Among the most popular chemotherapeutic agents, irinotecan, regarded as a prodrug belonging to the camptothecin family that inhibits topoisomerase I, is widely used to treat metastatic colorectal cancer (CRC). Although immunotherapy is promising for several cancer types, only microsatellite-instable (~7%) and not microsatellite-stable CRCs are responsive to it. Therefore, it is important to investigate the mechanism of irinotecan function to identify cellular proteins and/or pathways that could be targeted for combination therapy. Here, we have determined the effect of irinotecan treatment on the expression/activation of tumor suppressor genes (including p15Ink4b, p21Cip1, p27Kip1, and p53) and oncogenes (including OPN, IL8, PD-L1, NF-κB, ISG15, Cyclin D1, and c-Myc) using qRT-PCR, Western blotting, immunofluorescence (IF), and RNA sequencing of tumor specimens. We employed stable knockdown, neutralizing antibodies (Abs), and inhibitors of OPN, p53, and NF-κB to establish downstream signaling and sensitivity/resistance to the cytotoxic activities of irinotecan. Suppression of secretory OPN and NF-κB sensitized colon cancer cells to irinotecan. p53 inhibition or knockdown was not sufficient to block or potentiate SN38-regulated signaling, suggesting p53-independent effects. Irinotecan treatment inhibited tumor growth in syngeneic mice. Analyses of allograft tumors from irinotecan-treated mice validated the cell culture results. RNA-seq data suggested that irinotecan-mediated activation of NF-κB signaling modulated immune and inflammatory genes in mice, which may compromise drug efficacy and promote resistance. In sum, these results suggest that, for CRCs, targeting OPN, NF-κB, PD-L1, and/or ISG15 signaling may provide a potential strategy to overcome resistance to irinotecan-based chemotherapy.
在众多常用化疗药物中，伊立替康作为喜树碱家族的前体药物，通过抑制拓扑异构酶I活性，被广泛应用于转移性结直肠癌的治疗。尽管免疫疗法在多种癌症类型中展现出前景，但仅微卫星不稳定型（约占7%）而非微卫星稳定型结直肠癌对其产生应答。因此，探究伊立替康的作用机制，识别可作为联合治疗靶点的细胞蛋白和/或信号通路具有重要意义。本研究通过qRT-PCR、Western blotting、免疫荧光及肿瘤样本RNA测序技术，系统分析了伊立替康对抑癌基因（包括p15Ink4b、p21Cip1、p27Kip1和p53）及癌基因（包括OPN、IL8、PD-L1、NF-κB、ISG15、Cyclin D1和c-Myc）表达/活化的影响。采用稳定敲低技术、中和性抗体及OPN、p53和NF-κB抑制剂，明确了伊立替康细胞毒性作用的下游信号传导及敏感/耐药机制。抑制分泌型OPN与NF-κB可增强结肠癌细胞对伊立替康的敏感性。p53抑制或敲低未能有效阻断或增强SN38调控的信号通路，提示存在非p53依赖的作用机制。在同系小鼠模型中，伊立替康治疗显著抑制肿瘤生长。对给药小鼠异体移植瘤的分析验证了细胞培养实验结果。RNA测序数据显示，伊立替康介导的NF-κB信号通路活化可调控小鼠免疫与炎症相关基因，这可能影响药物疗效并促进耐药性产生。综上所述，针对结直肠癌，靶向OPN、NF-κB、PD-L1和/或ISG15信号通路可能为克服伊立替康化疗耐药提供潜在策略。
Overcoming Irinotecan Resistance by Targeting Its Downstream Signaling Pathways in Colon Cancer
肿瘤(癌症)患者之家