CSN5增强Sox2蛋白稳定性促进鼻咽癌放疗耐受及其分子机制

Radioresistance remains an obstacle to the innovation of nasopharyngeal carcinoma (NPC) treatment. Cancer stem cells (CSCs) may be the crucial cause of radiation tolerance and recurrence. Our previous study demonstrated that overexpressed CSN5 (COP9 signalosome subunit 5) promoted radioresistance in NPC cells. The K48-linked ubiquitination levels of Sox2 protein was upregulated after knockdown of CSN5 with siRNA, on the contrary, the expression level of Sox2 protein, the proportion of CD44 + cells, and the ability of tumor sphere formation and wound healing were attenuated. These results indicated that CSN5 may play a crucial role in sustaining the stability of Sox2 protein. Furthermore, we found that CSN5 and Sox2 proteins were highly expressed in relapsed NPC patients, which were positively correlated with poor prognosis. Therefore, CSN5 is a promising candidate target for dealing with radioresistance in NPC therapy, and it is essential to clarify the underlying mechanism of how Sox2 was ubiquitinated and then destined to proteasome degradation. .The further plans of this project are as follows: 1.to demonstrate how the ubiquitination of Sox2 was regulated by CSN5, and the impact of ubiquitination level on Sox2 stability. 2.To identify the certain ubiquitination-modified lysine loci of Sox2, and determine the function of the key lysine residue by dominant mutation. 3. to verify the impact of CSN5 mediated Sox2 ubiquitination on NPC cell radiation sensitivity, recurrence and prognosis..The goal of this project is to attempt a new way of the stability regulation of Sox2 to understand the mechanism of NPC radioresistance and relapse, provide a rationale for the CSN5 inhibitor in dealing with NPC radioresistance, and will help to find new strategies to NPC diagnosis and therapy.

放疗耐受是制约鼻咽癌疗效提升的关键，肿瘤干细胞（CSCs）可能是放疗耐受、肿瘤复发的重要原因。我们前期研究发现CSN5的高表达促进鼻咽癌放疗耐受，利用siRNA敲低CSN5的表达发现干细胞转录因子Sox2蛋白的K48型泛素化水平升高，蛋白水平下降，并且CD44+细胞比例、细胞成球能力、伤痕愈合能力也降低；CSN5与Sox2在复发型鼻咽癌患者组织内表达明显增高，且高表达与患者总生存时间成负相关性。本课题拟进一步明确Sox2泛素化调控的赖氨酸位点，阐明这种泛素化调控Sox2蛋白酶体途径降解的具体机制；研究CSN5介导的Sox2蛋白去泛素化作用所需的关键结构域及其对鼻咽癌细胞放疗耐受影响，探索维持Sox2蛋白稳定性的新机制。本研究将为CSN5靶向抑制剂的研发，靶向CSN5抑制肿瘤干细胞克服鼻咽癌放疗耐受提供理论基础和实验依据，为提高鼻咽癌的治疗水平提供新靶标和新策略。

放疗耐受是鼻咽癌治疗失败和复发的关键原因，而肿瘤干细胞被认为是导致放疗耐受和肿瘤复发的重要因素。我们前期工作发现CSN5在鼻咽癌放疗复发的患者中表达较高，且与预后不良相关。在本项目中，我们阐明了CSN5在鼻咽癌中发挥的重要作用，探讨了CSN5对鼻咽癌细胞干性的维持作用、对SOX2稳定性的影响以及其他可能促进鼻咽癌发生发展的作用机制。重要结果及关键数据如下：在鼻咽癌细胞中，放疗耐受的细胞株其CSN5和SOX2的表达水平较高，CD44+细胞比例也较高，表明放疗耐受与细胞干性间存在相关性。通过siRNA敲低CSN5的表达后我们发现鼻咽癌细胞的增殖能力、迁移能力和干性均发生明显下降，同时，SOX2的表达水平下降，半衰期随之缩短。而在敲低CSN5的基础上过表达SOX2可以恢复细胞的干性，表明CSN5可能通过稳定SOX2表达维持鼻咽癌干细胞特性从而介导放疗耐受。进一步通过蛋白质谱分析发现CSN5可以调控BPNT2的表达，通过调控BPNT2的泛素化水平增强其蛋白稳定性，促进鼻咽癌细胞的迁移能力。本研究发现了CSN5促进鼻咽癌发生发展的两大原因，一方面，CSN5可以通过增强SOX2稳定性维持鼻咽癌细胞干性介导放疗耐受，另一方面，CSN5可以通过增强BPNT2稳定性促进鼻咽癌细胞迁移介导转移复发，表明CSN5具有重要的生物学意义，可以为鼻咽癌的疾病进展、预后及治疗提供了新的预测指标。