RAS-Mitogen-Activated Protein Kinase Signal Is Required for Enhanced PD-L1 Expression in Human Lung Cancers.

Hidetoshi Sumimoto,Koji Teramoto,Atsushi Takano,Yataro Daigo,Srikumar Chellappan

doi:10.1371/journal.pone.0166626

Hidetoshi Sumimoto, Koji Teramoto + Show 3 more

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https://doi.org/10.1371/journal.pone.0166626

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Abstract

Ectopic programmed cell death ligand 1 (PD-L1) expression in non-small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Although some altered signals in NSCLCs are responsible for ectopic PD-L1 expression, the precise mechanisms remain obscure. Because we found a higher frequency of EGFR/KRAS mutations in NSCLC cell lines with high PD-L1 expression (p < 0.001), we evaluated the relationships between downstream signals and PD-L1 expression, particularly in three KRAS-mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 μM) significantly decreased the surface PD-L1 levels by 50–60% compared with dimethyl sulfoxide (p < 0.0001). Phorbol 12-myristate 13-acetate stimulation (100 nM, 15 min) increased (p < 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p < 0.05) PD-L1 expression. The transcriptional activity of the potential AP-1 site (+4785 to +5056 from the transcription start site) in the PD-L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP-1 site. Two STAT3 siRNAs decreased PD-L1 expression by 10–32% in two of the three KRAS-mutant lung adenocarcinoma cell lines (p < 0.05), while the PI3K inhibitor LY294002 (40 μM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD-L1-high and -low NSCLCs revealed a correlation between PD-L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD-L1 expression, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD-L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK, along with STAT3, is important for determining PD-L1 expression, which could be useful for targeted therapies against lung cancers.

Highlights

Recent advances in immune checkpoint therapies are rapidly changing the clinical applications of cancer therapies [1]
The mechanisms of ectopic Programmed cell death ligand 1 (PD-L1) expression in human cancers have been studied in lung cancers [10,11,12,13], melanomas [14,15], malignant lymphomas [16,17,18], myelomas [19], and gliomas [9]
It has been reported that various signals including MAPK [13,14,17,18,19], STAT3 [16], or PI3K [9, 13, 14] are relevant to PD-L1 expression; the contribution of each signal is inconsistent among different cancers

Summary

Introduction

Recent advances in immune checkpoint therapies are rapidly changing the clinical applications of cancer therapies [1]. Programmed cell death ligand 1 (PD-L1), known as cluster of differentiation 274 (CD274) and B7 homolog 1 (B7-H1), is widely expressed in normal tissues (natural killer cells, T and B cells, macrophages, dendritic cells, epithelial cells, and vascular endothelial cells). It is a ligand for programmed cell death 1 (PD-1) receptors expressed on activated T cells [2]. The PD-L1/PD-1 interaction inhibits signals from the Tcell receptor, leading to T cells that are exhausted, a state characterized by being unresponsive to antigens [3]

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