IL-8抑制miR-15b表达激活免疫检查点PD-L1在结肠癌发生及耐药中的作用及机制研究

Chemotherapy resistance is the main reason of treatment failures in therapy against colorectal cancer, immunotherapy with the immunological checkpoint PD-L1 inhibitor may provide a new idea for the treatment of cancer patients. Our preliminary study showed that miR-15b, as a tumor suppressor gene, is involved in the development and progression of colorectal cancer, level of miR-15 was significantly decreased during colitis-associated colon cancer, while the expression of PD-L1 was increased, and the expression of miR-15 was down-regulated by interleukin-8 (IL-8). Moreover, it is suggested that miR-15b target PD-L1 gene and inhibit PD-L1 expression from pre-experimental studies, but the detailed mechanism of is still unclear. Thus, we propose that the silencing miR-15 expression by IL-8 contribute to activate T-cell immune responses though promote PD-L1 expression, and thus regulate the progression and drug-resistance of colon cancer. Accordingly, as the potential regulatory factor of immune response, we will take advantage of relevant experimental methods such as the detection of T-cell apoptosis, and further investigate the biological function of miR-15-regulated the development and chemotherapy resistance of colon cancer, and provide clues for exploring the novel targets for treatment of colon cancer.

肿瘤耐药是结肠癌临床面临的重大瓶颈问题，免疫检查点（如程序性死亡配体1，PD-L1）抑制剂为肿瘤治疗提供了新思路。我们前期研究发现，miR-15b作为抑癌基因参与结肠癌演进，在炎-癌进程中逐级递减，且炎症因子IL-8能够诱导其表达下调；近期预实验发现miR-15b可靶向抑制PD-L1表达，且增加CD8+T细胞比例，但作用机制不清楚。基于此，我们提出炎症因子IL-8通过抑制miR-15b的表达激活免疫检查点PD-L1，进而促进结肠癌发生及耐药的假设。本研究拟在前期工作基础上，以揭示miR-15b作为潜在免疫应答调控因子为研究切入点，并拟通过免疫组化、定量PCR、T细胞凋亡检测等实验手段，从miR-15b调控T细胞免疫应答的角度探讨miR-15b与结肠癌发生及耐药的关系，为结肠癌的有效治疗提供新的靶点和策略。

在本项目的资助下，我们基于免疫检查点抑制剂（ICI），包括抗PD-1治疗，在微卫星稳定（MSS）结直肠癌（CRC）患者中疗效有限。寻找到导致免疫检查点耐药的关键分子IL-17A。本项目旨在探讨IL-17 A在MSS结直肠癌小鼠抗肿瘤免疫抵抗中的作用，并探讨抗IL-17 A联合抗PD-1治疗MSS结直肠癌的可行性。本团队明确了MSS结直肠癌细胞系和组织中程序性细胞死亡配体1（PD-L1）的表达及miR-15 b-5 p对其表达的调控。miR-15 b-5 p对肿瘤发生和抗PD-1治疗敏感性的影响在体外和结肠炎相关癌（CAC）和APCmin/+小鼠模型中得到验证。使用靶向IL-17 A和PD-1的抗体在荷皮下CT 26和MC 38肿瘤的小鼠中进行体内疗效和机制研究。临床病理标本的评估证实PD-L1 mRNA水平与CD 8 + T细胞浸润及较好的预后相关。miR-15 b-5 p在CAC和APCmin/+结直肠癌模型中可下调PD-L1的蛋白表达，抑制肿瘤的发生，增强抗PD-1的敏感性。IL-17 A可通过调节P65/NRF 1/miR-15 b-5 p轴而导致PD-L1在大肠癌细胞中的高表达。IL-17 A和PD-1联合阻断对CT 26和MC 38肿瘤有效，肿瘤中细胞毒性T淋巴细胞较多，髓系来源的抑制细胞较少。因此我们明确了IL-17 A通过p65/NRF 1/miR-15 b-5 p轴增加PD-L1的表达，增强抗PD-L1治疗的耐药性。阻断IL-17 A可提高抗PD-1治疗MSS CRC小鼠模型的疗效。IL-17 A可作为MSS CRC患者对ICI治疗敏感性的治疗靶点。. 在此课题的基础上，本团队探索了结肠癌免疫抑制微环境中肿瘤相关巨噬细胞对微卫星稳定（MSS）结直肠癌患者的免疫治疗的影响。其中使用CSF1R抑制剂靶向TAM具有治疗吸引力，但其抗肿瘤作用非常有限。在这里，我们初步确定了限制CSF1R靶向治疗效果的机制。我们证明了肿瘤细胞来源的CTGF促进肿瘤相关成纤维细胞激活形成免疫屏障。肿瘤细胞产生的CTGF导致CAF中CXCL12分泌增加，从而限制免疫细胞向肿瘤肿瘤中心的迁移。CSF1R抑制剂与CTGF单抗联合使用可阻断肿瘤微环境肿瘤相关成纤维新的激活，显示出强的抗肿瘤作用。