神经源性生长因子GDNF促进头颈鳞癌细胞PD-L1表达的作用及机制研究

PD-L1-mediated immunosuppression significantly contributes to the development of tumor. Characterizing information regarding PD-L1 expression and its regulatory mechanism is helpful to further understand the immune escape mechanism of tumor cells and improve the effect of anti-PD-L1 therapies. We have found that glial cell line-derived neurotrophic factor (GDNF) was significantly related to perineural invasion of HNSCC. According to GEO data analysis, we found that GDNF stimulation can significantly up-regulate the mRNA level of PD-L1. Further study found that GDNF can facilitate the expression of PD-L1. Hence, we hypothesized that GDNF can suppress the killing effect of immune cells on tumor cells by up-regulating the expression of PD-L1 in the HNSCC-nerve interaction microenvironment. This study will demonstrate the expression mechanism of PD-L1 regulated by GDNF by molecular biology and cytochemistry, and evaluate the role of GDNF signaling pathway in increasing tumor immune escape by up-regulate PD-L1 expression. We hope it can help to further develop GDNF signaling pathway as a therapeutic target, and establish new therapeutic strategies.

肿瘤细胞表达PD-L1是发生免疫逃逸的重要原因。研究PD-L1在肿瘤中表达的调控途径，有助于进一步认识肿瘤细胞的免疫逃逸机制，将提高抗PD-L1治疗效果。申请者前期已报道胶质细胞源性神经营养因子（GDNF）高表达与头颈鳞癌嗜神经浸润密切相关；进一步基于GEO数据分析发现GDNF与肿瘤细胞PD-L1 mRNA正相关；临床样本发现PD-L1在发生神经浸润的头颈鳞癌细胞中表达与GDNF正相关；细胞学也初步发现GDNF能促进PD-L1表达。因此我们提出假说：在头颈鳞癌神经-肿瘤微环境中，GDNF通过上调肿瘤细胞PD-L1表达从而影响免疫细胞对肿瘤细胞的杀伤。本项目将通过分子生物学和细胞化学手段在临床组织样本和细胞学水平验证假说，阐明GDNF促头颈鳞癌PD-L1表达的作用及机制，评估GDNF通过PD-L1增强肿瘤细胞逃避免疫细胞杀伤的影响，为开发以GDNF信号通路为靶点的新治疗策略提供研究基础。

肿瘤微环境中神经-肿瘤的交互调控在肿瘤的发生和进展中发挥重要作用。本项目着眼于口腔鳞癌-神经交互调控微环境，发现神经源性的胶质细胞源性神经营养因子（glial cell line derived neurotrophic factor，GDNF）、神经生长因子(Nerve growth factor，NGF)等关键性因子在肿瘤生长、侵袭、免疫逃逸中发挥重要用。通过口腔鳞癌临床样本分析发现，GDNF主要高表达于口腔鳞癌所侵袭的神经纤维及肿瘤细胞中，而程序性死亡配体-1（programmed cell death-ligand 1，PD-L1）在神经束周肿瘤细胞中高表达，GDNF与PD-L1表达呈正相关。细胞及分子生物学研究发现GDNF可通过激活其肿瘤细胞表面受体RET以及下游JAK2-STAT1信号通路，促进肿瘤细胞PD-L1表达，从而提高肿瘤细胞逃避NK细胞的杀伤作用，应用RET的抑制剂regorafenib可有效抑制GDNF诱导的肿瘤细胞PD-L1表达，提高NK细胞的杀伤作用。同时，在本项目资助下，我们还发现神经源性NGF-TrkA信号轴可促口腔鳞癌细胞上皮间质转化（Epithelial-Mesenchymal Transition，EMT），促进口腔鳞癌细胞的侵袭和转移；进一步研究发现，NGF可通过EMT诱导口腔鳞癌细胞对EGFR小分子抑制剂erlotinib的耐药，应用其受体TrkA的小分子抑制剂GNF5837可逆转NGF诱导的EMT作用，GNF5837和erlotinib具有协同抗肿瘤作用，细胞学水平及裸鼠皮下成瘤实验均发现联合应用GNF5837和erlotinib可显著提高对口腔鳞癌的抑制作用。本项目明确神经-肿瘤相互作用微环境中神经源性生长因子对肿瘤细胞生长微环境的塑造作用，为肿瘤发生发展的研究提供新角度。