STAMBP去泛素化E2F1激活Rb/E2F1通路促进膀胱癌恶性增殖的机制研究

Rb/E2F1 pathway is extremely important for tumorigenesis and development of bladder cancer. Recent study shows that K63-linked polyubiquitin chain can hold together E2F1 with Rb and HDAC1 as a scaffold. However, the mechanism of deuiquitination in this event remains unknown. We screened the deubiquitinating enzymes family, finding that STAMBP had great influence on the transcriptional activity of E2F1. Moreover, STAMBP can bind to E2F1 protein. Then we preliminaryly explored the role of STAMBP in bladder cancer, to find that overepression of STAMBP promoted proliferation of bladder cancer cells. What's more, we detected E2F1 recognition sequence in STAMBP promoter region. According to these findings, we hypothesis that, STAMBP deubiquitinated E2F1 by hydrolyzing the K63-linked polyubiquitin chain which maintained the stability of Rb/E2F1/HDAC1 complex, to relieve the transcriptional inhibition of Rb and HDAC1. On the other hand, E2F1 also transcriptionally activates the expression of STAMBP to form positive feedback and magnifies the signal to promote tumorigenesis and development of bladder cancer. The purpose of this project is to clarify the expression and function of STAMBP in bladder cancer, and to explore the molecular mechanism that STAMBP deubiquitinates E2F1 to activate the RB/E2F1 pathway, and how E2F1 transcriptionally activates STAMBP, which form a positive feedback loop. All these together would provide the basis for regarding STAMBP as a potential therapeutic target for bladder cancer.

Rb/E2F1通路在膀胱癌发生发展中极为重要。最新研究表明K63位多聚泛素链可作为脚手架维系E2F1与Rb、HDAC1之间的相互作用，但与之相对的去泛素化机制尚不明确，我们通过筛选去泛素酶家族发现STAMBP对E2F1转录活性影响极大，且该蛋白能与E2F1结合。初步探索STAMBP在膀胱癌中的功能发现过表达STAMBP促进增殖。此外，STAMBP启动子区发现E2F1识别序列。据此我们推测：STAMBP去泛素化E2F1，切除维系Rb/E2F1/HDAC1复合物稳定的K63泛素链，解除Rb和HDAC1对E2F1转录活性的抑制，E2F1还转录激活STAMBP形成正反馈而放大信号，促进膀胱癌发生发展。本项目旨在明确STAMBP在膀胱癌中的表达及功能，阐明STAMBP去泛素化E2F1激活Rb/E2F1通路及E2F1转录激活STAMBP的正反馈环路机制，为将STAMBP作为膀胱癌潜在治疗靶点提供依据。

本课题组研究发现K63位去泛素化酶STAMBP在膀胱癌中表达异常增高（相较正常膀胱组织和膀胱癌旁组织）。通过GAIN/LOSS of FUNCTION策略，无论体内外实验中，我们均证实STAMBP过表达STAMBP促进膀胱肿瘤细胞增殖、迁移，而敲除STAMBP则起抑制作用。本课题组进一步通过luciferase screening实验筛选到去泛素酶STAMBP对E2F1转录活性影响极大，RNAseq发现在膀胱癌细胞系中敲除STAMBP基因后，Rb/E2F1信号通路受到极大的抑制，Realtime PCR也进一步证实STAMBP ko之后下游信号通路的变化。进一步IP实验证实，STAMBP能与RB1、E2F1和HDAC1结合，且STAMBP能与HDAC1竞争性结合E2F1。本项目明确了STAMBP在膀胱癌中的表达及功能，阐明了STAMBP激活Rb/E2F1通路机制，为将STAMBP作为膀胱癌潜在治疗靶点提供依据。