CFLARL与GMEB1互调影响肺癌细胞凋亡的新机制及对化疗敏感性的作用研究

Chemotherapy is the cornerstone in the treatment of lung cancer. Further exploring the mechanisms of the chemotherapy-related apoptosis would be no doubt to improve clinical effects of chemotherapy. The degradation of CFLARL (c-FLIP), a key modulator of apoptosis, plays a very important role in mediating therapeutic chemical compound-induced apoptosis and we’ve digging in this field for almost 10 years. As a result, we recently discovered that CFLARL can be physically combined with GMEB1and this combination can facilitate an interaction between CFLARL and GMEB1, and can further impact chemotherapy sensitivity of lung cancer. We therefore propose that: on one hand, in the cytoplasm, the interaction between CFLARL and GMEB1could regulate the unbiquitination process of CFLARL: GMEB1 can play as an antagonist of ubiquitin ligase of CFLARL and GMEB1can also facilitate transporting the deubiquitinase to CFLARL; on the other hand, CFLARL can be translocated into the nucleus, where it can combine with GMEB1 and inhibit the transcriptional activity of GMEB1-FOXL2. Thus, the interaction between CFLARL and GMEN1can inhibit apoptosis via different mechanisms. This proposal will use molecular biological technique to ① detect the expression pattern of CFLARL and GMEG1 in tumor tissues, ② explore the mode of combination between CFLARL and GMEB1. ③ Clarify the molecular mechanism for GMEB1 to regulate CFLARL’s stability and degradation as well as the molecular mechanism for CFLARL to regulate GMEB1’s activity in the nucleus. The results of our study could be translated into discovering novel therapeutic targets and optimizing the strategy of treating lung cancer.

CFLARL是与化疗诱导肺癌细胞凋亡密切相关的蛋白，申请者从事相关研究近十年，首先发现CFLARL与GMEB1存在物理结合并相互调控，影响肺癌化疗敏感性，由此提出假说：一方面：在细胞质中，GMEB1通过与CFLARL的结合调控泛素连接酶和去泛素化酶与CFLARL的结合，降低CFLARL的泛素化，促进CFLARL的稳定性，从而抑制化疗药物诱导的肺癌细胞凋亡；另一方面：CFLARL在核内与GMEB1结合，降低GMEB1-FOXL2的转录活性，从而抑制肺癌细胞凋亡。本项目拟用分子生物学技术分析GMEB1和CFLARL在肿瘤组织中的表达情况，探讨GMEB1-CFLARL蛋白形成复合物的模式，明确GMEB1调控CFLARL稳定性和降解以及CFLARL入核降低GMEB1-FOXL2转录活性进而影响肺癌细胞功能（如凋亡）的分子机制,为设计高效的化疗方案和寻找新的治疗靶点提供理论依据。

本研究首次发现CFLAR与GMEB1可以形成复合物并相互调节，是调控细胞凋亡的新机制，并显著影响化学药物引起的肺癌细胞凋亡。GMEB1和CFLARL蛋白表达量在NSCLC细胞中正相关，且GMEB1中的47-324片段与CFLARL的P20和P12结构域可相互作用，GMEB1作为一个支架蛋白促进了USP40和CFLARL的相互作用，USP40才是CFLARL的去泛素化酶，且主要是去除CFLARL蛋白K48连接的多聚泛素化。敲低GMEB1可影响TRAIL诱导的DISC复合体中各组分的比例，CFLARL在其中的比例降低，FADD和CASP8的比例升高，敲低GMEB1还可提高TRAIL诱导的CASP8的活性，增强CASP8、CASP3和PARP的切割水平。当肿瘤细胞抵抗TRAIL诱导凋亡时，敲低GMEB1可促进对TRAIL的敏感性，肿瘤凋亡比率明显增加。敲低GMEB1同样促进SAHA诱导的CASP8、CASP3和PARP的切割，促进了肿瘤细胞凋亡的敏感性。CFLARL过表达能够抑制GMEB1敲低诱导的CASP8酶活性，抑制GMEB1敲低诱导的CASP8、CASP3和PARP的切割，抑制GMEB1和TRAIL处理诱导的细胞凋亡，抑制GMEB1和SAHA处理诱导的细胞凋亡，这些都表明GMEB1敲低促进凋亡是通过CFLARL实现的，当CFLARL降解被抑制时GMEB1敲低凋亡的促进作用将减弱。裸鼠移植瘤实验证实：小鼠移植瘤的生长于GMEB1的敲低效果密切相关，GMEB1敲低效果越好，移植瘤生长抑制越明显。从TCGA数据库中提取了492例肺腺癌病人的mRNA表达情况，GMEB1高表达的病人存活期相对较短，而GMEB1低表达的病人存活期相对较长。USP40对病人存活期的影响超过GMEB1，在USP40高表达的病人中，病人存活期将远低于USP40低表达的病人。