ERK抑制剂通过抑制核苷酸代谢增敏依维莫司治疗肾癌的研究

The clinical application of everolimus is limited due to no improvement in the overall survival of patients with renal cell carcinoma (RCC) and drug resistance. Our previous studies demonstrated that RCC cell was resistant to everolimus by activating the ERK signal pathway, up-regulating metabolism-associated proteins, and increasing purine and pyrimidine metabolites, which suggest that ERK-mediated nucleotide metabolism plays an important role in the resistance to everolimus. Currently, there was no report of related resistance mechanism. Further, we found that combination using everolimus and ERK inhibitor resulted in decreased expression of RRM1, CAD and transcription factor C/EBP-β in RCC. Therefore, we propose a scientific hypothesis that ERK inhibitors enhance the antitumor activity of everolimus against RCC through the MNK1-eIF4E-C/EBP-β-RRM1/CAD axis, which block DNA replication and induce G1 cell cycle arrest due to the reduced synthesis of nucleotides or deoxynucleotides. This project intends to investigate the mechanism hypothesis from multiple levels of cells, tissues, animals and molecules, and clarify the clinical significance of RRM1 and CAD in RCC; meanwhile, evaluate for efficacy of combination therapy on the patient derived tumor xenograft model after screening for the better ERK inhibitors in synergy with everolimus in RCC cells. This project will identify a new combination therapeutic strategy worthy of a clinical trial for the patients with RCC, which has an important clinical transforming significance.

依维莫司不改善肾癌患者总生存期，且容易产生耐药，限制了其临床应用。我们前期研究表明，肾癌依维莫司耐药株ERK通路激活，代谢相关蛋白上调，嘌呤及嘧啶代谢产物增多，提示ERK介导的核苷酸代谢对依维莫司耐药的重要作用。目前，尚无相关耐药机制报道。进一步我们发现，ERK抑制剂联合依维莫司导致肾癌细胞核苷酸代谢关键酶RRM1、CAD及转录因子C/EBP-β表达减少。由此，我们提出科学假设：ERK抑制剂通过MNK1-eIF4E-C/EBP-β-RRM1/CAD轴抑制核苷酸合成，导致DNA复制受阻，细胞G1期阻滞，从而增强肾癌对依维莫司的敏感性。本研究拟从细胞、组织、动物及分子等多层次明确该机制假说，并阐明RRM1和CAD在肾癌中的临床意义；同时，在肾癌细胞上筛选与依维莫司协同作用较好的ERK抑制剂，并在人源肿瘤异种移植瘤模型上评价药效。本研究为该联合方案进入临床研究提供依据，具有重要的临床转化意义。

靶向药物治疗临床响应率低、单药治疗效果有限、容易产生耐药性等是诸多药物临床应用面临的共性问题，也是制约患者临床获益的主要瓶颈。因此，进一步深入研究靶向药物的耐药相关分子机制并探索可能的联合治疗方案，对提高转移性肾癌患者的生存率至关重要。本项目聚焦依维莫司相关耐药机制，从细胞水平及动物模型等功能实验上评估了ERK抑制剂具有协同依维莫司治疗肾癌的作用，并通过qPCR、Western blot、ChIP等实验在分子水平上明确了ERK抑制剂通过E2F1-RRM1/RRM2转录抑制核苷酸代谢，导致G1细胞周期阻滞，从而增敏依维莫司抑制肾癌细胞增殖的作用机制。同时本研究还揭示了RRM2在肾癌中表达增高，并与分期、分级呈正比，与预后呈反比，抑制RRM2通过G1细胞周期阻滞显著抑制肾癌细胞的增殖，提示RRM2可作为肾细胞癌的潜在治疗靶点。本研究发现ERK抑制剂与依维莫司联合用药是具有进入临床研究潜力的肾癌靶向治疗方案，为ERK抑制剂上市后该联合治疗方案进入临床研究提供依据，具有一定的临床转化意义。