EGFR酪氨酸磷酸化修饰PD-L1蛋白调控肺腺癌细胞逃避肿瘤免疫的机制研究

Immune checkpoint inhibitors play an important role in the treatment of lung cancer. However, there are still a large number of patients who cannot benefit from it, especially lung adenocarcinomas patients with mutations in the EGFR gene, which accounts for about half of the total number of lung adenocarcinoma patients in China. The existing studies on the regulation of PD-L1 by EGFR mainly focus on the transcription level of this gene. No studies have been concerned with the regulation of post-translational modification and thus stability of PD-L1 protein by EGFR. Using BioID assay, we systematically screened PD-L1’s neighbor proteins in lung adenocarcinoma cell lines and we found that 15.7% (9/57) of the candidate proteins are enriched in the EGFR pathway, including the EGFR itself. At the same time, we and other researchers found that EGF stimulation can induce tyrosine phosphorylation of PD-L1. However, it is unclear how and by which protein(s) EGFR phosphorylates PD-L1. Moreover, it is still to be resolved whether EGFR-mediated PD-L1 tyrosine phosphorylation affects the stability and biological function of PD-L1. This study intends to answer the above questions using a range of biochemical, molecular biology and cell biology methods. This study will help us understand the mechanism(s) by which EGFR regulates lung adenocarcinoma cells to escape from immune system, and may provide additional guidance for the clinical application of immune checkpoint inhibitors, too.

免疫检查点抑制剂在肺癌治疗中发挥了重要作用。然而仍有大量患者未能从中获益，尤其是携带EGFR基因突变的肺腺癌患者，且该群体占我国肺腺癌患者总数的一半左右。现有EGFR对PD-L1调控的研究主要集中于对该基因转录水平的影响，尚未见文献涉及EGFR对PD-L1翻译后修饰及其稳定性的调控。通过前期在肺腺癌细胞系中利用临近蛋白标记技术筛选PD-L1临近蛋白，我们发现9/57的候选蛋白富集于EGFR通路，其中包括EGFR本身。同时，我们和其他研究者均发现EGF能够诱导PD-L1蛋白发生酪氨酸磷酸化修饰。然而EGFR通过何种蛋白以何种方式在哪个（些）位点修饰PD-L1尚不明确，同时，EGFR介导的PD-L1酪氨酸磷酸化是否影响该蛋白的稳定性及生物学功能也有待解决。本研究拟利用一系列生物化学、分子生物学和细胞生物学的研究方法首次回答上述问题。预期结果有助于理解EGFR调控肺腺癌细胞逃避肿瘤免疫的机制。

已有EGFR对PD-L1调控的研究集中于对该基因转录水平的影响，但EGFR是否调控PD-L1翻译后修饰及其稳定性尚待阐明。在我们发现EGFR为PD-L1的临近蛋白的基础之上，我们在携带野生及突变型EGFR的非小细胞肺癌细胞中均观察到EGFR的活化会导致PD-L1的酪氨酸磷酸化修饰。EGFR介导的PD-L1酪氨酸磷酸化对PD-L1自身蛋白稳定性及T细胞功能抑制的维持极为重要。在机制上，我们发现EGFR并非直接，而是借助中间蛋白EphA2磷酸化修饰PD-L1的112位酪氨酸。该位点磷酸化的缺失（Y112A），EphA2表达水平的下调及EphA2活性的抑制均能导致PD-L1糖基化修饰受阻、PD-L1蛋白稳定性降低，进而缓解或消除肿瘤细胞通过PD-1/PD-L1途径对T细胞功能的抑制。因此，EphA2抑制剂可能是潜在的提高EGFR突变肺癌患者T细胞抗肿瘤活性的重要药物。