OTUB1 inhibits the ubiquitination and degradation of FOXM1 in breast cancer and epirubicin resistance.
OTUB1 inhibits the ubiquitination and degradation of FOXM1 in breast cancer and epirubicin resistance.
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DOI：
10.1038/onc.2015.208
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发表时间：
2016-03-17
期刊：
影响因子：
8
通讯作者：
Lam EW
中科院分区：
文献类型：
--
作者：
Karunarathna U;Kongsema M;Zona S;Gong C;Cabrera E;Gomes AR;Man EP;Khongkow P;Tsang JW;Khoo US;Medema RH;Freire R;Lam EW
叉头转录因子FOXM1在DNA损伤反应中起关键作用，其非调控过表达与乳腺癌的遗传毒性耐药有关。然而，关于FOXM1表达受遗传毒性药物调控的翻译后机制以及它们在耐药细胞中是如何解除调控的，人们知之甚少。最初的免疫共沉淀研究证实了先前的蛋白质组分析，发现OTUB1是一种新的FOXM1相互作用蛋白。Western印迹分析显示，经基因毒剂处理后，MCF-7细胞中OTUB1和FOXM1的表达均降低，但在耐药细胞中的表达相对稳定。在MCF-7细胞中，当OTUB1被siRNA耗尽时，FOXM1的表达减少，而随着OTUB1的过度表达，FOXM1的表达增加，这表明OTUB1正向调节FOXM1的表达。一致的，免疫共沉淀实验表明FOXM1的表达与OTUB1结合有关，但与蛋白质降解相关的Lys-48连接的泛素链的结合呈负相关。野生型(WT)OTUB1的过表达，而不是OTUB1(C91S)突变体的过表达，扰乱了Lys48连接的泛素结合物在FOXM1上的形成。重要的是，在蛋白质合成抑制剂放线菌酮处理的MCF-7细胞中，siRNA下调OTUB1导致FOXM1的周转增加，而WT OTUB1的过度表达，而不是OTUB1(C91S)突变体，显著延长FOXM1的半衰期。此外，增殖和克隆形成实验也表明OTUB1可以通过靶向FOXM1来提高细胞的增殖率和对表阿霉素的耐药性，因为OTUB1对FOXM1缺陷细胞的作用很小。在乳腺癌患者样本中FOXM1和OTUB1表达之间的显著相关性进一步强调了OTUB1调节FOXM1的生理学相关性。COX回归生存分析表明，OTUB1的过度表达与较差的预后有关，特别是在接受化疗的患者。总之，这些数据表明，OTUB1限制了乳腺癌中FOXM1的泛素化和降解，并在遗传毒剂耐药性中发挥了关键作用。
The forkhead transcription factor FOXM1 has a key role in DNA damage response, and its deregulated overexpression is associated with genotoxic drug resistance in breast cancer. However, little is known about the posttranslational mechanisms by which FOXM1 expression is regulated by genotoxic agents and how they are deregulated in resistant cells. Initial co-immunoprecipitation studies verified previous proteomic analysis finding that the OTUB1 is a novel FOXM1-interacting protein. Western blot analysis showed that both OTUB1 and FOXM1 expression reduced upon genotoxic agent treatment in MCF-7 cells, but remained relatively constant in resistant cells. FOXM1 expression reduced upon OTUB1 depletion by siRNA and increased with OTUB1 overexpression in MCF-7 cells, arguing that OTUB1 positively regulates FOXM1 expression. In agreement, co-immunoprecipitation experiments demonstrated that FOXM1 expression is associated with OTUB1 binding but inversely correlates with conjugation to the protein degradation-associated Lys-48-linked ubiquitin-chains. Overexpression of wild-type (WT) OTUB1, but not the OTUB1(C91S) mutant, disrupted the formation of Lys48-linked ubiquitin-conjugates on FOXM1. Importantly, knockdown of OTUB1 by siRNA resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide, whereas overexpression of WT OTUB1, but not the OTUB1(C91S) mutant, significantly enhances the half-life of FOXM1. In addition, proliferative and clonogenic assays also show that OTUB1 can enhance the proliferative rate and epirubicin resistance through targeting FOXM1, as OTUB1 has little effect on FOXM1-deficient cells. The physiological relevance of the regulation of FOXM1 by OTUB1 is further underscored by the significant correlations between FOXM1 and OTUB1 expression in breast cancer patient samples. Cox-regression survival analysis indicates that OTUB1 overexpression is linked to poorer outcome in particular in patients treated with chemotherapy. Collectively, these data suggest that OTUB1 limits the ubiquitination and degradation of FOXM1 in breast cancer and has a key role in genotoxic agent resistance.