ARHGAP24 represses β-catenin transactivation-induced invasiveness in hepatocellular carcinoma mainly by acting as a GTPase-independent scaffold.
ARHGAP24 represses β-catenin transactivation-induced invasiveness in hepatocellular carcinoma mainly by acting as a GTPase-independent scaffold.
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ARHGAP24 主要通过充当不依赖 GTP 的支架来抑制 β-catenin 反式激活诱导的肝细胞癌侵袭性
DOI：
10.7150/thno.72134
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发表时间：
2022
期刊：
影响因子：
12.4
通讯作者：
Guo, Wei
中科院分区：
文献类型：
--
作者：
Yang, Wenjing;Wang, Beili;Yu, Qian;Liu, Te;Li, Tong;Tian, Tongtong;Jin, Anli;Ding, Lin;Chen, Wei;Wang, Hao;Xian, Jingrong;Pan, Baishen;Zhou, Jian;Fan, Jia;Yang, Xinrong;Guo, Wei
基本原理：越来越多的证据表明Rho-GT3-activating proteins（RhoGAPs）对肿瘤细胞的增殖和转移具有抑制作用。然而，目前还没有系统研究RhoGAP的临床意义以及ARHGAP 24在肝细胞癌（HCC）中的作用。研究方法：通过使用癌症基因组图谱和基因表达Omnibus数据库研究RhoGAP表达与HCC预后之间的关系。逆转录-聚合酶链反应、免疫印迹和免疫组化染色检测ARHGAP 24的表达。此外，进行体外测定，包括细胞计数试剂盒-8、集落形成、伤口愈合和Transwell测定，以及体内肿瘤生长和肺转移评估，以评估ARHGAP 24在HCC中的生物学功能。通过液相色谱-串联质谱、免疫共沉淀、GT3激活、泛素化和荧光素酶报告基因分析以及生物信息学分析，深入了解ARHGAP 24的肿瘤抑制功能的机制。结果如下：ARHGAP 24在HCC组织中的表达显著降低，低ARHGAP 24表达是HCC患者无进展生存的独立预后不良指标。ARHGAP 24过表达显著抑制细胞增殖、迁移和侵袭，而ARHGAP 24敲低则产生相反的效果。通过基因集富集分析（Gene Set Enrichment Analysis，GSEA）发现ARHGAP 24主要通过减弱β-catenin的反式激活来抑制肝癌细胞的增殖和侵袭，阻断β-catenin信号通路可以有效地消除ARHGAP 24敲低对肝癌细胞的促进作用。值得注意的是，ARHGAP 24的GAP缺陷突变体表现出与野生型相似的抑制作用，表明ARHGAP 24的抑制功能不依赖于其RhoGAP活性。此外，我们确定了丙酮酸激酶M2（PKM 2）作为ARHGAP 24的新结合伴侣，其招募了一种新的E3连接酶（WWP 1），随后促进PKM 2降解。WWP 1敲低显著降低了ARHGAP 24的抑制功能，并且ARHGAP 24的C末端片段（氨基酸329 - 430和631 - 748）分别直接结合WWP 1和PKM 2（氨基酸388 - 531）。结论：我们的数据表明，ARHGAP 24可能是一个独立的肝癌预后指标。它是HCC的关键抑制因子，募集WWP 1用于PKM 2降解。靶向ARHGAP 24/WWP 1/PKM 2/β-catenin轴可能为HCC的预防和治疗提供新的见解。
Rationale: Accumulating evidence shows that Rho-GTPase-activating proteins (RhoGAPs) exert suppressive roles in cancer cell proliferation and metastasis. However, no study has systematically investigated the clinical significance of RhoGAPs and analyzed the functions of ARHGAP24 in hepatocellular carcinoma (HCC). Methods: The relationship between RhoGAP expression and HCC prognosis was investigated via using The Cancer Genome Atlas and Gene Expression Omnibus databases. ARHGAP24 expression was detected by reverse transcription-polymerase chain reaction, western blot and immunohistochemistry staining assays. Moreover, in vitro assays including cell counting kit-8, colony formation, wound healing and Transwell assays, and in vivo tumor growth and pulmonary metastases evaluations were conducted to evaluate the biological function of ARHGAP24 in HCC. Liquid chromatography-tandem mass spectrometry, co-immunoprecipitation, GTPase activation, ubiquitination, and luciferase reporter assays and bioinformatics analysis were carried out to gain insights into the mechanisms underlying the tumor-suppressive function of ARHGAP24. Results: ARHGAP24 expression was dramatically decreased in HCC tissues, and low ARHGAP24 expression was an independent poor prognostic indicator for progression-free survival in HCC patients. ARHGAP24 overexpression significantly inhibited cell proliferation, migration and invasion, while knockdown of ARHGAP24 exerted the opposite effects. Through Gene Set Enrichment Analysis (GSEA), we found ARHGAP24 mainly suppressed HCC cell proliferation and invasion by attenuating β-catenin transactivation and blocking β-catenin signaling could effectively abolish the promotional effects of ARHGAP24 knockdown in HCC cells. Notably, GAP-deficient mutant of ARHGAP24 exerted similar inhibitory effects as the wild-type did, indicating suppressive function of ARHGAP24 was independent of its RhoGAP activity. Moreover, we identified pyruvate kinase M2 (PKM2) as a new binding partner of ARHGAP24, which recruited a novel E3 ligase (WWP1) and subsequently promoted PKM2 degradation. WWP1 knockdown significantly reduced the inhibitory function of ARHGAP24, and the C-terminal fragments of ARHGAP24 (amino acids 329 - 430 and 631 - 748) bound directly to WWP1 and PKM2 (amino acids 388 - 531), respectively. Conclusions: Our data indicate that ARHGAP24 may be an independent prognostic indicator for HCC. It is a critical suppressor of HCC that recruits WWP1 for PKM2 degradation. Targeting the ARHGAP24/WWP1/PKM2/β-catenin axis may provide new insights into HCC prevention and treatment.
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