MED1 MUTATIONS IN COLORECTAL CANCER

转移性结直肠癌的治疗在很大程度上是不令人满意的，并且非常需要新的治疗或针对当前可用的治疗的改进的患者选择。 DNA修复基因代表了作为新的预后和预测因子的可行候选者，因为它们与结直肠癌生物学和发病机制密切相关，并且还可能影响对DNA损伤化疗的反应。以错配修复蛋白MLH 1为诱饵，通过酵母双杂交筛选，我们克隆了一个新的人类DNA修复基因MED 1。 MED 1蛋白在人类细胞中与MLH 1功能性相互作用，与细菌DNA修复糖基化酶/裂解酶同源，并与CpG甲基化DNA结合，表明胞嘧啶甲基化在哺乳动物DNA修复中起着新的作用。MED 1显示胸腺嘧啶和尿嘧啶糖基化酶活性，从G：T和G：U错配中去除这些碱基。 这表明MED 1可能通过5-甲基胞嘧啶和胞嘧啶分别自发脱氨基为胸腺嘧啶和尿嘧啶来抵消诱变。 我们还发现MED 1可以有效地从DNA中去除5-氟尿嘧啶，这是结直肠癌全身治疗的支柱。最近，我们获得的证据表明，MED 1基因经常突变（25%的情况下），在人类结直肠癌和结肠外癌表现出微卫星不稳定性。 在结直肠癌标本中，我们还检测到MED 1基因座的杂合性丢失。本项目的基本假设是：MED 1是确保人类细胞基因组保真度的机制的重要组成部分; MED 1突变与结直肠癌的发病机制相关;并且它可能在对化疗药物的耐药性/敏感性中发挥作用。实验旨在解决：1）结直肠癌中MED 1突变的性质和频率; 2）MED 1突变是否是预后因素并影响对治疗的反应。

The therapy of metastatic colorectal cancer is largely unsatisfactory and new treatments or improved patient selection for the currently available treatments are greatly needed. DNA repair genes represent viable candidates as novel prognostic and predictive factors, because they are intimately connected with colorectal cancer biology and pathogenesis, and may also affect response to DNA-damaging chemotherapy. In a yeast two-hybrid screening with the mismatch repair protein MLH1 as "bait", we cloned MED1, a novel human DNA repair gene. The MED1 protein functionally interacts with MLH1 in human cells, is homologous to bacterial DNA repair glycosylases/lyases, and binds to CpG-methylated DNA, suggesting that cytosine methylation plays a novel role in mammalian DNA repair. MED1 displays thymine and uracil glycosylase activity, removing these bases from G:T and G:U mismatches. This suggests that MED1 may counteract mutagenesis by spontaneous deamination of 5-methylcytosine and cytosine to thymine and uracil, respectively. We also found that MED1 efficiently removes from DNA 5-fluorouracil, the mainstay of systemic therapy of colorectal cancer. Recently, we obtained evidence that the MED1 gene is frequently mutated (25% of the cases) in human colorectal and extracolonic carcinomas exhibiting microsatellite instability. In colorectal cancer specimens, we also detected loss of heterozygosity at the MED1 locus. The hypotheses underlying the present project are: that MED1 is an important component of the machinery ensuring genomic fidelity in human cells; that MED1 mutations are relevant to the pathogenesis of colorectal cancer; and that it may play a role in resistance/sensitivity to chemotherapeutic agents. Experiments are proposed to address: 1) the nature and frequency of MED1 mutations in colorectal cancer; 2) whether MED1 mutations are a prognostic factor and influence response to treatment.