FBXW7与LSD1非降解结合调节DNA损伤修复和肺癌形成的作用及机理

FBXW7 is the substrate recognizing subunit of SCF (SKP1-Cullin-F-box protein) E3 ubiquitin ligase, and mainly known as a tumor suppressor by promoting ubiquitylation and degradation of oncogenic proteins (e.g. c-Myc, Notch, c-Jun, cyclin E) via K48-linked polyubiquitylation. Our recent study (Mol Cell 61: 419-433, 2016) showed that FBXW7 also promotes K63-linked polyubiquitylatoin of XRCC4 to facilitate non-homologous end-joining (NHEJ) repair. Whether FBXW7 has any ubiquitylation-independent function and its role in lung tumorigenesis are totally unknown. Here we showed that FBXW7 binds to LSD1, a protein demethylase, under physiological conditions, not for targeted ubiquitylation and degradation, but for functional modulation of each other. In this proposal, we will 1) show, for the first time, that FBXW7 indeed has ligase-independent function and elucidate mechanistically a) how FBXW7 is involved in epigenetic regulation via modulating LSD1 demethylase activity, and on the other hand, b) how LSD1 demethylates FBXW7 to regulate its NHEJ repair, and c) how LSD1, acting as a non-degradable pseudo-substrate, inhibits ligase activity of FBXW7 towards other real substrates; 2) use conditional KO in combination with KrasG12D-driven lung tumorigenesis model in mouse to demonstrate that Fbxw7 is a tumor suppressor, whereas LDS1 promotes tumorigenesis in the lung; 3) use clinical lung cancer tissues to show that hyper-methylation in the promoter of FBXW7 gene is responsible for its reduced expression, and FBXW7 down-regulation is associated with better patient response to chemoradiation therapy due to reduced NHEJ repair. Thus, FBXW7 level can serve as an efficacy biomarker. Our study, ranging from basic research to animal models, then to clinical study, not only elucidates mechanistically the novel functions of FBXW7-LSD1 in regulation of epigenetics, DNA damage repair, and lung tumorigenesis, but also has significant translational potential in precision medicine for individual based effective cancer therapy as well as for patient prognosis.

FBXW7是SCF泛素连接酶的底物识别受体，通过促进多个原癌蛋白的K48泛素化降解来发挥肿瘤抑制功能。我们最近研究发现它也能介导XRCC4的K63泛素化促进非同源末端连接修复DNA损伤。但FBXW7是否发挥非蛋白泛素降解的功能和它在肺癌发生中的作用完全未知。我们前期工作表明FBXW7能与去甲基化酶LSD1结合，不促其降解，但影响各自的功能。本项目将1）首次揭示FBXW7具有非蛋白降解的全新功能，可通过与LSD1结合，影响LSD1去甲基化酶活性来参与表观遗传调控；而LSD1通过FBXW7的去甲基化来调控DNA修复；以非降解底物竞争性抑制FBXW7底物泛素化活性；2）运用基因敲除鼠肺癌模型证明FBXW7是肺的抑癌基因，而LSD1是促癌基因；3）运用临床样本证明FBXW7启动子超甲基化与其低表达和肺癌发生发展密切相关；FBXW7低表达可作为选择临床联用放化疗方案的标志物，具有潜在的转化应用价值。

SCF泛素连接酶底物识别受体FBXW7通过识别和促进多个原癌蛋白的K48泛素化降解来发挥肿瘤抑制功能。但FBXW7是否发挥非蛋白泛素降解的功能和它在肺癌发生中的作用完全未知。本项目围绕FBXW7在肺癌发生和发展中的作用和机制，在以下几方面取得了重要进展：1）阐明了FBXW7与LSD1非降解性结合通过阻断FBXW7二聚体的形成促进FBXW7的自身泛素化，继而通过蛋白酶体和p62介导的自噬溶酶体两条途径降解；2）LSD1通过与FBXW7底物竞争性结合FBXW7影响FBXW7的泛素化连接酶活性；3）LSD1通过降低FBXW7的稳定性抑制FBXW7的生物学功能，包括细胞增殖抑制，NHEJ修复和辐射保护功能等；4）利用条件性敲除鼠肺癌模型，本项目在生理水平证明Fbxw7能抑制KrasG12D突变诱导的肺癌发生，而Lsd1则具有促肺癌活性，同时敲除Fbxw7和Lsd1能逆转Fbxw7或Lsd1敲除对肺癌发生的影响；5）FBXW7还能与负责组蛋白H3K4甲基化的KMT2复合体中的RbBP5结合，但不影响RbBP5的降解；6）FBXW7与KMT2组分竞争性结合RbBP5，抑制KMT2复合体甲基转移酶活性，进而调控下游靶基因转录表达；7）FBXW7通过影响炎症相关细胞因子的转录表达抑制肺癌细胞生长、迁移和侵袭，抑制KMT2甲基转移酶活性能逆转FBXW7敲除对炎症相关基因表达的上调和肺癌细胞增殖、迁移和侵袭的促进作用；8）通过分析肺癌临床病理组织样品，本项目发现肺癌中FBXW7的表达下调可能与其启动子的甲基化相关。因此，本项目一方面揭示了LSD1以其去甲基化酶非依赖性方式靶向调控FBXW7的降解从而促进肺癌发生发展的功能，提示LSD1可能是一个肺癌治疗的新靶点；另一方面，本项目从体外细胞水平、基因敲除鼠肺癌模型和肺癌临床样本三个层次，研究了FBXW7抑制体内肺癌发生的作用，发现FBXW7可通过其非降解作用调控表观遗传和基因转录抑制癌症发生的新机制，进一步拓展人们对FBXW7功能调控以及LSD1和FBXW7在肺癌发生发展中作用的认识，为指导靶向LSD1-FBXW7轴的肺癌个性化治疗提供了理论基础，具有重要的转化潜能。