Cullin2-FEM1B泛素连接酶介导抑癌蛋白RhoB泛素化降解促进乳腺癌恶性表型的机制研究

The degradation of tumor suppressor regulated by ubiquitin-proteasome system is an important mechanism for tumor development. As a tumor suppressor, RhoB is significantly down-regulated in breast cancer, and its loss promotes the progression of breast cancer. However, the underlying mechanism of RhoB down-regulation in breast cancer has not been fully elucidated. We demonstrate that CRL2 ubiquitin E3 ligase, a member of Cullin-RING Ligase family, promotes the ubiquitination and degradation of RhoB. Moreover, knockdown of FEM1B, a substrate receptor protein of CRL2, inhibits the degradation of RhoB. Accordingly, we hypothesize that FEM1B specifically binds to RhoB and promotes its ubiquitination and degradation, which results in the downregulation of p27 expression, thereby promoting the malignant progression of breast cancer. On the contrary, the inhibition of Cullin2-FEM1B pathway would induce the accumulation of RhoB and inhibit the malignancy of breast cancer cells, leading to a potential anticancer effect. To test this hypothesis, we will carry out intensive studies in this project to elucidate the underlying mechanism of RhoB degradation mediated by Cullin2-FEM1B and its regulatory role in breast cancer development. Thus our project would provide a clue to the identification of anti-cancer targets for breast cancer.

泛素化修饰介导的抑癌蛋白降解是促进肿瘤发生发展的重要机制。研究表明，抑癌蛋白RhoB在乳腺癌中显著低表达，其缺失促进乳腺癌发生发展，但RhoB在乳腺癌中表达下调的分子机制尚未阐明。我们发现，CRL2（Cullin-RING Ligase 2）多亚基泛素连接酶可促进RhoB的泛素化降解，但该复合物中直接结合RhoB的底物识别亚基有待鉴定。进一步研究发现，干扰CRL2复合体新型底物识别蛋白FEM1B可诱导RhoB降解受阻。据此，我们提出假说：FEM1B特异性结合RhoB并促进其泛素化修饰与降解，继而下调其下游效应分子p27的表达，从而促进乳腺癌的恶性进展；相反，靶向灭活FEM1B则可诱导RhoB蛋白积聚，抑制乳腺癌细胞恶性表型，发挥抗癌效应。本项目旨在检验该假说，阐明Cullin2-FEM1B促进RhoB泛素化降解的机制及其对乳腺癌发生发展的调控作用，为乳腺癌抗癌分子靶点鉴定提供科学依据。

CRL泛素连接酶是细胞内最大的多亚基泛素连接酶家族，调控约20%经泛素-蛋白酶体系统介导的底物的降解。CRL泛素连接酶异常活化促进抑癌蛋白降解是重要的促癌机制之一。CRL2泛素连接酶复合体由Cullin2、RBX1、接头蛋白Elongin B/Elongin C（EloB/EloC）以及不同的底物识别蛋白组成，参与调控肿瘤发生发展。FEM1B被报道能够作为CRL2的底物识别蛋白发挥作用，但Cullin2-FEM1B泛素连接酶调控抑癌蛋白底物降解却尚未有报道。RhoB是重要的抑癌蛋白，在多种肿瘤中显著下调。RhoB缺失在乳腺癌发生发展过程中发挥重要的促进作用，但在乳腺癌中RhoB表达下调的分子机制及促癌机理尚未充分阐明。.我们通过siRNA筛选发现，FEM1B能够作为介导RhoB降解的特异性底物识别蛋白发挥作用，并通过开展研究探索Cullin2-FEM1B调控RhoB降解的分子机制及其对乳腺癌恶性表型的影响。研究发现：（1）下调FEM1B诱导RhoB蛋白积聚，抑制RhoB蛋白降解；（2）FEM1B能直接结合RhoB，并介导其发生泛素化修饰；（3）FEM1B在乳腺癌中高表达且其高表达提示预后不良；（4）下调FEM1B抑制乳腺癌细胞增殖及小鼠皮下瘤生长；（5）下调FEM1B抑制乳腺癌细胞增殖是通过RhoB介导的。.综上所述，本项目鉴定Cullin2-FEM1B泛素连接酶介导抑癌蛋白RhoB泛素化降解，而在乳腺癌中靶向灭活FEM1B能够通过上调RhoB表达从而抑制乳腺癌增殖。本项目为靶向灭活Cullin2-FEM1B泛素连接酶诱导抑癌蛋白RhoB积聚从而杀伤乳腺癌细胞的治疗策略提供了科学依据。