PCK1介导丝氨酸代谢重编程在肝癌干细胞化疗耐药中的作用及机理研究

Chemotherapy is one of the most common treatments for patients whom are diagnosed with cancer. However, the efficiency for hepatocellular carcinoma patients is less than 20%. Therefore, it is urgent to investigate the mechanism of chemotherapy resistance and explore effective treatment strategies for hepatocellular carcinoma. Our previous results showed that the rate-limiting enzymes of gluconeogenesis are downregulated in HCC tissues, which are well known as phosphoenolpyruvate carboxykinase(PEPCK),a key enzyme in gluconeogenesis. However, we recently found that cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), also known as PCK1, was upregulated in liver cancer stem like cells (TRCs). Surprisingly, up-regulated PCK1 in TRCs did not mediate gluconeogenesis but promoted glucose side-branch metabolism, including glycolysis and serine metabolic rearrangements. As we know, serine metabolism is associated with synthesis of NADPH and GSH. Based on those findings, we attempt to further investigate the chemotherapy is one of the most common treatments for patients whom are diagnosed with cancer. However, the efficiency for hepatocellular carcinoma patients is less than 20%. Therefore, it is urgent to investigate the mechanism of chemotherapy resistance and explore effective treatment strategies for hepatocellular carcinoma. Our previous results showed that the rate-limiting enzymes of gluconeogenesis are downregulated in HCC tissues, which are well known as phosphoenolpyruvate carboxykinase(PEPCK),a key enzyme in gluconeogenesis. However, we recently found that cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), also known as PCK1, was upregulated in liver cancer stem like cells (TRCs). Surprisingly, up-regulated PCK1 in TRCs did not mediate gluconeogenesis but promoted glucose side-branch metabolism, including glycolysis and serine metabolic rearrangements. As we know, serine metabolism is associated with synthesis of NADPH and GSH. Based on those findings, we attempt to further investigate the mechanism of serine metabolic rearrangements promoting chemotherapy resistance in TICs: during serine metabolism, some metabolites such as NADPH and GSH can increase the repair of DNA damage, reduce the toxicity of reactive oxygen species, promote the survival of tumor cells, and finally acquired the ability of chemotherapy resistance. This study highlights the novel mechanism of chemotherapy resistance in TRCs，which provide a new perspective view of chemotherapy for HCC through regulating serine metabolic rearrangement.

化疗作为肿瘤治疗最为常用的方法之一，对肝癌的有效率却不足20%。积极探索化疗耐药机理和寻找有效干预措施对于肝癌治疗十分重要。我们前期研究发现肝癌组织中糖异生的限速酶磷酸烯醇式丙酮酸羧化激酶（PEPCK）低表达，但在肝癌干细胞中PEPCK的胞浆型PCK1表达却上升，然而升高的PCK1并不能增强糖异生活动，反而促进糖酵解和增强丝氨酸合成代谢。丝氨酸代谢与烟酰胺腺嘌呤二核苷磷酸（NADPH）、还原谷胱甘肽（GSH）合成相关。在前期研究基础上，本项目拟进一步研究肝癌干细胞通过丝氨酸的代谢重编程使其获得耐药性的可能性机制：丝氨酸合成代谢增强将产生更多的核苷酸合成底物和NADPH，而这些代谢产物有助于DNA损伤修复和消除活性氧（ROS）的毒害作用，促进肿瘤细胞存活，最终达到对化疗药物耐药的目的。本研究从一个新的视角探索肝癌干细胞化疗耐药的机制，以期通过干预丝氨酸代谢重编程增强化疗药物的敏感性。

研究背景:胆管癌的发生发展常伴有代谢重编程。目前针对胆管癌的治疗方法的局限性凸显了破译胆管癌代谢复杂性的重要性。越来越多的证据表明，糖代谢和脂质合成之间的动态转换赋予了肿瘤细胞对铁死亡的抗性。然而，CCA甚至其他肿瘤中铁死亡抵抗发生的具体机制尚未明确。.方法:本研究在体外(CCA细胞系和患者来源类器官)和体内(转基因小鼠模型)中研究了胞质磷酸烯醇丙酮酸羧激酶1(PCK1)(糖异生限速酶)Ser90位点磷酸化在糖脂代谢转换及其影响铁死亡抵抗的作用机制。PI3K-AKT过度激活是胆管癌中普遍存在的。PCK1 S90磷酸化显著促进糖酵解、脂代谢增强和随后的铁死亡抵抗，而这些在2-DG(一种糖酵解抑制剂)或GSK2837808A (LDHA特异性抑制剂)处理下被消除。机制上，AKT依赖的PCK1磷酸化直接结合并磷酸化LDHA Y10位点，促进有氧糖酵解和总蛋白赖氨酸乳酸化上调。通过LC-MS/MS，我们发现PCK1-LDHA轴促进SREBP调节基因(SPRING) Lys82位点的乳酸化，促进SCAP向高尔基体转运，随后激活SREBP依赖的脂代谢。此外，我们发现由SREBP2驱动的甲羟戊酸通路激活介导了SPRING的铁死亡抵抗的发生，我们最终证明PCK1-LDHA-SPRING轴是PCK1-INSIG1/2轴介导的脂肪生成所必需的。具体来说，磷酸化的PCK1作为一个 “牛仔”，利用两条缰绳(PCK1- LDHA - SPRING和PCK1- INSIG1 /2)促进甲羟戊酸通路的后续激活，导致CoQ10-FSP1轴和GSH-GPX4轴同时激活，最终导致铁死亡抵抗的发生。最后，我们使用一种穿膜肽（特异性靶向PCK1 S90位点抑制其磷酸化）在胆管癌转基因小鼠模型中通过消除PCK1磷酸化促进T细胞介导的肿瘤细胞铁死亡从而增敏化疗联合免疫治疗疗效，并在临床样本中证实PCK1的磷酸化与胆管癌患者化疗联合免疫治疗的不良预后相关。.结论:本研究揭示了PCK1 S90磷酸化在胆管癌中通过调节糖脂代谢转换从而导致铁死亡抵抗发生的机制，即通过PCK1介导LDHA Y10磷酸化和随后SPRING K82乳酸化促进甲羟戊酸通路的激活。这些发现完善了PI3K-AKT过度激活背景下PCK1 S90磷酸化促进脂代谢的机制，靶向PCK1 S90磷酸化可能是改善CCA化疗联合免疫治疗效果的有前途的策略。