Melatonin Downregulates PD-L1 Expression and Modulates Tumor Immunity in KRAS-Mutant Non-Small Cell Lung Cancer

Abstract

Non-small cell lung cancer (NSCLC) patients harboring a KRAS mutation have unfavorable therapeutic outcomes with chemotherapies, and the mutation also renders tolerance to immunotherapies. There is an unmet need for a new strategy for overcoming immunosuppression in KRAS-mutant NSCLC. The recently discovered role of melatonin demonstrates a wide spectrum of anticancer impacts; however, the effect of melatonin on modulating tumor immunity is largely unknown. In the present study, melatonin treatment significantly reduced cell viability accompanied by inducing cell apoptosis in KRAS-mutant NSCLC cell lines including A549, H460, and LLC1 cells. Mechanistically, we found that lung cancer cells harboring the KRAS mutation exhibited a higher level of programmed death ligand 1 (PD-L1). However, treatment with melatonin substantially downregulated PD-L1 expressions in both the presence and absence of interferon (IFN)-γ stimulation. Moreover, KRAS-mutant lung cancer cells exhibited higher Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) levels, and PD-L1 expression was positively correlated with YAP and TAZ in lung cancer cells. Treatment with melatonin effectively suppressed YAP and TAZ, which was accompanied by downregulation of YAP/TAZ downstream gene expressions. The combination of melatonin and an inhibitor of YAP/TAZ robustly decreased YAP and PD-L1 expressions. Clinical analysis using public databases revealed that PD-L1 expression was positively correlated with YAP and TAZ in patients with lung cancer, and PD-L1 overexpression suggested poor survival probability. An animal study further revealed that administration of melatonin significantly inhibited tumor growth and modulated tumor immunity in a syngeneic mouse model. Together, our data revealed a novel antitumor mechanism of melatonin in modulating the immunosuppressive tumor microenvironment by suppressing the YAP/PD-L1 axis and suggest the therapeutic potential of melatonin for treating NSCLC.

Keywords: Hippo pathway; PD-L1; lung cancer; melatonin; tumor immunity.

Figure 1

Effect of melatonin on the viability of NSCLC. (A) H460, A549, and LLC1 cells were treated with various concentrations of melatonin (Mel: 0, 1.25, 2.5, 5, 10, 15 mM) for 24 and 48 h, and cell viability was measured with a CCK8 assay. Data are expressed as a percentage of the control. Values are expressed as the mean ± standard deviation. (B) Colony formation of H460, A549, and LLC1 cells in the presence of melatonin (Mel: 0, 1, 2.5, 5 mM) for 7 days. (C) Microscopic observation of cell morphologies of H460, A549, and LLC1 cells in the presence of melatonin (Mel: 0, 1, 2.5, 5 mM) for 24 h. (D) Flow cytometry analysis of Annexin V/PI staining in response to melatonin (Mel: 0, 1, 2.5, 5 mM) for 24 h. Quantifications of the total apoptotic cell population (Annexin V + /PI- and Annexin V + /PI + ) were obtained from three independent experiments (right panel). Data are presented as the mean ± standard deviation (SD).* p < 0.05, ** p < 0.01, as determined by an unpaired t-test. (E) Western blot analysis of apoptosis-related proteins in H460, A549, and LLC1 cells in response to melatonin (Mel: 0, 1, 2.5, 5 mM) for 24 or 48 h.

Figure 2

Melatonin downregulates PD-L1 in NSCLC. (A) Box plot with Tukey whisker of PD-L1 mRNA level in KRAS mutant and wild-type lung cancer cells. ** p < 0.01 as determined by an unpaired t-test. (B) Western blot analysis of PD-L1 protein level in NSCLC cell lines. KRAS-mutant cell lines were labeled in red color. (C) H460 and A549 cells were treated with various concentrations of melatonin (Mel: 0, 1, 2.5, 5 mM) for 24 h (upper panel), or treated with 2.5 mM melatonin at different time intervals (lower panel), and protein lysates were subjected to Western blot analysis. (D) Real-time PCR analysis of PD-L1 mRNA expression in response to melatonin (Mel: 2.5 mM) for 6 and 12 h in A549 and H460 cells, respectively. Values are expressed as the mean ± standard deviation. ** p < 0.01 was regarded as indicating a significant difference. (E and F) Serum-starved H460 and A549 cells were pretreated with melatonin (2.5 mM) for 60 min followed by stimulation with IFN γ (50 ng/mL) for a further 24 h. (E) Protein level of PD-L1 in total cell lysate was measured by Western blot, and (F) histogram plot of PD-L1 expression was performed by flow cytometry analyses. Quantifications of PD-L1 positive percentages from three-independent experiments are shown (lower panel). ** p < 0.01, as determined by an unpaired t-test.

Figure 3

Melatonin inhibits the YAP/PD-L1 axis. (A) Box plots with Tukey whisker of YAP (YAP1) and TAZ (WWTR1) mRNA levels in KRAS-mutant and wild-type lung cancer cell lines. * p < 0.05 and ** p < 0.01 as determined by an unpaired t-test. (B) Scatterplot of PD-L1 (CD274) and YAP (YAP1) in lung cancer cell lines (left panel). Correlation coefficient was performed by Pearson’s test. Box blot of PD-L1 (CD274) levels in lung cancer cell lines stratified by YAP (YAP1) expression levels (right panel). (C) Western blot analysis of YAP and TAZ protein levels in NSCLC cell lines. KRAS-mutant cell lines were labeled in red color. (D) H460 and A549 cells were treated with various concentrations of melatonin (Mel: 0, 1, 2.5, 5 mM) for 24 h, and protein lysates were subjected to Western blot analysis. (E) RT-qPCR analysis of the expressions of CTGF and Cyr61 in melatonin-treated H460 and A549 cells. ** p < 0.01 as determined by an unpaired t-test. (F) Western blot analysis of YAP and PD-L1 protein levels in H460 and A549 cells treated with verteporfin (VP: 1 μM) and melatonin (Mel: 2.5 mM). (G) Flow cytometry analysis of PD-L1 expression in H460 and A549 treated with verteprofin (VP: 1 µM) and melatonin (Mel: 2.5 mM). Quantifications of PD-L1 positive percentage from three-independent experiments are shown. * p < 0.05 and ** p < 0.01, as determined by a one-way ANOVA. (H) Scatterplot of PD-L1 (CD274) and YAP (YAP1), TAZ (WWTR1), and YAP/TAZ signature in TCGA_LUAD cohort. Correlation coefficients were determined by Pearson’s test. (I) Kaplan-Meier curve analysis of the overall survival probability of lung cancer patients stratified by PD-L1 (CD274) expression level. LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma. HR: hazard ratio.

Figure 3

Melatonin inhibits the YAP/PD-L1 axis. (A) Box plots with Tukey whisker of YAP (YAP1) and TAZ (WWTR1) mRNA levels in KRAS-mutant and wild-type lung cancer cell lines. * p < 0.05 and ** p < 0.01 as determined by an unpaired t-test. (B) Scatterplot of PD-L1 (CD274) and YAP (YAP1) in lung cancer cell lines (left panel). Correlation coefficient was performed by Pearson’s test. Box blot of PD-L1 (CD274) levels in lung cancer cell lines stratified by YAP (YAP1) expression levels (right panel). (C) Western blot analysis of YAP and TAZ protein levels in NSCLC cell lines. KRAS-mutant cell lines were labeled in red color. (D) H460 and A549 cells were treated with various concentrations of melatonin (Mel: 0, 1, 2.5, 5 mM) for 24 h, and protein lysates were subjected to Western blot analysis. (E) RT-qPCR analysis of the expressions of CTGF and Cyr61 in melatonin-treated H460 and A549 cells. ** p < 0.01 as determined by an unpaired t-test. (F) Western blot analysis of YAP and PD-L1 protein levels in H460 and A549 cells treated with verteporfin (VP: 1 μM) and melatonin (Mel: 2.5 mM). (G) Flow cytometry analysis of PD-L1 expression in H460 and A549 treated with verteprofin (VP: 1 µM) and melatonin (Mel: 2.5 mM). Quantifications of PD-L1 positive percentage from three-independent experiments are shown. * p < 0.05 and ** p < 0.01, as determined by a one-way ANOVA. (H) Scatterplot of PD-L1 (CD274) and YAP (YAP1), TAZ (WWTR1), and YAP/TAZ signature in TCGA_LUAD cohort. Correlation coefficients were determined by Pearson’s test. (I) Kaplan-Meier curve analysis of the overall survival probability of lung cancer patients stratified by PD-L1 (CD274) expression level. LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma. HR: hazard ratio.

Figure 4

Effects of melatonin on tumor growth and tumor immunity. (A) Growth curve of LLC1 tumor-bearing mice administrated PBS (n = 6) or melatonin (30 mg/kg, three times per week, n = 7) for 4 weeks. Tumor volumes were measured every other day. (B) Tumor weights were measured at the end of the experiment. (C) Mouse body weight changes under different treatments. (D) Quantification of tumor-infiltrating lymphocytes in PBS- and melatonin-treated tumor tissues, as measured by flow cytometry analysis. Values are expressed as the mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001 indicating a significant difference.