ASAP2 interrupts c-MET-CIN85 interaction to sustain HGF/c-MET-induced malignant potentials in hepatocellular carcinoma.
ASAP2 interrupts c-MET-CIN85 interaction to sustain HGF/c-MET-induced malignant potentials in hepatocellular carcinoma.
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ASAP2阻断c-MET-CIN85相互作用以维持HGF/c- met诱导的肝细胞癌的恶性潜能。
DOI：
10.1186/s40164-023-00393-3
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发表时间：
2023-04-15
影响因子：
10.9
通讯作者：
Lu, Ren-Quan
中科院分区：
文献类型：
--
作者：
Ma, Xiao-Lu;Nie, Yan-Yan;Xie, Su-Hong;Zheng, Hui;Tong, Ying;Wang, Yan-Chun;Yan, Tian-Qing;Meng, Xin;Cao, Jia-Zhen;Tang, Wei-Guo;Guo, Lin;Lu, Ren-Quan
肝细胞生长因子（HGF）/c-MET信号的持续激活是肝细胞癌（HCC）进展的主要驱动因素，但其潜在机制尚不清楚。具有SH 3结构域、锚蛋白重复序列和PH结构域2（ASAP 2）的ArfGAP据报道可以激活GTP酶并促进受体酪氨酸激酶信号传导。然而，ASAP 2在HCC中的确切作用，特别是c-MET活化，仍然是难以捉摸的。使用qRT-PCR、蛋白质印迹（WB）分析和免疫组织化学染色定量HCC组织和细胞中的ASAP 2表达水平。细胞计数试剂盒-8（CCK-8）和集落形成试验用于评估细胞增殖率。进行流式细胞术测定以评估凋亡率。进行伤口愈合和Transwell测定以确定细胞迁移和侵袭能力。还检查了上皮-间质转化（EMT）相关标志物表达水平。皮下植入和尾静脉注射模型分别用于体内生长和转移评估。进行癌症基因组图谱和STRING数据集的生物信息学分析以探索ASAP 2下游信号传导。分别进行免疫共沉淀和环己酰亚胺追踪实验来评估蛋白质-蛋白质相互作用和蛋白质半衰期。ASAP 2在肝癌组织中的表达水平高于正常肝组织，且预后不良。敲低ASAP 2显著损害细胞增殖、迁移和侵袭能力，但促进体外HCC细胞凋亡。然而，ASAP 2的过表达实现了相反的效果。体内实验证实ASAP 2能促进肝癌细胞生长和肺转移。有趣的是，ASAP 2是触发EMT的关键。基因集富集分析表明，c-MET信号转导在ASAP 2高HCC病例中大大富集。此外，c-MET信号传导活性在ASAP敲低后显著降低，通过c-MET、p-AKT和p-ERK 1/2蛋白水平降低证明。重要的是，ASAP 2敲低有效地减弱了HGF/c-MET信号诱导的恶性表型。c-MET和ASAP 2表达水平在我们的队列中呈正相关。从机制上讲，ASAP 2可以直接结合CIN 85，从而破坏其与c-MET的相互作用，并因此可以拮抗CIN 85诱导的c-MET内化和溶酶体介导的降解。值得注意的是，敲低CIN 85可以挽救所观察到的由ASAP 2敲低引起的抑制作用。这项研究强调了ASAP 2在维持c-MET信号传导中的重要性，这可以促进HCC的进展。在线版本包含补充材料，可通过10.1186/s40164-023-00393-3获得。
Sustained activation of hepatocyte growth factor (HGF)/c-MET signaling is a major driver of hepatocellular carcinoma (HCC) progression, but underlying mechanism is unclear. ArfGAP With SH3 Domain, Ankyrin Repeat And PH Domain 2 (ASAP2) can reportedly activate GTPases and promote receptor tyrosine kinase signaling. However, the exact role of ASAP2 in HCC, especially for c-MET activation, also remains elusive. ASAP2 expression levels in HCC tissues and cells were quantified using qRT-PCR, western blot (WB) analysis, and immunohistochemistry staining. Cell counting kit-8 (CCK-8) and colony formation assays were performed to evaluate cell proliferation rates. Flow cytometry assays were conducted to assess apoptosis rates. Wound healing and Transwell assays were performed to determine cell migration and invasion capacities. Epithelial-mesenchymal transition (EMT)-related marker expression levels were also examined. Subcutaneous implantation and tail vein injection models were applied for in vivo growth and metastasis evaluations, respectively. Bioinformatics analyses of The Cancer Genome Atlas and STRING datasets were performed to explore ASAP2 downstream signaling. Co-immunoprecipitation and Cycloheximide chasing experiments were performed to assess protein–protein interactions and protein half-life, respectively. ASAP2 had higher expression levels in HCC tissues than in normal liver, and also predicted poor prognosis. Knocking down ASAP2 significantly impaired cell proliferation, migration, and invasion capacities, but promoted apoptosis in HCC cells in vitro. However, overexpression of ASAP2 achieved the opposite effects. In vivo experiments confirmed that ASAP2 could promote HCC cell growth and facilitate lung metastasis. Interestingly, ASAP2 was essential for triggering EMT. Gene Set Enrichment Analysis demonstrated that c-MET signaling was greatly enriched in ASAP2-high HCC cases. Additionally, c-MET signaling activity was significantly decreased following ASAP knockdown, evidenced by reduced c-MET, p-AKT, and p-ERK1/2 protein levels. Importantly, ASAP2 knockdown effectively attenuated HGF/c-MET signaling-induced malignant phenotypes. c-MET and ASAP2 expression levels were positively correlated in our cohort. Mechanistically, ASAP2 can directly bind to CIN85, thereby disrupting its interaction with c-MET, and can thus antagonize CIN85-induced c-MET internalization and lysosome-mediated degradation. Notably, knocking down CIN85 can rescue the observed inhibitory effects caused by ASAP2 knockdown. This study highlights the importance of ASAP2 in sustaining c-MET signaling, which can facilitate HCC progression. The online version contains supplementary material available at 10.1186/s40164-023-00393-3.
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