Gao Zheng, Chen Jia-Feng, Li Xiao-Gang, Shi Ying-Hong, Tang Zheng, Liu Wei-Ren, Zhang Xin, Huang Ao, Luo Xuan-Ming, Gao Qiang, Shi Guo-Ming, Ke Ai-Wu, Zhou Jian, Fan Jia, Fu Xiu-Tao, Ding Zhen-Bin
Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China.
Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Shanghai, China.
Cancer Cell Int. 2022 Mar 19;22(1):128. doi: 10.1186/s12935-022-02550-w.
While the correlation between PD-L1 expression and KRAS mutation has been previously reported in other solid tumors such as non-small cell lung cancer (NSCLC), whether PD-L1 can be modulated by ERK signaling downstream of KRAS in intrahepatic cholangiocarcinoma (iCCA) and the underlying molecular regulatory mechanism remain unclear.
The expression of ERK, p-ERK, PD-L1 and autophagy markers following KRAS knockdown or Ras/Raf/MEK/ERK signaling inhibitors treatment was examined in two human iCCA cell lines (HuCCT1 and RBE) using western blotting and immunofluorescence. Both pharmacological autophagy inhibitors and short-interfering RNA against ATG7 were applied to inhibit autophagy. The apoptosis rates of iCCA cell lines were detected by flow cytometry and CCK-8 after co-culturing with CD3/CD28-activated human CD8 T lymphocytes. Immunohistochemistry was applied to detect the correlation of ERK, p-ERK and PD-L1 in 92 iCCA tissues.
The present study demonstrated that the PD-L1 expression level was distinctly reduced in KRAS-mutated iCCA cell lines when ERK signaling was inhibited and ERK phosphorylation levels were lowered. The positive association between p-ERK and PD-L1 was also verified in 92 iCCA tissue samples. Moreover, ERK inhibition induced autophagy activation. Both inhibiting autophagy via autophagy inhibitors and genetically silencing the ATG7 expression partially reversed the reduced PD-L1 expression caused by ERK inhibition. In addition, ERK-mediated down-regulation of PD-L1 via autophagy pathways induced the apoptosis of iCCA cells when co-cultured with CD3/CD28-activated human CD8 T lymphocytes in vitro.
Our results suggest that ERK signaling inhibition contributes to the reduction of PD-L1 expression through the autophagy pathway in iCCA. As a supplement to anti-PD-1/PD-L1 immunotherapy, ERK-targeted therapy may serve as a potentially novel treatment strategy for human KRAS-mutated iCCA.
虽然此前在其他实体瘤如非小细胞肺癌（NSCLC）中已报道了PD-L1表达与KRAS突变之间的相关性，但在肝内胆管癌（iCCA）中，KRAS下游的ERK信号是否能调节PD-L1以及潜在的分子调控机制仍不清楚。
使用蛋白质免疫印迹法和免疫荧光法，检测了两种人iCCA细胞系（HuCCT1和RBE）在KRAS基因敲低或Ras/Raf/MEK/ERK信号抑制剂处理后ERK、p-ERK、PD-L1和自噬标志物的表达。应用药理学自噬抑制剂和针对ATG7的小干扰RNA来抑制自噬。与经CD3/CD28激活的人CD8 T淋巴细胞共培养后，通过流式细胞术和CCK-8检测iCCA细胞系的凋亡率。应用免疫组织化学法检测92例iCCA组织中ERK、p-ERK和PD-L1的相关性。
本研究表明，当ERK信号被抑制且ERK磷酸化水平降低时，KRAS突变的iCCA细胞系中PD-L1表达水平明显降低。在92例iCCA组织样本中也证实了p-ERK与PD-L1之间存在正相关。此外，ERK抑制诱导自噬激活。通过自噬抑制剂抑制自噬和通过基因沉默ATG7表达，均部分逆转了ERK抑制导致的PD-L1表达降低。此外，在体外与经CD3/CD28激活的人CD8 T淋巴细胞共培养时，ERK通过自噬途径介导的PD-L1下调诱导了iCCA细胞凋亡。
我们的结果表明，ERK信号抑制通过自噬途径导致iCCA中PD-L1表达降低。作为抗PD-1/PD-L1免疫治疗的补充，靶向ERK的治疗可能成为人KRAS突变型iCCA的一种潜在新治疗策略。
