SPOP基因突变通过调控FANCI-FANCD2复合物多聚泛素化影响前列腺癌基因组稳定性相关机制研究

Prostate cancer is a common tumor in male, but the pathogenesis is still not known clearly. The latest research has found that SPOP gene mutation can disrupt genomic stability, but the mechanism remains an ongoing topic of study. The Fanconi anemia (FA) pathway, as a classic pathway for DNA damage repair, regulates the stability of the genome. The FA pathway is a pathway involving many FA proteins, in which the ubiquitination of FANCI-FANCD2 complexes is the key to the opening of DNA damage repair. Our previous work has confirmed that SPOP protein combined with FANCI, and induced FANCI polyubiquitination, then promoted the DNA cross-links injury repair. Therefore，we hypothesize that the SPOP gene mutation affects the genomic stability of the prostate cancer by regulating the polyubiquitination of ID complexes and promotes the development of the tumor. So, in this study, we plan to use GST pull-down, ubiquitination and phosphorylation suppression, and other molecular biology experiment to figure out the binding site of SPOP and ID complexes, the processes and fuctions of ubiquitination and phosphorylation in this regulation of SPOP on ID complex; to clearly clarify the effects of SPOP gene mutation on DNA damage repair and the development of prostate cancer by immunofluorescence, quantitative analysis of DNA and FCM; in human peripheral blood cells and prostate cancer tissues, using nsSNV, chromosome structure analysis, and so on, to confirm that SPOP gene mutation influences the genomic stability of the human prostate cancer and promotes the development of the tumor. This study is expected to provide a theoretical basis for the mechanism of prostate cancer and to shed light on developing more effective treatment for prostate cancer.

前列腺癌是男性常见肿瘤，但发病机制仍不详知。最新发现SPOP基因突变影响了前列腺癌基因组稳定性，但机制有待研究。范可尼贫血(FA)通路中FANCI-FANCD2（ID）复合物的泛素化是调控DNA损伤修复的关键。前期我们发现SPOP与FANCI在蛋白水平结合，泛素化FANCI，并参与DNA损伤修复。基于此我们提出假说"SPOP基因突变通过调控ID复合物多聚泛素化影响前列腺癌基因组稳定性促进肿瘤发生"。课题组拟应用GSTpull-down、泛素化及磷酸化抑制等方法研究SPOP与ID复合物的结合位点、泛素化、磷酸化修饰;应用免疫荧光、DNA定量、FCM等明确SPOP基因突变对DNA损伤修复的影响及对前列腺癌发生的作用;在人类外周血及前列腺癌组织中运用nsSNV、染色体结构分析等手段证明SPOP基因突变对于人类前列腺癌基因组稳定性的影响。本研究有望为前列腺癌发生机制及新治疗方案的提出提供理论依据。

前列腺癌是最常见的实体肿瘤之一，但发病机制仍不详知，对其治疗手段有限。最新发现SPOP基因突变影响了前列腺癌基因组稳定性，但机制有待研究。范可尼贫血(FA)通路中FANCI-FANCD2（ID）复合物的泛素化是调控DNA损伤修复的关键。前期我们发现SPOP与FANCI在蛋白水平结合，泛素化 FANCI，并参与DNA损伤修复。基于此我们提出假说"SPOP基因突变通过调控ID复合物多聚泛素化影响前列腺癌基因组稳定性促进肿瘤发生"。本课题通过体内、外试验，进一步证明，在前列腺癌细胞中， SPOP与FA通路中的FANCI结合，但是并不与FANCD2结合。SPOP不影响FANCI的表达，但通过K63位依赖方式介导FANCI多聚泛素化。 而前列腺癌相关SPOP突变体不能调节FANCI泛素化。SPOP促进FANCI-FANCD2-FAN1细胞核聚合体（foci）形成，参与维持基因组的稳定性；DNA损伤通过调节ATR磷酸化FANCI促进SPOP泛素化修饰FANCI。研究还发现，在前列腺癌中，敲除SPOP的前列腺癌细胞对于MMC治疗更敏感。研究成果有助于更深入了解前列腺癌发病机制；更有助于确立前列腺癌的新的治疗靶点，为前列腺癌治疗提供新的思路。