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.

The protein RB1CC1 (retinoblastoma 1 (RB1)-inducible coiled-coil 1) has been identified as a key regulator of the tumor-suppressor gene RB1 (ref. 1). RB1CC1 is localized in the nucleus and has been proposed to be a transcription factor because of its nuclear localization signal, leucine zipper motif and coiled-coil structure. The gene RB1CC1 is localized to a region of chromosome 8q11 (ref. 2) containing several loci of putative tumor-suppressor genes; however, its role in human cancers remains to be determined. Here we report that 20% (7 of 35) of primary breast cancers examined contained mutations in RB1CC1, including nine large interstitial deletions predicted to yield markedly truncated RB1CC1 proteins. Wildtype RB1CC1 and RB1 were absent or significantly less abundant than normal in the seven cancers with mutations in RB1CC1, but were abundant in cancers without such mutations. In all seven cancers, both RB1CC1 alleles were inactivated; two showed compound heterozygous deletions. Thus, RB1CC1 is frequently mutated in breast cancer and shows characteristics of a classical tumor-suppressor gene.

Given the close interaction between tumor cells and stromal cells in the tumor microenvironment (TME), TME-targeted strategies would be promising for developing integrated cancer immunotherapy. Cancer-associated fibroblasts (CAFs) are the dominant stromal component, playing critical roles in generation of the pro-tumorigenic TME. We focused on the immunosuppressive trait of CAFs, and systematically explored the alteration of tumor-associated immune responses by CAF-targeted therapy. C57BL/6 mice s.c. bearing syngeneic E.G7 lymphoma, LLC1 Lewis lung cancer, or B16F1 melanoma were treated with an anti-fibrotic agent, tranilast, to inhibit CAF function. The infiltration of immune suppressor cell types, including regulatory T cells and myeloid-derived suppressor cells, in the TME was effectively decreased through reduction of stromal cell-derived factor-1, prostaglandin E2, and transforming growth factor-β. In tumor-draining lymph nodes, these immune suppressor cell types were significantly decreased, leading to activation of tumor-associated antigen-specific CD8+ T cells. In addition, CAF-targeted therapy synergistically enhanced multiple types of systemic antitumor immune responses such as the cytotoxic CD8+ T cell response, natural killer activity, and antitumor humoral immunity in combination with dendritic cell-based vaccines; however, the suppressive effect on tumor growth was not observed in tumor-bearing SCID mice. These data indicate that systemic antitumor immune responses by various immunologic cell types are required to bring out the efficacy of CAF-targeted therapy, and these effects are enhanced when combined with effector-stimulatory immunotherapy such as dendritic cell-based vaccines. Our mouse model provides a novel rationale with TME-targeted strategy for the development of cell-based cancer immunotherapy.

IntroductionThe contribution of programmed cell death ligand-1 (PD-L1) immune checkpoint molecule toward progression of non-small cell lung cancer (NSCLC) has not yet been elucidated, in part, because of lack of a standardised method to evaluate PD-L1 expression. In this study, we developed a novel method for the evaluation of PD-L1 expression on NSCLC cells and examined its correlation with clinicopathological characteristics.MethodsAfter immunohistochemical examination of PD-L1 expression for surgically resected pulmonary adenocarcinomas (n=106), based on the findings that PD-L1 are consistently expressed on alveolar macrophages, PD-L1 staining intensity of tumour cells was classified into four levels relative to PD-L1 staining intensity in alveolar macrophages; PD-L1 expression scores (range, 0–300) were semiquantitatively assessed. An analysis of statistical association between PD-L1 expression score and clinicopathological characteristics was performed.ResultsAlmost all of the alveolar macrophages in the specimens were moderately to strongly stained with PD-L1, serving as an internal positive control in the immunohistochemistry of PD-L1. PD-L1 expression score (median, 52.3) was significantly higher in tumours with G2/3 differentiation than in those with G1 (p=0.022) and higher in those with lymphatic invasion than in those without invasion (p=0.032). Postoperative relapse-free survival was significantly shorter in patients with a high PD-L1 expression score than in those with low PD-L1 expression score (p=0.035). Smoking habits, histological subtype, and epidermal growth factor receptor mutation status were not associated with PD-L1 expression score.ConclusionsGiven the heterogeneous distribution of PD-L1 expression in pulmonary adenocarcinoma cells, the scoring of PD-L1 expression on tumour cells relative to that in alveolar macrophages appears to be a valid indicator of PD-L1 status of patients with pulmonary adenocarcinomas, demonstrating a significant correlation with several factors associated with tumour progression.