基于MAP3K3通路干预PD-L1表达探讨LBP在改善肺腺癌免疫抑制微环境中的作用

Immunotherapy brings gospel to the cancer patients, however, the presence of an immunosuppressive micro-environment in tumor limits its full potential, therefore, improving the immune response is the key to the success of the treatment. MAP3K3 signaling pathway is involved in normal immune regulation, with its highly tumor-related activation being revealed just in the recent years. Our previous work using bioinformatics has informed us the strong relationship between MAP3K3 expression and prognosis of patients with lung adenocarcinoma, and this result was verified at School of Medicine of the University of Michigan in 101 cases of lung adenocarcinoma patients. At the same time, we found out that MAP3K3 expression is also associated with interstitial lymphocytes infiltration. After silenced the MAP3K3 gene expression in lung adenocarcinoma cell lines , the expression of PD-L1 can be downregulated also, this indicate that MAP3K3 molecule can communicate between the tumor cells and the immune cells, thus, it can worked as a potential target of immunotherapy. LBP, as the main component of Chinese wolfberry, has the function of immunoregulation. Our study has pithily confirmed that LBP regulates the expression of MAP3K3 and PD-L1 in lung adenocarcinoma cell lines. this proposal will employ internal and external experiments to further explore the role of LBP on improving immune micro-environment in lung adenocarinoma via regulating the MAP3K3 and PD-L1 signals . To conclude, this project can open up a new perspective for explaining the anti-tumor immune mechanism of LBP, as well as providing experimental basis for the development of Chinese medicine adjuvant immunotherapy of lung cancer.

免疫治疗为肿瘤患者带来福音，而微环境免疫抑制状态限制了其全部潜能发挥，故改善免疫应答环境是治疗成败的关键。MAP3K3信号通路参与正常机体免疫调控，近年来发现其激活与肿瘤关系密切。前期工作采用生物信息学数据分析发现MAP3K3表达与肺腺癌预后相关，此结果在101例临床肺腺癌患者中得到验证；MAP3K3表达与间质淋巴细胞浸润程度相关，且肺腺癌细胞株MAP3K3基因沉默后PD-L1表达下调，均提示MAP3K3信号参与肿瘤和免疫细胞对话，具有免疫治疗潜能。LBP为枸杞子的主要化学成分，我们已证实LBP具有明显的抗肿瘤免疫效应，并可抑制肺腺癌细胞株MAP3K3及PD-L1表达。本项目拟通过体外及动物实验探讨LBP通过MAP3K3信号通路干预PD-L1表达，具有改善肺腺癌免疫微环境作用。此项目完成可为阐释LBP抗肿瘤免疫机制开辟新的视角，为开发肺癌免疫治疗中药佐剂提供实验依据。

项目前期研究发现MAP3K3蛋白表达与临床肺腺癌患者预后关系密切，MAP3K3信号通路影响肺腺癌免疫微环境状态。体外实验，通过肺腺癌细胞株MAP3K3基因过表达和敲减，基因工程正反两方面验证了MAP3K3信号通路可以影响肺腺癌细胞PD-L1表达。H1299肺腺癌细胞经枸杞多糖处理后，转录组测序分析发现基因表达差异主要集中在数条免疫信号通路和MAPK通路，Western Blot法确认了枸杞多糖可下调H1299、A549肺腺癌细胞中MAP3K3和PD-L1蛋白表达。体外实验发现枸杞多糖对人肺腺癌细胞H1299、A549的生长增殖活性无明显影响；但体外将肺腺癌细胞与刺激激活的健康人外周血PBMC共培养后，却发现枸杞多糖可促进PBMC对肿瘤细胞的杀伤作用，而过表达MAP3K3的肺腺癌细胞则可抵抗PBMC的杀伤作用。进一步实验发现枸杞多糖可下调肺腺癌细胞p-p65蛋白表达，增加IκBα蛋白表达，从而抑制NF-κB 信号通路活性，因此推断枸杞多糖促进肺腺癌细胞的免疫杀伤作用与抑制了MAP3K3及下游NF-κB 信号通路，进而下调了肺腺癌细胞PD-L1表达有关。通过建立小鼠肺腺癌皮下移植瘤模型，随机分组为模型组、LBP低、中、高剂量用药，联合化疗药物组(CTX+LBP100mg/kg)，观察发现枸杞多糖可在体内抑制肺腺癌小鼠移植瘤生长，减少移植瘤体积和重量，提高脾脏指数和胸腺指数，提高单细胞悬液CD3+CD4+T、CD3+CD8+T细胞数量，并促进脾单个核细胞IL-2表达，降低PD-1表达，明确了枸杞多糖可激活整体免疫细胞功能。本研究为阐释枸杞多糖抗肿瘤免疫机制开辟了一个新的视角，为开发肺癌靶向药物及中药免疫治疗佐剂提供新的理论和实验依据。