化阳性和阴性标准品，建立基于甲基化敏感的高分辨率熔解曲线（MS-HRM）标准曲线的检测方法，并对收集的57例EMPD病人样本进行了检测，证实了MS-HRM分析方法定量检测MSH2启动子甲基化水平的可行性。.鉴于MSH2异常在EMPD患者中的高检出率，本项目进一步研究其功能对于患者5-氟尿嘧啶（5-FU）化疗敏感性的影响机制。通过构建不同MSH2状态的细胞系，研究MSH2功能与5-FU敏感性的联系。研究发现MSH2功能缺失会激活ATR-Chk1信号通路，抑制细胞凋亡。5-FU药物作用后引发的G2周期阻滞依赖于MSH2功能，但同时也取决于TP53蛋白的表达，当TP53突变或表达降低情况下，MSH2增高能够更加显著阻滞G2期。评估MSH2及p53状态具有预测5-FU敏感性的价值。