USP2介导RagA去泛素化稳定肿瘤细胞“Do not eat me”信号的机制研究

To clarify the mechanism of tumor immune escape is the core to understand the initiation and progression of lung cancer, and investigate the novel strategy for immunotherapy. Our preliminary studies showed that ubiquitin specific protease 2 (USP2) played a crucial role in the activation of “Do not eat me” signaling by regulating the stable expression of lung cancer-protective "coat" CD47, which is dependent on protein degradation pathway. However, the detailed mechanism remains unclear. Further evidence suggested that both USP2 and CD47 were binding proteins for RagA and these three proteins coexisted in tumor cell endosome, and USP2 is a deubiquitination enzyme of RagA. The project proposed the hypothesis that USP2 mediates RagA deubiquitination to inhibit CD47 endocytosis and degradation, CD47 is stably expressed and anchored to the cell membrane, and subsequently lead to evade immune killing. Multiple molecular techniques including IP, GST pull-down and deletion mutation will be applied to decipher the role of USP2 in CD47 endocytosis and lysosomal degradation, the influence of USP2 ubiquitination modification on the ability of RagA binding to GTP/GDP, and elucidate the mechanism involved in RagA deubiquitination of USP2 regulating stable expression of CD47 and its clinical significance. The results are expected to provide new ideas and targets for further understanding the original mechanism of lung cancer immune escape and exploring new strategies for lung cancer diagnosis and treatment.

阐明肿瘤免疫逃逸的机制是理解肺癌发生与演进、探索免疫治疗新策略的核心基础。我们前期发现泛素特异性蛋白酶2(USP2)通过蛋白质降解途径调控肺癌细胞保护性“外衣”—CD47的稳定表达，在激活“Do not eat me”信号中发挥重要作用，但机制不清；进一步证据表明USP2和CD47是RagA的结合蛋白且三者共存于细胞内体，USP2是RagA的去泛素化酶。本项目提出“USP2介导RagA去泛素化抑制CD47内吞和降解，稳定CD47表达并锚定于细胞膜，从而逃逸免疫杀伤”的假说，拟采用Co-IP、GST pull-down、基因定点突变等技术明确USP2在CD47内吞-溶酶体降解中的作用及USP2泛素化修饰影响RagA GTP/GDP的结合能力，揭示RagA去泛素化在USP2调控CD47稳定表达中的机制及临床学意义；结果可望为深入认识肺癌免疫逃逸的源动力机制及探索肺癌诊疗新策略提供新思路和靶点。

肺癌是威胁人类健康的重大恶性疾病，是最常见的恶性肿瘤之一，其死亡率占我国恶性肿瘤死亡率的第一位。肿瘤免疫逃逸是导致肺癌恶性进展的重要原因,基于免疫抑制检查点的肿瘤免疫治疗是肺癌治疗的主要研究方向。由于肿瘤细胞与微环境免疫细胞的通讯互作存在复杂性和多样性，人们对肿瘤免疫逃逸的调控机制的认识尚不清晰。已有证据表明，CD47是介导巨噬细胞吞噬和清除的巨噬细胞特异性免疫检查点，肿瘤细胞膜表面稳定高表达CD47蛋白与巨噬细胞表面配体SIRPα 组成细胞间通讯系统（CD47-SIRPα系统）释放负调节的“Do not eat me”（“别吃我”）信号从而逃避免疫细胞吞噬杀伤。本项目围绕肺腺癌及其微环境巨噬细胞的互作机制，主要研究内容包括阐明USP2/RAGA在肺腺癌巨噬细胞微环境调控和免疫逃逸中的作用；明确肺腺癌USP2去泛素化RAGA信号通路在CD47内吞-溶酶体降解过程中的作用机制；解析USP2调控RAGA GTP/GDP结合能力及其对下游的内吞分子机制和巨噬细胞吞噬的影响；以及探讨 CD47溶酶体降解途径的临床意义。实验结果表明溶酶体蛋白RAGA在肺腺癌中抑制“Do not eat me”信号促进巨噬细胞吞噬肿瘤细胞，从而杀伤肿瘤；并进一步解析了RAGA通过促进免疫检查点蛋白CD47经内吞-溶酶体降解激活巨噬细胞杀伤力的分子机制；同时揭示了RAGA与CD47在临床样本中的表达负相关性及其在肺腺癌病人生存期及治疗中的意义。此外， 发现了RAGA的上游调控分子泛素化酶SKP2与去泛素化酶USP2,重点研究发现USP2与RAGA相互作用，泛素化RAGA，抑制CD47蛋白降解，从而促进“Do not eat me”信号抑制巨噬细胞对肿瘤细胞的吞噬杀伤作用。我们的研究成果不但揭示了免疫检查点蛋白CD47降解的分子调控机制，以及 RAGA/CD47蛋白在肺腺癌中的表达情况、相互关系及其对肺癌诊断、预后评估的临床意义，还阐明了基于RAGA蛋白表达水平的肿瘤治疗预测模型，并在动物中实现了靶向RAGA与CD47的肿瘤协同治疗策略，提供了增强CD47抗体治疗敏感性的治疗方向，在此基础上进行转化和应用有可能促进肺癌肿瘤免疫治疗策略的发展。

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