ERK1/2/OTUB1/GPX4通路抑制铁死亡引起肾癌对sunitinib耐药的机制研究

The activation of alternative pro-angiogenic ERK1/2 pathway and the epithelial-mesenchymal transition are related to the resistance of sunitinib in renal cell carcinoma. However, the molecular mechanism of the drug resistance of renal mesenchymal-like cells is not clear. Previous studies showed that phospholipid glutathione peroxidase GPX4 inhibited the ferroptosis and maintained the mesenchyme state of drug-resistant cells. Knockdown of GPX4 enhanced the sensitivity of renal cell carcinoma to sunitinib. GPX4 was positively correlated the deubiquitination activity of OTUB1, and OTUB1 was regulated by transcription factor SP1 which was the downstream signalling molecule of ERK1/2. We propose the hypothesis that ERK1/2/OTUB1/GPX4 pathway inhibits ferroptosis and then promotes sunitinib resistance of renal cell carcinoma. This project is aimed to detect the expression of p-ERK1/2, OTUB1 and GPX4 in sunitinib sensitive and resistant renal cancer specimens. The sunitinib-resistant cell lines and lentivirus knockout stable cell lines are constructed to observed the regulation of ferroptosis and sunitinib resistance by this pathway in renal cell carcinoma cells. Luciferase reporter gene, chromatin immunoprecipitation and ubiquitination assay are performed to evaluate the mechanisms of ERK1/2/OTUB1/GPX4 pathway. It provides an important theoretical basis for predicting the therapeutic effect of sunitinib and reversing sunitinib resistance in the treatment of renal cancer.

旁路促血管生成途径ERK1/2激活和上皮间充质转化与肾癌sunitinib耐药相关。但肾癌间充质样细胞耐药的具体分子机制尚不明确。申请者前期研究发现，GPX4抑制铁死亡维持耐药细胞间充质状态，敲减其表达增强肾癌对sunitinib敏感性，GPX4和OTUB1去泛素化活性正相关，OTUB1被ERK1/2下游转录因子SP1调控。据此提出ERK1/2/OTUB1/GPX4通路抑制铁死亡引起肾癌对sunitinib耐药的假说。本项目拟检测sunitinib敏感和耐药肾癌标本中p-ERK1/2、OTUB1和GPX4表达，构建耐药细胞系及慢病毒敲减稳定株，观察该通路抑制铁死亡引起肾癌细胞sunitinib耐药，并通过荧光素酶报告基因、染色质免疫共沉淀、泛素化实验等手段检测ERK1/2/OTUB1/GPX4通路调控机制，以证明该假说，为预测sunitinib治疗疗效和逆转肾癌耐药治疗提供重要的理论依据。

旁路促血管生成途径ERK1/2激活和上皮间充质转化与肾癌sunitinib耐药相关。申请者前期研究发现，GPX4抑制铁死亡维持耐药细间充质状态，敲减其表达增强肾癌对sunitinib敏感性，GPX4和OTUB1去泛素化活性正相关，OTUB1被ERK1/2下游转录因子SP1调控。这些结果初步表明ERK1/2/OTUB1/GPX4通路抑制铁死亡引起肾癌对sunitinib耐药。本项目在前期工作基础上构建耐药细胞系及慢病毒敲减稳定株，观察该通路抑制铁死亡引起肾癌细胞sunitinib耐药，并通过免疫共沉淀、泛素化实验、裸鼠成瘤实验等手段检测ERK1/2/OTUB1/GPX4通路调控机制，结果发现sunitinib耐药肾癌细胞内ERK1/2信号通路促进OTUB1转录，OTUB1通过去泛素化调节GPX4蛋白稳定性，GPX4的谷胱甘肽过氧化物酶活性能抑制铁死亡，维持耐药细胞高度间充质状态，发挥对sunitinib耐药作用。此研究发现了一种逆转肾癌sunitinib耐药的新型机制，为肾癌靶向治疗失败的二线治疗提供了一种新型选择，为新型药物研发提供了重要的理论基础。