PHGDH触发丝氨酸代谢重排在卵巢癌顺铂耐药中的作用机制研究

Conquering cisplatin resistance is the critical aim for treatment of ovarian cancer, therefore exploring the molecular mechanisms underlying cisplatin resistance, as well as reversing drug-resistance, is of great importance in ovarian cancer therapy. Recently we found that key enzyme of serine biosynthesis, as well as serine biosynthesis pathway was increased in cisplatin resistance ovarian cancer cells. Mechanically, we found that ubiquitylation of PHGDH was decreased and its protein stability was increased in cisplatin resistance ovarian cancer cells. We screened the potential E3 ligase involved in ubiquitylation of PHGDH. Our data indicated that HUWE1 might be the potential E3 ligase responsible for PHGDH ubiquitylation. In addition, we found that HUWE1 mRNA was destabilized in cisplatin resistance ovarian cancer cells, possibly via HuR downregulation. Collectively, our preliminary data indicated that downregulation of HuR resulted in HUWE1 mRNA destabilization by decreasing HuR recruitment, while increasing TTP recruitment to HUWE1 mRNA. Subsequently, downregulation of HUWE1 suppressed ubiquitylation of PHGDH and increased stability of PHGDH protein. Finally, upregulation of PHGDH triggered serine biosynthesis, resulting promotion of NADPH and GSH generation responsible for cisplatin resistance. The current study aimed to investigate the mechanisms underlying regulation of HUWE1 by HuR and TTP, as well as ubiquitylation of PHGDH by HUWE1 in cisplatin resistance in ovarian cancer cells. The current study might provide potential targets for reversing cisplatin resistance or enhancing cisplatin therapeutic efficiency in treatment of ovarian cancer.

探索顺铂耐药机制，克服或逆转耐药是卵巢癌治疗的关键目标。我们前期研究发现顺铂耐药细胞中丝氨酸合成代谢旺盛，关键酶PHGDH泛素化水平降低而蛋白质稳定性增加，并且PHGDH抑制剂部分逆转顺铂耐药。在耐药细胞中E3连接酶HUWE1降低，并且PHGDH可能是HUWE1尚未报道的泛素化底物。我们还发现耐药细胞中HUWE1 mRNA稳定性降低，可能与HuR表达降低向HUWE1 mRNA富集减少，而TTP富集增多促进mRNA降解有关。因此，我们推测耐药细胞中HuR表达降低使HUWE1 mRNA稳定性降低而表达减少，导致PHGDH泛素化水平降低而稳定，促进丝氨酸代谢和GSH产生，从而产生顺铂耐药。本项目拟深入研究HuR和TTP在HUWE1表达调控中的作用，以及HUWE1对PHGDH泛素化调节在顺铂耐药性的作用。本项目的深入解析将为寻求有效的耐药逆转剂或化疗增敏剂提供潜在的新靶点和方向。

由于卵巢癌发病隐匿和缺乏完善的早期诊断方法，约70%患者确诊时已为晚期，其死亡率居妇科恶性肿瘤的首位。以顺铂为基础的联合化疗方案在卵巢癌综合治疗中占有重要的位置，但由于卵巢癌细胞对顺铂的原发性和/或获得性耐药，限制了其临床应用，导致晚期患者的5年生存率仅为20-30%。因此，更好地了解顺铂耐药机制具有重要的临床意义。代谢重编程是肿瘤细胞的特征之一，与肿瘤的发生、发展和耐药性密切相关。关于细胞内糖代谢途径，除了糖酵解和线粒体氧化磷酸化外，还有一些分支途径，如丝氨酸合成途径。PHGDH是丝氨酸生物合成途径中的关键酶，在癌细胞丝氨酸生物合成和线粒体氧化还原稳态中起关键作用。本项目发现顺铂耐药组织和细胞中，PHGDH高表达且与不良预后有关。在顺铂耐药卵巢癌细胞中，敲除PHGDH基因增加顺铂敏感性，降低铂耐药卵巢癌的侵袭能力和球体形成能力；过表达PHGDH则抑制对顺铂的反应性，促进铂敏感卵巢癌细胞的侵袭和球体形成。此外，我们发现耐药细胞中PHGDH mRNA水平无变化，且经典的溶酶体和泛素蛋白酶体途径抑制剂并不影响PHGDH的蛋白水平,提示PHGDH的合成而非降解途径增加了其在耐药细胞中的水平。进一步研究发现，顺铂耐药细胞中PHGDH的翻译被激活，并且依赖于DDX3X在PHGDH转录物的3’-UTR区的募集。接下来，我们的研究发现lncRNA RMRP在顺铂耐药卵巢癌细胞中上调，并促进DDX3X在PHGDH mRNA上的富集。之后研究PHGDH的下游调节机制，发现PHGDH抑制剂CBR-584通过激活ROS/Wnt/β-catenin途径抑制卵巢癌的增殖、迁移和侵袭，并与PARP抑制剂奥拉帕利发挥协同作用，这为PHGDH抑制物CBR-5884在临床治疗中提供了理论基础为卵巢癌耐药的诊治提供了重要的实验依据。综上，本研究表明以PHGDH为靶点可能为克服卵巢癌顺铂耐药性提供潜在机会。