Zhao Saili, Wang Xuran, Wu Rui, Wang Fenglan, Tang Xiaoxuan, Chen Junhui, Jiang Runqiu, Kang Wei, Xu Guifang, Wang Lei, Wang Zhangding, Zou Xiaoping, Zhang Bin
Department of Gastroenterology, Nanjing Drum Tower Hospital Clinical College of Xuzhou Medical University, Nanjing China.
Medical School of Nanjing University, Nanjing, China.
J Adv Res. 2024 Dec 11. doi: 10.1016/j.jare.2024.12.017.
We have previously demonstrated that RRP15 (Ribosomal RNA Processing 15 Homolog) was significantly elevated in hepatocellular carcinoma (HCC) and correlated directly with poor prognosis. RRP15 suppression curtails HCC progression through induction of cellular senescence and apoptosis. However, the impact of RRP15 on the precise therapeutic potential of lenvatinib has remained underexplored.
To investigate the relationship between RRP15 expression and sensitivity of lenvatinib in HCC treatment, and also explore the potential of targeting RRP15 by lenvatinib to inhibit HCC progression.
RRP15 and KBTBD8 (Kelch Repeat and BTB Domain Containing 8) expression was examined using western blot and immunohistochemistry. Cell viability, proliferation, migration and invasion as well as apoptosis were assessed using CCK-8, clonogenic assays, transwell, TUNEL (Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling) and Annexin V staining assays. The interaction between RRP15 and KBTBD8 was identified through pull-down and mass spectrometry analysis and further validated by immunofluorescence and co-immunoprecipitation assays. RRP15 ubiquitination and degradation were assessed using cycloheximide treatment, plasmid transfection and co-immunoprecipitation, followed by western blot analysis. Tail vein injection lung metastasis model was performed to determine tumor metastasis in vivo.
We reveled a correlation between RRP15 downregulation and enhanced sensitivity to lenvatinib, presenting marked suppression of metastasis and invasiveness. Proteomic analyses and subsequent validation disclosed the pivotal role of the E3 ubiquitin ligase KBTBD8 in mediating the ubiquitination and subsequent degradation of RRP15 protein post-lenvatinib treatment in HCC cells. KBTBD8 inhibition stalled RRP15 ubiquitination and degradation, while its overexpression accelerated these processes. Moreover, RRP15 overexpression fosters HCC cell proliferation and metastasis, a pathological effect mitigated by KBTBD8 overexpression. In vivo experiments further validate the role of lenvatinib in promoting RRP15 degradation via KBTBD8 upregulation.
Our study elucidated a previously unidentified mechanism of lenvatinib action and identified the RRP15-KBTBD8 axis as a novel therapeutic target in HCC, offering new avenues for treatment strategies in combating HCC.
我们之前已经证明，核糖体RNA加工15同源物（RRP15）在肝细胞癌（HCC）中显著升高，并且与不良预后直接相关。RRP15的抑制通过诱导细胞衰老和凋亡来抑制HCC进展。然而，RRP15对乐伐替尼确切治疗潜力的影响尚未得到充分研究。
研究RRP15表达与乐伐替尼在HCC治疗中的敏感性之间的关系，并探讨乐伐替尼靶向RRP15抑制HCC进展的潜力。
采用蛋白质免疫印迹法和免疫组织化学法检测RRP15和含kelch重复序列和BTB结构域蛋白8（KBTBD8）的表达。使用CCK-8、克隆形成试验、Transwell试验、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）和膜联蛋白V染色试验评估细胞活力、增殖、迁移、侵袭以及凋亡情况。通过下拉和质谱分析鉴定RRP15与KBTBD8之间的相互作用，并通过免疫荧光和免疫共沉淀试验进一步验证。使用放线菌酮处理、质粒转染和免疫共沉淀，随后进行蛋白质免疫印迹分析，评估RRP15的泛素化和降解情况。通过尾静脉注射建立肺转移模型以确定体内肿瘤转移情况。
我们发现RRP15下调与对乐伐替尼的敏感性增强之间存在相关性，表现为转移和侵袭性受到显著抑制。蛋白质组学分析及后续验证揭示了E3泛素连接酶KBTBD8在介导乐伐替尼处理后HCC细胞中RRP15蛋白的泛素化及随后的降解过程中起关键作用。抑制KBTBD8会阻止RRP15的泛素化和降解，而其过表达则会加速这些过程。此外，RRP15过表达促进HCC细胞增殖和转移，而KBTBD8过表达可减轻这种病理效应。体内实验进一步验证了乐伐替尼通过上调KBTBD8促进RRP15降解的作用。
我们的研究阐明了乐伐替尼作用的一种先前未被识别的机制，并确定RRP15-KBTBD8轴是HCC中的一个新的治疗靶点，为对抗HCC的治疗策略提供了新途径。
