PD-L2介导的K-Ras突变型结直肠癌免疫逃逸及其调控机制研究

Programmed death 1 (PD-1) protein, a T-cell coinhibitory receptor, and its ligands, PD-L1 and PD-L2, play a pivotal role in the ability of tumor cells to evade the host's immune system. Blockade of interactions between PD-1 and PD-L1 enhances immune function in vitro and mediates antitumor activity in preclinical models. Our preliminary studies suggest that 1)In colorectal cancer tissue and cell lines, the positive rate of PD-L2 is higher than that of PD-Ll;2) The protein expression is higher in K-Ras mutant cells than in K-Ras wild type cells; 3) Down-regulation of PD-L2 can sensitize K-Ras mutant cells to the TRAIL indused apoptosis. All these data suggest that the PD-L2 plays an important role in immune suppression of colorectal cancer. Here we hypothesize that up-regulation of PD-L2 pathway may contribute to the immune escape of K-Ras-driven colorectal tumor. The aims of this study are: 1.to explore the relationship between PD-L2 expression and the pathologic diagnosis and prognosis of the colorectal cancer; 2.to understand if blockade of PD-L2/PD-1 pathway could effectely restore the CD8+ T cell function in tumor; 3.to identify the correlation ship between K-Ras mutation and the immunosuppression manifected by PD-L2 upregulation in colorectal cancer. In summary, a better understanding of PD-L2 will provide a novel insight into the mechanisms of immune escape associated K-Ras mutation and in the end may serve to shift paradigms relating to Ras signaling and immunotherapy.

PD-1及配体PD-L1/2是T细胞活化过程中重要的负性共刺激分子，与肿瘤免疫耐受和逃逸密切相关，阻断PD-1及配体的靶向药物是目前肿瘤免疫治疗的研究热点之一。我们在前期研究中发现：PD-L2在结直肠癌细胞和组织中阳性表达率高于PD-L1；K-Ras突变细胞中PD-L2蛋白水平高于野生型，抑制PD-L2可以增加K-Ras突变细胞对TRAIL诱导凋亡的敏感性。据此我们提出假说"PD-1/PD-L2介导的免疫抑制可能是K-Ras突变结直肠癌免疫逃逸的重要机制"。本项目将研究PD-L2与K-Ras突变结直肠癌临床病理及预后的关系，揭示PD-L2/PD-1在以CD8+为主的抗肿瘤免疫中的作用，阐明PD-L2表达是否对K-Ras突变存在依赖性及分子机制。研究结果将明确K-Ras突变调控结直肠癌中PD-L2表达并促进肿瘤细胞免疫逃逸作用和分子机制，为该类型肿瘤靶向免疫治疗提供坚实的理论基础。

结直肠癌是肠粘膜上皮在环境或遗传等多种因素作用下发生的恶性病变。 作为结直肠癌早期出现的基因改变，KRAS突变激活下游生存通路促进细胞异常增殖，参与了肿瘤发生、发展和耐药的过程。在肿瘤免疫微环境中，PD-1-L1/2作为协同刺激信号调控T细胞活化，在肿瘤耐受方面起着主导作用。虽然阻断PD-1-L1/2策略被认为是肿瘤免疫治疗的里程碑，但目前并没有明确PD-1及其配体在结肠癌中表达的治疗意义。因此结肠癌患者肠道免疫微环境是否处于抑制状态，突变的KRAS如何调控免疫抑制微环境形成，是本项目研究的主要目的。. 在课题实施中，我们证实PD-L1/L2在结直肠癌患者外周血中相对于正常人高表达，且在癌组织相对于癌旁组织过表达而且其表达和肿瘤分化程度呈正相关，有望成为新的结直肠癌的生物标记。同时我们发现KRAS突变结直肠癌免疫微环境呈抑制状态。体外细胞培养实验发现，在抑制KRAS突变细胞生存依赖的下游分子TAK1后，KRAS突变细胞发生凋亡，但是并没有影响KRAS野生型和CD3+免疫细胞的增殖。在小鼠KRAS突变原位结直肠癌模型中，我们发现TAK1抑制剂诱导KRAS突变细胞发生凋亡的效果有赖于抗肿瘤免疫的存在。在KRAS突变肿瘤细胞-CD3+淋巴细胞混合培养中，TAK1抑制剂可促使CD8+T细胞增加，CD4+T细胞减少，肿瘤细胞表面PD-L1及淋巴细胞表面PD-L1表达降低。进一步研究发现TAK1抑制剂通过抑制NF-κB信号通路下调PD-L1表达。. 综上所述，TAK1抑制剂在诱导KRAS突变型结直肠癌细胞凋亡的同时， 通过下调KRAS突变肿瘤细胞及CD3+细胞PD-L1表达，激活肿瘤免疫杀伤作用。本研究结果首次证实TAK1具有抑制KRAS突变细胞异常增殖和激活免疫微环境的双重效应，是KRAS突变型结直肠癌靶向治疗和免疫联合治疗的有力靶点。.