背景:奥沙利铂耐药通常会导致结直肠癌(CRC)的治疗失败和预后不良,而潜在的机制还没有完全理解。代谢重编程与耐药性密切相关,然而,代谢重编程在奥沙利铂耐药中的作用和机制尚不清楚。这里,本研究旨在探讨嘌呤代谢在奥沙利铂诱导CRC细胞凋亡中的作用及机制。
方法:产生了耐奥沙利铂的CRC细胞系,并进行了非靶向代谢组学分析。通过定量实时聚合酶链反应(qPCR)和蛋白质印迹分析确定CRC细胞系中肌苷5'-单磷酸脱氢酶II型(IMPDH2)的表达。IMPDH2过表达的影响,通过体内和体外细胞凋亡的流式细胞术分析,评估了对CRC中奥沙利铂耐药性的敲除和药理学抑制。
结果:代谢分析显示,嘌呤代谢物的水平,尤其是鸟苷一磷酸(GMP),在奥沙利铂耐药的CRC细胞中显著升高。嘌呤代谢产物的积累主要来自IMPDH2表达的上调。基因集富集分析(GSEA)表明CRC中IMPDH2高表达与嘌呤代谢和多药耐药途径相关。具有较高的IMPDH2表达的CRC细胞对奥沙利铂诱导的凋亡更具抗性。CRC细胞中IMPDH2的过表达导致奥沙利铂治疗后细胞死亡减少,而IMPDH2的敲除通过影响Caspase7/8/9和PARP1蛋白对细胞凋亡的激活而导致对奥沙利铂的敏感性增加。霉酚酸(MPA)或霉酚酸酯(MMF)对IMPDH2的靶向抑制可增强体外细胞凋亡,并与奥沙利铂联合治疗时降低体内肿瘤负荷。机械上,Wnt/β-catenin信号在奥沙利铂耐药的CRC细胞中过度激活,Wnt/β-catenin与IMPDH2之间存在相互正向调控机制。阻断Wnt/β-catenin通路可使耐药细胞对奥沙利铂重新敏感,这可以通过添加GMP来恢复。
结论:IMPDH2是CRC中奥沙利铂耐药的预测生物标志物和治疗靶点。
Oxaliplatin resistance usually leads to therapeutic failure and poor prognosis in colorectal cancer (CRC), while the underlying mechanisms are not yet fully understood. Metabolic reprogramming is strongly linked to drug resistance, however, the role and mechanism of metabolic reprogramming in oxaliplatin resistance remain unclear. Here, we aim to explore the functions and mechanisms of purine metabolism on the oxaliplatin-induced apoptosis of CRC.
An oxaliplatin-resistant CRC cell line was generated, and untargeted metabolomics analysis was conducted. The inosine 5\'-monophosphate dehydrogenase type II (IMPDH2) expression in CRC cell lines was determined by quantitative real-time polymerase chain reaction (qPCR) and western blotting analysis. The effects of IMPDH2 overexpression, knockdown and pharmacological inhibition on oxaliplatin resistance in CRC were assessed by flow cytometry analysis of cell apoptosis in vivo and in vitro.
Metabolic analysis revealed that the levels of purine metabolites, especially guanosine monophosphate (GMP), were markedly elevated in oxaliplatin-resistant CRC cells. The accumulation of purine metabolites mainly arose from the upregulation of IMPDH2 expression. Gene set enrichment analysis (GSEA) indicated high IMPDH2 expression in CRC correlates with PURINE_METABOLISM and MULTIPLE-DRUG-RESISTANCE pathways. CRC cells with higher IMPDH2 expression were more resistant to oxaliplatin-induced apoptosis. Overexpression of IMPDH2 in CRC cells resulted in reduced cell death upon treatment with oxaliplatin, whereas knockdown of IMPDH2 led to increased sensitivity to oxaliplatin through influencing the activation of the Caspase 7/8/9 and PARP1 proteins on cell apoptosis. Targeted inhibition of IMPDH2 by mycophenolic acid (MPA) or mycophenolate mofetil (MMF) enhanced cell apoptosis in vitro and decreased in vivo tumour burden when combined with oxaliplatin treatment. Mechanistically, the Wnt/β-catenin signalling was hyperactivated in oxaliplatin-resistant CRC cells, and a reciprocal positive regulatory mechanism existed between Wnt/β-catenin and IMPDH2. Blocking the Wnt/β-catenin pathway could resensitize resistant cells to oxaliplatin, which could be restored by the addition of GMP.
IMPDH2 is a predictive biomarker and therapeutic target for oxaliplatin resistance in CRC.