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2024年10月24日,浙江大学生命科学学院易文/朱强团队在PNAS杂志在线发表题为 “O-GlcNAcylation of enolase 1 serves as a dual regulator of aerobic glycolysis and immune evasion in colorectal cancer” 的研究论文。浙江大学生命科学学院百人计划研究员朱强是本文的第一作者;朱强和易文教授为共同通讯作者。该研究揭示了O-GlcNAc 糖基化通过调控烯醇化酶(enolase 1, ENO1)来促进结直肠癌有氧糖酵解以及免疫逃逸的机制,为结直肠癌的治疗提供可能的新靶点。
代谢重编程是肿瘤的基本特征之一。肿瘤细胞即使在氧气充足的条件下仍优先通过糖酵解途径产生能量,这种现象被称为Warburg效应。研究表明结直肠癌是一种具有典型Warburg 效应的肿瘤,在其发生发展过程中,有氧糖酵解起着非常重要的作用,抑制Warburg 效应显著抑制了结直肠癌细胞增殖以及肿瘤生长。因此,靶向有氧糖酵解选择性杀灭肿瘤细胞的治疗策略成为抗肿瘤药物研发的重要方向。
逃避免疫系统监视的能力也是肿瘤的重要特征之一。程序性死亡配体1(PD-L1)是一种在癌细胞表面高度表达的关键免疫检查点蛋白。PD-L1能与细胞毒性T淋巴细胞表面的PD-1相结合,从而抑制T细胞的抗肿瘤活性。最近,以PD-1/PD-L1抑制剂为典型代表的免疫检查点阻断疗法已成为治疗多种肿瘤的有效策略。然而,PD-1/PD-L1抑制剂治疗晚期结直肠癌的疗效并不理想,仅部分亚型的结直肠癌患者从中获益。PD-L1蛋白表达水平被认为是评估抗PD-L1/PD-1治疗的临床反应性的关键生物标志物。因此,深入探究PD-L1的表达调控机制以及开发新的联合治疗药物靶点对于结直肠癌的免疫治疗至关重要。
氧连-N-乙酰葡萄糖胺修饰(O-GlcNAcylation)是一种发生在细胞内蛋白丝氨酸或者苏氨酸残基上的单糖修饰,其失调与肿瘤等人类疾病的发生发展密切相关。研究表明O-GlcNAc糖基化修饰在结直肠癌等多种肿瘤中显著上调,然而其促进肿瘤发生发展的机制有待进一步的研究。
研究人员首先通过临床结直肠癌组织切片染色发现相比于癌旁组织, ENO1的表达在结直肠癌组织中显著上调(图1A)。敲低ENO1显著性抑制了结直肠癌细胞系HCT116在免疫缺陷的小鼠体内的成瘤。在ENO1敲低的细胞系中回补表达野生型(WT)的ENO1能完全恢复其肿瘤生长速率,而回补表达酶活缺失突变体(S40A)只能轻微拯救肿瘤生长(图1B),表明ENO1促进肠癌生长主要依赖于代谢酶活性。研究人员接下来通过化学酶联标记的方法对ENO1蛋白的O-GlcNAc糖基化修饰进行了检测,添加OGA抑制剂TMG以及过表达OGT 均能提高ENO1蛋白的O-GlcNAc糖基化信号,证实了ENO1具有O-GlcNAc糖基化修饰(图1C)。同时通过质谱检测结合点突变的生化手段,研究人员确定了T19以及S249为ENO1蛋白上的主要的糖基化修饰位点(图1D)。体外酶活实验表OGA抑制剂TMG处理或者过表达OGT均能显著性提高WT以及S249 ENO1的酶活,而T19A突变体的酶活性不受OGT以及TMG所调控(图1E),提示T19 位糖基化促进了ENO1酶活性。细胞外酸度分析(ECAR)表明ENO1野生型细胞的糖酵解速率显著高于T19A的细胞(图1F)。裸鼠成瘤实验表明与T19A 的HCT116细胞相比,野生型细胞有着更强的肿瘤形成能力。过表达OGT显著性提高了野生型细胞的肿瘤生长速率,而不影响T19A细胞肿瘤生长(图1G-1H)。这些结果表明ENO1的T19位糖基化通过激活有氧糖酵解从而促进结直肠癌增殖。
图1. ENO1的T19位糖基化促进有氧糖酵解
之前的研究表明ENO1能够与PD-L1相互作用并促进PD-L1蛋白酶体降解,从而抑制肿瘤免疫逃逸。研究人员根据ENO1(PDB ID:2PSN)和PD-L1(PDB ID: 3BIS)的晶体结构信息,通过AlphaFold2构建了ENO1与PD-L1的互作模型,发现ENO1的 E250氨基酸残基位于两者的互作界面上(图2A)。研究者推测S249位糖基化很有可能通过影响E250互作界面,从而进一步影响ENO1与PD-L1的相互作用。免疫共沉淀实验发现TMG处理显著性抑制了WT 以及T19A的 ENO1与PD-L1的相互作用。而在S249A 的RKO细胞中ENO1与PD-L1的相互作用不受TMG所调控(图2B),提示S249位的糖基化抑制了ENO1与PD-L1的互作。此外,TMG处理显著抑制了WT细胞中PD-L1与泛素连接酶STUB1的相互作用以及PD-L1的泛素化,而S249A 的RKO细胞中PD-L1的泛素化不受TMG所调控(图2C),提示ENO1的S249位糖基化抑制STUB1介导的PD-L1蛋白降解。研究人员接下来检测ENO1 糖基化与肿瘤免疫之间的关系。在常规培养条件下, WT与S249A细胞具有相同的增殖速率(图2D)。在与激活的CD8+T细胞共培养的条件下,S249A的细胞毒性相比较于WT细胞显著升高。回补PD-L1能够完全拯救T细胞对S249A 细胞的杀伤(图2E)。在免疫完全的C57BL/6J小鼠中也得到了相一致的结果(图2F-2G)。最后,研究人员发现ENO1的糖基化在临床结直肠癌样品中显著升高,而OGT的表达与PD-L1的表达呈显著正相关,与CD8+T细胞的浸润水平呈显著负相关(图2H-2K)。
图2. ENO1的S249位糖基化促进PDL1表达以及肿瘤免疫逃逸
总之,该研究揭示了ENO1的O-GlcNAc 糖基化调控结直肠癌代谢重编程以及免疫逃逸的新机制(图3),强调了干预ENO1糖基化可以作为治疗结肠癌的潜在策略。
图3. 代谢酶ENO1的O-GlcNAc糖基化调控结肠癌代谢和免疫逃逸工作模式图
摘要:Abstract
Aerobic glycolysis and immune evasion are two key hallmarks of cancer. However, how these two features are mechanistically linked to promote tumor growth is not well understood. Here, we show that the glycolytic enzyme enolase-1 (ENO1) is dynamically modified with an O-linked β-N-acetylglucosamine (O-GlcNAcylation), and simultaneously regulates aerobic glycolysis and immune evasion via differential glycosylation. Glycosylation of threonine 19 (T19) on ENO1 promotes its glycolytic activity via the formation of active dimers. On the other hand, glycosylation of serine 249 (S249) on ENO1 inhibits its interaction with PD-L1, decreases association of PD-L1 with the E3 ligase STUB1, resulting in stabilization of PD-L1. Consequently, blockade of T19 glycosylation on ENO1 inhibits glycolysis, and decreases cell proliferation and tumor growth. Blockade of S249 glycosylation on ENO1 reduces PD-L1 expression and enhances T cell–mediated immunity against tumor cells. Notably, elimination of glycosylation at both sites synergizes with PD-L1 monoclonal antibody therapy to promote antitumor immune response. Clinically, ENO1 glycosylation levels are up-regulated and show a positive correlation with PD-L1 levels in human colorectal cancers. Thus, our findings provide a mechanistic understanding of how O-GlcNAcylation bridges aerobic glycolysis and immune evasion to promote tumor growth, suggesting effective therapeutic opportunities.
Significance
Colorectal cancer cells display key features such as metabolizing glucose at high rates, and escaping immune surveillance. However, how these two features are linked at molecular levels is largely unknown. Here, we show that an important glycolytic enzyme (enolase 1, ENO1) is dynamically modified by N-acetylglucosamine (O-GlcNAc) at multiple residues in colorectal cancer cells. The modification on threonine 19 of ENO1 increases glucose metabolism, while the modification on serine 249 promotes immune escape. The elimination of modifications on both sites synergistically inhibits colorectal cancer growth. Thus, our study reveals that O-GlcNAc modifications of ENO1 could serve as a dual regulator for glucose metabolism and immune evasion to promote colorectal cancer growth.
DOI: https://doi.org/10.1073/pnas.2408354121
原文链接:https://www.pnas.org/doi/10.1073/pnas.2408354121
参考资料:Zhu, Q., Li, J., Sun, H., Fan, Z., Hu, J., Chai, S., ... & Yi, W. (2024). O-GlcNAcylation of enolase 1 serves as a dual regulator of aerobic glycolysis and immune evasion in colorectal cancer. Proceedings of the National Academy of Sciences, 121(44), e2408354121.
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