The role of CENP-E inhibitors in lung cancer cells
To demonstrate the effect of CENP-E on A549 proliferation, colony formation and the cell cycle, we inhibited CENP-E using the CENP-E-specific inhibitor GSK923295. The IC50 of GSK923295 at 72 h was 43.79 nmol/L (Fig. 1A). Therefore, we treated A549 and H522 cells with GSK923295 for the CCK8 assays, colony formation assays and PI staining. Our data showed that CENP-E inhibition with GSK923295 inhibited the proliferation of A549 and H522 cells, especially at 72 h and at 50 and 100 nmol/L (Fig. 1B-C). Significantly decreased colony formation numbers were observed in A549 and H522 cells treated with GSK923295 (Fig. 1D). The inhibition of CENP-E arrested the cell cycle of A549 and H522 cells at the G2/M phase (Fig. 1E). Therefore, our data indicated that CENP-E inhibition exhibited anti-tumor activity by directly suppressing the proliferation of tumor cells and arresting the cell cycle.
CENP-E inhibition upregulates PDL1 expression in lung cancer cells.
To elucidate the influence of CENP-E inhibition on immunity, we conducted RNA sequencing. We investigated the expression of a total of 17135 genes and found that 429 genes were upregulated and 331 genes were downregulated in A 549 cells treated with GSK923295 (Fig. 2A). We further investigated the distinctive genes related with immunity and PD-L1. We found that PD-L1 was obviously highly expressed after CENP-E inhibition. The expression of immunosuppressive molecules, including IL1β, IL-6, IL-33 and PTGS2, were also elevated, and genes that participate in the regulation of PD-L1, including IFNGR, CXCL8, IL-6, JAK3 and JUN, were upregulated (Fig. 2B).
To verify the influence of CENP-E on PD-L1 expression in lung cancer, we evaluated the expression of PD-L1 in A549 and H522 cells treated with the CENP-E-specific inhibitor GSK923295. Our data showed that GSK923295 induced PD-L1 expression at the mRNA and protein level at 50 and 100 nmol/L (Fig. 2C and D). GSK923295 also increased the expression of MHC I in lung cancer cells at 50 and 100 nmol/L (Fig. 2E). We further confirmed the expression of CENP-E and PD-L1 in lung cancer tissues from patients. The results indicated a negative correlation between CENP-E and PD-L1 in lung cancer tissues (Fig. 2F). Therefore, the inhibition of CENP-E in lung cancer induced the expression of PD-L1 at the mRNA and protein level.
CENP-E signal inhibition induces T-cell suppression in vitro and in vivo.
To explore whether the CENP-E inhibition-induced upregulation of PD-L1 in A549 cells affected antitumor immunity, we cultured mononuclear cells isolated from MPE and A549 cells treated with GSK923295. A549 cells treated with GSK923295 exhibited a dose-dependent reduction in the ratio of CD8+ T cells to CD4+ T cells and an increase in the proportion of apoptotic CD8+ T cells (7-AAD+CD8+) and apoptotic CD4+ T cells (7-AAD+CD4+) (Fig. 3A and B). We also found that A549 cells treated with 50 and 100 nmol/L GSK923295 resulted in the elevation of Tregs (Fig. 3C).
To verify the in vivo influence of CENP-E on lung tumor growth, an A549 nude mouse model was established and treated with GSK923295. GSK923295 significantly suppressed A549 growth in nude mice and prolonged the survival of tumor-bearing nude mice (Fig. 3D). We further established a CENP-E knockdown Lewis cells line and a subcutaneous Lewis lung cancer C57BL/6 mice model. Our results indicated that the knockdown of CENP-E slightly suppressed tumor growth in Lewis lung tumor-bearing C57BL/6 mice, but it did not affect the survival of immunocompetent tumor bearing mice (Fig. 3E and F). CENP-E knockdown reduced the infiltration of CD8+ T cells and the secretion of IFN-γ in CD8+ T cells and increased the proportion of Tregs compared to control mice (Fig. 3G). Therefore, the inhibition of CENP-E in lung cancer reduced CD8+ T cells and elevated Tregs, which led to immunosuppression.
The inhibition of CENP-E stabilized PD-L1 mRNA by TTP targeting of the 3’UTR.
To determine whether CENP-E regulated the stability of PD-L1 mRNA and its mechanism, we conducted mRNA stability assays. The inhibition or knockdown of CENP-E retarded the degradation of PD-L1 mRNA and enhanced the stability of PD-L1 mRNA (Fig. 4A). A luciferase reporter gene assay was performed to further investigate whether the 3’ UTR mediated this influence on the stability of PD-L1 mRNA. The inhibition of CENP-E using GSK923295 in PD-L1 3’UTR wild-type A549 cells enhanced the luciferase activity and stabilized the mRNA. After mutation in the PD-L1 3’UTR, the inhibition of CENP-E did not increase the luciferase activity (Fig. 4B). Therefore, the influence of CENP-E on the stability of PD-L1 mRNA was mediated by the PD-L1 3’UTR. To identify the proteins regulating the stability of PD-L1 mRNA, we performed a selected screen of likely candidate genes TTP and UPF1 (both genes regulate mRNA stability via the 3’UTR). PD-L1 expression was increased after the knockdown of TTP in A549 cells, not UPF1 knockdown (Fig. 4C). The inhibition of CENP-E in A549 cells downregulated TTP expression, and the high expression of TTP inhibited PD-L1 expression. The CENPE inhibitor-induced upregulation of PD-L1 was rescued with the upregulation of TTP (Fig. 4D). The CENP-E protein was bound to the TTP promoter (Fig. 4E). We used a TTP primary antibody to immunoprecipitate the bound RNA. The results demonstrated considerable mRNA expression of PD-L1 in IP samples, which indicates the interaction between TTP and the mRNA of PD-L1 (Fig. 4F). Therefore, CENP-E stabilized PD-L1 mRNA via the 3’UTR, which was mediated by TTP. All of these findings show that CENP-E bound and induced of the transcription of TTP, and the inhibition of CENP-E stabilized PD-L1 mRNA by TTP targeting of the 3’UTR.
Synergy between CENP-E inhibition and PD-L1 mAbs enhanced the antitumor effect.
Our previous data demonstrated that GSK923295 induced PD-L1 expression in A549 cells and resulted in immunosuppression. This pathway may be the mechanism for future resistance, and the high expression of PD-L1 may indicate that the patient would benefit from an anti-PD-L1 antibody. We cultured A549 cells with mononuclear cells isolated from non-small cell lung cancer patient MPE to investigate the efficacy of the combination therapy of GSK923295 and an anti-PD-L1 antibody atezolizumab. The results showed no difference between A549 cells treated with an anti-PD-L1 antibody and no treatment. GSk923295 slightly suppressed the colony formation ability of A549 cells, and the treatment of GSK923295 combined with anti-PD-L1 antibody inhibited colony formation activity more efficiently (Fig. 5A). Control and CENP-E knockdown Lewis lung cancer-bearing mice were treated with PD-L1 antibodies. CENP-E knockdown without anti-PD-L1 antibody treatment and PD-L1 treatment alone in control mice suppressed tumor growth to some extent, and anti-PD-L1 antibody treatment in CENP-E knockdown mice inhibited the tumor growth more obviously (Fig. 5C and D). Survival was also significantly improved by the combination treatment of PD-L1 antibodies with CENP-E knockdown (Fig. 5E). Tumors treated with the anti-PD-L1 antibody enhanced the effector CD8 + T cell response, and the tumor-infiltrating Treg response was impaired. (Fig. 5F and G).
High expression of CENP-E indicated poor prognosis in lung cancer.
We analyzed the expression of CENP-E in lung cancer using the website UALCAN and found obvious high expression of CENP-E in lung adenocarcinoma and squamous cell carcinoma. The expression of CENP-E was 5.8-fold higher in lung adenocarcinoma tissues than normal tissues (0.304 vs. 2.073, p < 0.0001). The expression of CENP-E was 13.5-fold higher in lung squamous cell carcinoma tissues than normal tissues (0.385 vs. 5.571, p < 0.0001)(Fig. 6A). We used the TCGA database to analyze the expression levels of various tumor CENP-E proteins in patients. We found high expression of CENP-E in lung cancer, colon cancer, breast cancer, stomach cancer, urothelial cancer, testicular cancer and melanoma (Fig. 6B). However, further analysis found that CENP-E was only related to the prognosis of lung cancer (Fig. S1).
To explore the prognostic significance of CENP-E in lung cancer patients, bioinformatic analysis was conducted using the website Kaplan-Meier Plotter. Lung cancer patients with high CENP-E expression had better outcome than patients with low CENP-E expression, including FP (first progression) (10 months vs. 28 months, p < 0.0001), OS (overall survival) (45 months vs. 96 months, p < 0.0001) and PPS (post progression survival) (10.6 months vs. 19.47 months, p = 0.012) (Fig. 6C). Collectively, all of the TCGA data suggested the prognostic value of CENP-E in lung adenocarcinoma patients.
Bioinformatic analysis was performed using the website R2: Genomics Analysis and Visualization Platform to explore the relationship between the expression of CENP-E and immune molecules. Surprisingly, the results indicated that CENP-E in lung cancer tissues negatively correlated with checkpoint PD-L1 (CD274), the ligand of TIM3 galectin-9 and the ligand of BTLA HVEM. CENP-E in lung cancer tissues positively correlated with the immunostimulating cytokines IL2 and IL12B and negatively correlated with the immunosuppressive factor TGFB (Fig. 6D). Therefore, our results demonstrated the relationship between CENP-E and the outcome of patients and the expression of immune checkpoint and cytokines in lung cancer. All of these results suggest that a CENP-E inhibitor would exhibit anti-tumor effects and upregulate PD-L1-induced immunosuppression in lung cancer. Elevated PD-L1 levels provide a possible strategy for combination immunotherapy, and this combination of immunotherapeutic strategies may offset the negative effects of a CENP-E inhibitor on its immune-mediated antitumor abilities in lung cancer treatment.