phorbol and Lung-Neoplasms

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.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; B7-H1 Antigen; Butadienes; Cell Adhesion; Cell Movement; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Mutation; Nitriles; Phorbols; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); RNA, Small Interfering; STAT3 Transcription Factor; Transcription Factor AP-1; Transcription Initiation Site

We have undertaken studies of the regulation of the secretion and cytoplasmic mRNA of chromogranin-A (CgA) and calcitonin (CT) in cells that secrete both substances in order to determine if they are regulated through the same mechanisms. Studies were conducted in cell lines derived from a human medullary thyroid carcinoma (MTC) and a human lung cancer (M103). Treatment with phorbol-12-myristate-13-acetate (PMA) resulted in a dose-dependent increase in the secretion of both CT and CgA by the two cell lines. Phorbol at 1000 nM resulted in 4- and 10-fold increases in CgA secretion and 3- and 5-fold increases in CT secretion by the MTC and M103 cells, respectively. The secretory patterns were similar for CgA and CT. In the M103 cells the same treatment resulted in a 100% increase in CgA-specific cytoplasmic RNA and a 70% increase in CT-specific cytoplasmic RNA. The secretion of CgA and CT was coordinately regulated by phorbol in both MTC and M103 cells, and related mRNA changes could be detected in the M103 cells. These observations support the hypothesis that CT and CgA secretion and gene expression are under similar regulatory controls.

Topics: Calcitonin; Cell Line; Chromogranin A; Chromogranins; Dose-Response Relationship, Drug; Humans; Lung Neoplasms; Nerve Tissue Proteins; Nucleic Acid Hybridization; Phorbol Esters; Phorbols; RNA; Tetradecanoylphorbol Acetate; Thyroid Neoplasms; Tumor Cells, Cultured