GBM中自噬诱导HERC3泛素化降解Smad7激活TGF-β通路并介导EMT和放化疗抵抗的机制研究

Epithelial-to-mesenchymal transition (EMT) is the most important reason for migration, invasion and chemoradio-therapy resistant of glioblastoma (GBM). Our preliminary work proved that Temozolomide (TMZ) induced autophagy and EMT in GBM cell line U87, which accompanyed with the downregulation of the Smad7 expression (Inhibitory-smad), and upregulation of the Smad3 (Receptory-smad) level. Meanwhile, the E3 ubiquitin ligase HERC3 expression was upregulated significantly as well. On the other hand, patient-derived adult GBM cells were separately isolated from peripheral “normal” tissue (PNCs) and enhancing core lesions (ECs) of T1+Gadolinium sequence. Single clone culture was conducted in ECs and U87 cells to isolate clones with distinct EMT phenotypes. As results, cell lines with higher and lower EMT stauts were established. Proteomics assay displayed the same tendency between EMT status and the protein level of HERC3 and Smads. Further detection revealed that HERC3 might be able to promote the Smad7 degradation via ubiquitination, and then upregulated the p-Smad2/3 level. The 3-MA and Chloroquine was able to inhibite this function, but MG132 proteasome inhibitor couldn’t. Kaplan-meier survival analysis of 46 GBM patients indicated that HERC3 higher expression cases were correlated with poorer clinical prognosis for TMZ chemoradio-theray. So we proposed the hypothesis that under the high autophagic condition induced by TMZ, the ubiquitination function of HERC3 to the Smad7 was promoted to activate the TGF-β/Smad3 pathway, and consequently, EMT and chemoradio-resistance occurred. So this stduy took the aim to investigate and prove the above molecular mechanism in details, and was hoping to provide the experimental evidence and molecular-based therapeutic targets for GBM chemoradio-therapy resistant.

上皮间质化（EMT）是GBM迁移侵袭和放化疗抵抗的重要原因，我们已发现替莫唑胺（TMZ）能诱导U87细胞自噬并促使EMT发生，同时Smad7（I-smad）表达下调，Smad3（R-smad）上调，而E3泛素连接酶HERC3也明显上调。另对U87和GBM中心及瘤周细胞行单克隆培养，挑选EMT强和弱的细胞株，对比不同EMT状态细胞显示了相似的HERC3和Smads变化趋势。进一步发现HERC3可能以Smad7为泛素化底物，抑制其表达并上调p-Smad2/3水平，而3-MA和氯喹能逆转该作用，但蛋白酶体抑制剂MG132不能。生存分析也显示HERC3高表达GBM病例，TMZ联合放化疗的预后较差。故我们推测：在TMZ诱导的高自噬条件下，HERC3加速对Smad7的泛素化降解，激活TGF-β/Smad3通路，促进了EMT和治疗抵抗的发生。本课题拟探明上述具体机制，以期为GBM放化疗抵抗提供治疗靶点。

上皮间质化(EMT)是GBM迁移侵袭和放化疗抵抗的重要原因，本项目中我们发现替莫唑胺(TMZ )能诱导U87细胞自噬并促使EMT发生，同时Smad7(I-smad)表达下调，Smad3(R-smad)上调，而E3泛素连接酶HERC3也明显上调。在对U87和GBM中心及瘤周细胞行单克隆培养，挑选EMT强和弱的细胞株，对比不同EMT状态细胞显示了相似的HERC3和Smads变化趋势。进一步发现HERC3可能以Smad7为泛素化底物，抑制其表达并上调p-Smad2/3水平，而3-MA和氯喹能逆转该作用，但蛋白酶体抑制剂MG132不能。生存分析也显示HERC3高表达GBM病例，TMZ联合放化疗的预后较差。进一步我发现在TMZ诱导的高自噬条件下，HERC3加速对Smad7的泛素化降解，而其降解通路是通过自噬溶酶体途径，从而激活了TGF-β/Smad3通路，并促进了EMT和治疗抵抗的发生。而动物体内实验也证实，HERC3高表达增加了GBM肿瘤细胞的TMZ耐药性，使得肿瘤更大，细胞侵袭能力更强。同时结合临床患者和TCGA数据分析，发现联合MGMT表达情况，HERC3能预测患者的临床预后。本课题的完成，揭示了GBM中自噬诱导EMT是放化疗抵抗的重要机制，而HERC3/Smad7通路在其中起到了关键性的分子作用，并为GBM放化疗抵抗的增敏治疗提供了靶点。