The E3 ligase RBCK1 reduces the sensitivity of ccRCC to sunitinib through the ANKRD35-MITD1-ANXA1 axis

Despite the promise of targeted tyrosine kinase inhibitors (TKIs), such as sunitinib, in the extension of survival time in patients with clear cell renal cell carcinoma (ccRCC) progression or metastasis, the patients eventually succumb to inevitable drug resistance. Protein degradation executed by the ubiquitin-dependent proteasome system played an important role in determining the sensitivity of ccRCC to sunitinib. Here, we applied the bioinformatic analysis to identify that E3 ligase RBCK1 was elevated in the sunitinib-resistant renal cancer cell lines or patient specimens. The subsequent in vitro or in vivo studies demonstrated that RBCK1 contributed to decreasing the sensitivity of ccRCC to sunitinib. Then, we showed that inhibition of RBCK1 activated the AKT and MAPK signaling pathways, which might be one of the main reasons why RBCK1 induces sunitinib resistance in ccRCC cells. Mechanistically, our results indicated that RBCK1 promotes the degradation of ANKRD35 and that ANKRD35 destabilizes MITD1 by binding with SUMO2 in ccRCC cells. In addition, we showed that the RBCK1-ANKRD35-MITD1-ANXA1 axis regulates the phosphorylation of AKT and ERK and contributes to the dysregulation of sunitinib in ccRCC cells. Therefore, we identified a novel mechanism for regulating the sensitivity of sunitinib in ccRCC. Therefore, we elucidated a novel mechanism by which RBCK1 regulates sunitinib sensitivity in ccRCC. Overall design: Comparative gene expression profiling analysis of RNA-seq data for the ccRCC cell after kncokdown of RBCK1, ANKRD35 or MITD1 respectively.

尽管舒尼替尼（sunitinib）这类酪氨酸激酶抑制剂（TKIs）在延长透明细胞肾细胞癌（ccRCC）进展或转移患者的生存期方面颇具应用前景，但此类患者最终仍会出现不可避免的耐药性。由泛素依赖性蛋白酶体系统（ubiquitin-dependent proteasome system）介导的蛋白质降解，在决定ccRCC对舒尼替尼的敏感性过程中发挥关键作用。本研究通过生物信息学分析，发现在舒尼替尼耐药肾癌细胞系及患者标本中，E3泛素连接酶（E3 ligase）RBCK1的表达水平显著升高。后续的体外（in vitro）与体内（in vivo）实验证实，RBCK1会降低ccRCC对舒尼替尼的敏感性。研究表明，抑制RBCK1可激活AKT与丝裂原活化蛋白激酶（MAPK）信号通路，这可能是RBCK1诱导ccRCC细胞产生舒尼替尼耐药的核心机制之一。从机制层面分析，本研究结果显示，RBCK1可促进ANKRD35的降解，而ANKRD35可通过与小泛素相关修饰物2（SUMO2）结合，降低ccRCC细胞内MITD1的稳定性。此外，本研究证实RBCK1-ANKRD35-MITD1-膜联蛋白A1（ANXA1）信号轴可调控AKT及ERK的磷酸化水平，参与ccRCC细胞对舒尼替尼的应答异常。综上，本研究阐明了一种调控ccRCC对舒尼替尼敏感性的全新机制。整体实验设计：分别敲低RBCK1、ANKRD35或MITD1后，对ccRCC细胞的RNA测序（RNA-seq）数据开展比较基因表达谱分析。