MUC3A induces PD-L1 and reduces tyrosine kinase inhibitors effects in EGFR-mutant non-small cell lung cancer

Abstract

The immune checkpoint ligand programmed death-ligand 1 (PD-L1) and the transmembrane mucin (MUC) 3A are upregulated in non-small cell lung cancer (NSCLC), contributing to the aggressive pathogenesis and poor prognosis. Here, we report that knocking down the oncogenic MUC3A suppresses the PD-L1 expression in NSCLC cells. MUC3A is a potent regulator of epidermal growth factor receptor (EGFR) stability, and MUC3A deficiency downregulates the activation of the PI3K/Akt and MAPK pathways, which subsequently reduces the expression of PD-L1. Furthermore, knockdown of MUC3A and tyrosine kinase inhibitors (TKIs) in EGFR-mutant NSCLC cells play a synergistic effect on inhibited proliferation and promoted apoptosis in vitro. In the BALB/c nude mice xenograft model, MUC3A deficiency enhances EGFR-mutated NSCLC sensitivity to TKIs. Our study shows that transmembrane mucin MUC3A induces PD-L1, thereby promoting immune escape in NSCLC, while downregulation of MUC3A enhances TKIs effects in EGFR-mutant NSCLC. These findings offer insights into the design of novel combination treatment for NSCLC.

Keywords: EGFR; MUC3A; PD-L1.; non-small cell lung cancer.

Figure 1

MUC3A was positively correlated with PD-L1 expression. (A) Representative IHC images of PD-L1 expression. Scoring was measured by the percentage of positive cells with the following staining intensities: less than 5% scored “0”; 5-24% scored “1”; 25-49% scored “2”; 50-74% scored “3”; and more than 74% scored “4”. (B) and (C) The level of MUC3A was positively correlated with PD-L1 expression. (D) High levels of MUC3A and PD-L1 were related to poor clinical outcomes. (E) Relative MUC3A mRNA expression in BEAS-2B, H1975, PC9, H1299 and H292. *, p < 0.05.

Figure 2

MUC3A induced PD-L1 in EGFR-mutant NSCLC cell lines. (A) The mRNA levels of PD-L1 were independent of the mRNA levels of EGFR. (B) The mRNA levels of PD-L1 were correlated with EGFR mutation. Data of lung cancer were downloaded from the TCGA database. 544 non-mutation, 32 mutation. (C, D) The PD-L1 mRNA levels were determined by real-time PCR analysis after EGF stimulation for 24 hours. (E, F) The PD-L1 protein levels were determined by flow cytometry analysis after EGF stimulation for 24 hours. (G) The PD-L1 mRNA levels were determined by real-time PCR analysis after EGF stimulation for 24 hours. EGF, 50 ng/mL. *, p < 0.05; **, p < 0.01; ns: not significant. NC: negative control.

Figure 3

MUC3A promoted the activity of PI3K/Akt and MAPK pathway. (A) and (B) MUC3A promoted the activity of PI3K/Akt and MAPK pathway, and knockdown of MUC3A decreased EGFR protein levels. Representative immunoblotting of total and phosphorylated EGFR, AKT, MEK and ERK in H1975 and PC9 cells. (C) Quantifications of phosphorylated AKT, MEK and ERK. (D) MUC3A knockdown had no effect on EGFR mRNA levels, but decreased EGFR protein levels. (E) Immunoblotting of PI3K/Akt and MAPK pathway proteins with MEK and PI3K inhibitor treatment for 1 hour. (F) Flow cytometry of PD-L1 expression on the surface of H1975 cells treated with EGF, MEK and PI3K inhibitor for 24 hours. (G) Immunoblotting of PI3K/Akt and MAPK pathway proteins with AKT and ERK activator treatment for 1 hour. (H) Flow cytometry of PD-L1 expression on the surface of H1975 and PC9 cells treated with SC79 or honokiol for 24 hours. Abbreviations are as follows: MFI, mean fluorescence intensity; EGF, 50 ng/mL; MEK inhibitor GSK1120212, 25 nM; PI3K inhibitor GDC-0941, 500 nM; AKT activator SC79, 5 μg/mL; ERK activator honokiol, 20 μM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns: not significant. NC: negative control.

Figure 4

MUC3A deficiency improved NSCLC sensitivity to TKIs in vitro. (A) and (B) MUC3A deficiency potentiated gefitinib and AZD-9291-induced growth inhibition in H1975 cells. (C) and (D) Cell growth inhibition after exposed to AZD-9291 (0.1 μM) and Gefitinib (10 μM) for 24, 48 and 72 hours. (E) and (F) MUC3A knockdown increases NSCLC cell apoptosis induced by Gefitinib (10 μM) and AZD-9291 (0.1 μM). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns: not significant. NC: negative control.

Figure 5

MUC3A deficiency improved NSCLC sensitivity to TKIs in vivo. (A) The overall scheme of animal experiments. (B) Twenty-four nude mice were sacrificed on day 38. (C) Gross view of tumor. N = 6. (D) Growth curves of tumor volume indicate that the combination therapy significantly inhibits tumor growth in vivo compared with single treatment groups. N = 6. (E) Tumor weight. N = 6. (F) Representative H&E staining images of tumor, and representative IHC images of p-EGFR, p-AKT and p-ERK, scale bar: 100 μm. (G) and (H) Representative IHC images of MUC3A and PD-L1 in tumor tissues. The MUC3A deficiency group had less MUC3A and PD-L1 expression than that of control. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns: not significant. NC: negative control.

Figure 6

Schematic model proposed for the role of MUC3A inducing PD-L1 and reducing TKI effects in EGFR-mutant NSCLC. MUC3A induced PD-L1 via PI3K/AKT and MAPK pathway. MEK and PI3K inhibitors (trametinib and pictilisib) blocked EGF-induced PD-L1 expression. TKIs (AZD-9291) inhibited the cell proliferation.