靶向CACYBP活化RNF41/ErbB3通路抑制肝癌生长的分子机制及其潜在应用

Despite the available treatments including traditional therapies and multiple kinase inhibitor-Sorafenib, the overall survival of hepatocellular carcinoma (HCC) patients remains poor. Therefore, new biomarkers and therapeutic options are urgently required for improving the survival of HCC patients. Recently, we examined four microarray datasets from Oncomine and TCGA database, a large number of human tissue samples, and cohort with long-term follow-up. We found CACYBP (Calcyclin-binding protein/Siah-1-interacting protein) was significantly overexpressed in the majority of cancer tissues compared with adjacent non-neoplastic controls. It was also significantly associated with poor prognosis. Silencing CACYBP in liver cancer cells decreased cellular growth, clonal formation, and epithelial mesenchymal transition. Analyses from proteomics and immunoprecipitation indicated CACYBP could bond to E3 ubiquitin ligase RNF41, and thus upregulated its downstream oncogene ErbB3. The inactivation of RNF41/ErbB3 pathway is often implicated in carcinogenesis. These findings suggest that blockage of CACYBP to activate RNF41/ErbB3 pathway may enhance the protein degradation of oncogenes, and may represent a novel therapeutic potential. However, the oncogenic mechanisms remain unknown. In this study, we aim to (1) identify the role of CACYBP/RNF41/ErbB3 in HCC development and tumor progression as well as the related target genes, (2) elucidate the molecular mechanism for inhibiting cell growth and transition upon activating RNF41/ErbB3 pathway in CACYBP-silencing cancer cells, and (3) evaluate the anticancer effect of CACYBP inhibitor. The successful completion of this work will not only identify a novel marker and cancer pathway for HCC, but also facilitate the development of a novel strategy based on anti-CACYBP inhibitor monotherapy or combination therapy.

发现和鉴定新治疗靶点是改善肝癌预后的关键，最近，我们通过生物信息大数据、临床肝癌样本、长期随访队列发现并证实了CACYBP在肝癌中高表达，是肝癌不良预后的独立相关因素；体外敲除CACYBP可显著抑制癌细胞生长和降低克隆形成及侵袭转移；蛋白组学研究提示CACYBP与E3泛素连接酶RNF41结合并上调其下游ErbB3，而RNF41/ErbB3通路失调在许多肿瘤发生发展起着重要作用；这些发现提示阻断CACYBP活化RNF41/ErbB3通路有助于癌基因降解，但其分子机制及其体内抗肿瘤作用未阐明。本项目中，我们将鉴定CACYBP/RNF41/ErbB3通路在肝癌中的作用，阐明靶向CACYBP活化RNF41/ErbB3通路进而抑制肿瘤生长的分子机制，并在体内评价阻断CACYBP的抗肿瘤作用。项目成功完成不仅阐明肝癌新发基因和分子通路，并为探索靶向CACYBP单药或联合治疗新策略提供科学依据。

发现和鉴定新的诊疗靶点有助于改善肝癌患者临床结局，在本项目中，我们利用生物信息数据库挖掘及肝癌病人组织队列验证发现了CACYBP表达在肝癌中显著上调，通过关联分析和生存分析发现CACYBP高表达肝癌病人具有更高的AFP、AST、ALB水平，且与患者死亡和肿瘤复发显著相关，其总生存时间及无进展生存时间显著缩短，表明CACYBP可能成为肝癌预后预测的重要指标。随后我们选择SK-Hep-1和Huh7分别建立CACYBP稳定敲低的肝癌细胞株，通过体内外肿瘤研究模型证实了敲低CACYBP能显著抑制肝癌细胞的生长增殖能力。机制上，我们发现E3泛素连接酶RNF41通过其C端结构域招募CACYBP，并通过泛素化促进其蛋白降解，该效应能被蛋白酶体抑制剂MG-132或酶活性缺失突变RNF41-D56V所回复。更进一步，CACYBP表达能够促进P27Kip1 Ser10磷酸化水平，使其更多的定位于细胞质中，而RNF41共表达则能够逆转该作用；同时，在CACYBP敲低肝癌细胞中过表达胞质定位的P27Kip1-S10D突变能够部分逆转细胞凋亡，而胞核定位P27Kip1-S10A突变则不能，表明RNF41/CACYBP轴通过Ser10磷酸化状态参与P27Kip1细胞定位调控，削弱其抑制细胞周期的能力，从而导致肝癌细胞快速增殖。综合上述结果，本研究证实了CACYBP对于肝癌预后预测具有一定作用且阐明了其调控肝癌细胞生长增殖的分子机制，有望为开发新型肝癌诊疗策略提供科学依据。

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