OTUD1 downregulates PD-L1 expression by deubiquitinating STAT3 and promotes the immune response in ccRCC

Renal cell carcinoma (RCC) is a common urinary malignancy. It has the highest mortality rate among urologic malignancies, and its incidence has been increasing in recent years, and its common subtype is renal clear cell carcinoma (ccRCC). Ovarian tumor domain protease 1 (OTUD1) is known as a member of the OTUD deubiquitinase family and has the ability to remove ubiquitin modifications from target proteins. The OTUD family consists of 10 members, including OTUD1, all of which have the function of deubiquitinating target proteins. In this study, we determined the correlation between OTUD1 and the immune pathway through bioinformatics analysis. Subsequently, by mass spectrometry analysis and immunoprecipitation experiments, we demonstrated that OTUD1 can interact with STAT3 and regulate the expression of PD-L1 in kidney cancer cells through STAT3. OTUD1 can also inhibit the activity and nuclear translocation of STAT3 by regulating the K63 ubiquitination modification of STAT3. Finally, we found that OTUD1 can deubiquitinate STAT3 and inhibit its activity and nuclear translocation, thereby regulating the PD-1 signaling pathway, and ultimately promoting the immune response of renal cancer tumors. Overall design: Comparative gene expression profiling analysis of RNA-seq data for the ACHN cells after kncokdown of OTUD1

肾细胞癌（Renal cell carcinoma, RCC）是常见的泌尿系统恶性肿瘤，在泌尿生殖系统恶性肿瘤中死亡率最高，且近年来发病率呈上升趋势，其最常见的亚型为肾透明细胞癌（renal clear cell carcinoma, ccRCC）。卵巢肿瘤结构域蛋白酶1（Ovarian tumor domain protease 1, OTUD1）属于OTUD去泛素化酶家族成员，具备从靶蛋白上移除泛素修饰的能力。该家族共计10个成员（包含OTUD1），所有成员均具有去泛素化修饰靶蛋白的功能。本研究通过生物信息学分析明确了OTUD1与免疫通路的相关性；随后借助质谱分析与免疫沉淀实验，证实OTUD1可与信号转导与转录激活因子3（STAT3）相互作用，并通过STAT3调控肾癌细胞中程序性死亡受体配体1（PD-L1）的表达。OTUD1还可通过调控STAT3的K63位泛素修饰，抑制STAT3的活性及其核转位。最终研究发现，OTUD1能够对STAT3进行去泛素化修饰，进而抑制其活性与核转位，从而调控PD-1信号通路，最终促进肾癌肿瘤的免疫应答。实验整体设计：对敲低OTUD1后的ACHN细胞的RNA测序（RNA-seq）数据进行比较基因表达谱分析。