新型WNT通路抑制剂LGK974对MLH1缺失型肿瘤放射增敏作用及机制的研究

Activation of WNT signaling has been linked to tumor initiation, progression and altered therapeutic responses to treatment. Of particular, the WNT pathway regulates cellular radiosensitivity, making it a potential target for radiosensitization. The long-term goal of this study is to identify a novel molecular based radiosensitizer that can be used for clinical radiotherapy. In our preliminary study, we found that a novel WNT inhibitor, LGK974, worked synergistically with radiation in the MLH1-deficient background. Therefore the central hypothesis of this proposal is that LGK974 can enhance radiosensitivity by inhibiting the WNT signaling pathway in the presence of MLH1-deficiency. In this proposal, three aims were proposed: 1) In Aim 1 we will investigate the mechanism by which LGK794 to affect radio sensitivity by looking at effects on activation of DNA damage repair and cell cycle checkpoints. Also, based on the kinomic analysis in the preliminary studies, we will validate two critical targets (PRKACb and PRKACa) of LGK974 inhibition to see whether the changes of the enzymatic activities are dependent on MLH1-deficiency. 2) Aim 2 is to establish a series primary tumor cell lines from colorectal cancer tissues, and use the cell lines to validate MLH1-deficiency dependent LGK974 radiosensitization. 3) Aim 3 will use a nude mouse model with HCT116 colorectal cancer xerographs either with a vector-control or MLH1 complementation to assess the in vivo activity of LGK974. Completion of the proposed studies will provide solid foundation for exploring a new molecular radiosensitizer in clinic in the near future.

WNT通路与肿瘤的发生发展密切相关且参与调控细胞的放射敏感性。我们发现新型WNT通路抑制剂LGK974对MLH1缺失肿瘤有放疗增敏作用并通过激酶组学找到两个磷酸化靶点，本课题将验证LGK974作为分子放射增敏剂的潜能及机制。假设LGK974能提高MLH1表达缺失肿瘤细胞的放射敏感性。课题将①研究LGK974对DNA损伤修复、细胞周期检测点的激活机制及MLH1导入MLH1缺失的细胞后LGK974合并放疗对磷酸化靶点的影响，了解该变化是否依赖于MLH1功能的缺失。②建立一系列原代培养结直肠癌细胞系以用来进一步验证LGK974的放射增敏作用与MLH1缺失的关系。③建立稳转的HCT116细胞株来表达野生型MLH1，在荷瘤小鼠模型中检测在体水平LGK974合并放射治疗在MLH1基因缺失状态下的增敏作用。以上研究结果将为开发新型分子放射增敏剂、放疗方案的个体化选择和分子靶向放疗提供重要的理论依据。

Mut L homolog -1 (MLH1)是一种关键的DNA错配修复蛋白，参与对DNA损伤因子的敏感性。然而，它在肿瘤细胞放射敏感性中的作用还不太清楚。在本研究中，我们研究了MLH1在细胞对电离辐射(IR)反应中的作用，并探索了相关的信号分子。将人结直肠癌HCT116细胞MLH1精通(MLH1+)和缺失(MLH1-)的同基因对在3 cGy剂量下暴露于IR 24 h。克隆形成实验检测克隆生存。流式细胞仪分析细胞周期分布。Western blotting检测MLH1、DNA损伤标志物γH2AX和抗肿瘤药物常见靶点蛋白激酶A催化亚基(protein kinase A catalytic subunit, PRKAC)蛋白水平的变化。结果表明,HCT116(一种+)细胞与人口增加,证明增加radio-resistance G2人口减少,降低γH2AX和磷酸化的比率PRKACαβ总PRKAC,和一个高水平的总PRKAC和磷酸化PRKACβ二世后红外光谱相比HCT116(种)细胞。重要的是，沉默HCT116 (MLH1+)细胞中的PRKAC增加了细胞的放射敏感性。综上所述，MLH1可能通过激活PRKAC增强细胞对IR的耐药性。我们的发现首次证明了PRKACPKA在mlh1介导的放射敏感性中的重要作用，这表明PRKACPKA有潜力作为生物标志物和增加放射敏感性的治疗靶点。