School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, China 325035; Nelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, NY 10987, USA.
School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, China 325035.
Neoplasia. 2017 Sep;19(9):672-683. doi: 10.1016/j.neo.2017.06.002. Epub 2017 Aug 1.
BACKGROUND: Since invasive bladder cancer (BC) is one of the most lethal urological malignant tumors worldwide, understanding the molecular mechanisms that trigger the migration, invasion, and metastasis of BC has great significance in reducing the mortality of this disease. Although RelA/p65, a member of the NF-kappa B transcription factor family, has been reported to be upregulated in human BCs, its regulation of BC motility and mechanisms have not been explored yet. METHODS: NF-κBp65 expression was evaluated in N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced high invasive BCs by immunohistochemistry staining and in human BC cell lines demonstrated by Western Blot. The effects of NF-κBp65 knockdown on BC cell migration and invasion, as well as its regulated RhoGDIα and FBW7, were also evaluated in T24T cells by using loss- and gain-function approaches. Moreover, the interaction of FBW7 with RhoGDIα was determined with immunoprecipitation assay, while critical role of ubiquitination of RhoGDIα by FBW7 was also demonstrated in the studies. RESULTS: p65 protein was remarkably upregulated in the BBN-induced high invasive BCs and in human BC cell lines. We also observed that p65 overexpression promoted BC cell migration by inhibiting RhoGDIα expression. The regulatory effect of p65 on RhoGDIα expression is mediated by its upregulation of FBW7, which specifically interacted with RhoGDIα and promoted RhoGDIα ubiquitination and degradation. Mechanistic studies revealed that p65 stabilizing the E3 ligase FBW7 protein was mediated by its attenuating pten mRNA transcription. CONCLUSIONS: We demonstrate that p65 overexpression inhibits pten mRNA transcription, which stabilizes the protein expression of ubiquitin E3 ligase FBW7, in turn increasing the ubiquitination and degradation of RhoGDIα protein and finally promoting human BC migration. The novel identification of p65/PTEN/FBW7/RhoGDIα axis provides a significant insight into understanding the nature of BC migration, further offering a new theoretical support for cancer therapy.
背景：由于浸润性膀胱癌（BC）是全球最致命的泌尿系统恶性肿瘤之一，因此了解触发 BC 迁移、侵袭和转移的分子机制对于降低该疾病的死亡率具有重要意义。虽然 NF-κB 转录因子家族的成员 RelA/p65 已在人类 BC 中上调，但尚未探索其对 BC 运动性的调节机制。
方法：通过免疫组织化学染色评估 N-丁基-N-（4-羟丁基）亚硝胺（BBN）诱导的高侵袭性 BC 中的 NF-κBp65 表达，并通过 Western Blot 评估人 BC 细胞系中的 NF-κBp65 表达。还通过使用缺失和获得功能方法，在 T24T 细胞中评估 NF-κBp65 敲低对 BC 细胞迁移和侵袭的影响，以及其对 RhoGDIα 和 FBW7 的调节作用。此外，通过免疫沉淀测定确定 FBW7 与 RhoGDIα 的相互作用，并且还在研究中证明了 FBW7 对 RhoGDIα 的泛素化的关键作用。
结果：p65 蛋白在 BBN 诱导的高侵袭性 BC 和人 BC 细胞系中显著上调。我们还观察到，p65 过表达通过抑制 RhoGDIα 表达促进 BC 细胞迁移。p65 对 RhoGDIα 表达的调节作用是由其对 FBW7 的上调介导的，FBW7 特异性与 RhoGDIα 相互作用并促进 RhoGDIα 的泛素化和降解。机制研究表明，p65 通过减弱 pten mRNA 转录来稳定 E3 连接酶 FBW7 蛋白的表达。
结论：我们证明 p65 过表达抑制 pten mRNA 转录，从而稳定泛素 E3 连接酶 FBW7 的蛋白表达，进而增加 RhoGDIα 蛋白的泛素化和降解，最终促进人 BC 迁移。p65/PTEN/FBW7/RhoGDIα 轴的新鉴定为理解 BC 迁移的本质提供了重要的见解，为癌症治疗提供了新的理论支持。
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