MET Receptor Tyrosine Kinase Inhibition Reduces Interferon-Gamma (IFN-γ)-Stimulated PD-L1 Expression through the STAT3 Pathway in Melanoma Cells

Abstract

Melanoma is the leading cause of death from cutaneous malignancy. While targeted therapy and immunotherapy with checkpoint inhibitors have significantly decreased the mortality rate of this disease, advanced melanoma remains a therapeutic challenge. Here, we confirmed that interferon-gamma (IFN-γ)-induced PD-L1 expression in melanoma cell lines. This increased expression was down-regulated by the reduction in phosphorylated STAT3 signaling via MET tyrosine kinase inhibitor treatment. Furthermore, immunoprecipitation and confocal immunofluorescence microscopy analysis reveals MET and PD-L1 protein-protein interaction and colocalization on the cell surface membrane of melanoma cells. Together, these findings demonstrate that the IFN-γ-induced PD-L1 expression in melanoma cells is negatively regulated by MET inhibition through the JAK/STAT3 signaling pathway and establish the colocalization and interaction between an RTK and a checkpoint protein in melanoma cells.

Keywords: MET; PD-L1; STAT3; immunotherapy; melanoma; tyrosine kinase inhibitors.

Figure 1

Interferon-γ treatment upregulates PD-L1 expression in melanoma cells, and MET inhibitors down-regulate interferon-γ induced PD-L1 expression in the melanoma cell lines. (A) Flow cytometry analysis of PD-L1 expression on the cell membrane of melanoma cells, treated with interferon-γ. Mean fluorescent intensity (MFI) values in the graph are mean ± SD calculated from three independent experiments. (B) Western blot analysis of PD-L1 expression in total protein extracts from melanoma cells. All experiments were treated with interferon-γ (50 ng/mL) for 48 h. (C) Flow cytometry analysis from one independent experiment showing the labeled cells in the absence (−) or presence (+) of IFN-γ with the indicated concentration of MET inhibitor (Crizotinib). (D) Flow cytometry analysis of PD-L1 protein on the cell membrane of melanoma cells upon 48 h treatment with IFN-γ alone or in combination with the MET inhibitors as indicated concentration. Western blot analysis of PD-L1 expression on total protein extracts from melanoma cells treated with 48 h with IFN-γ alone or in combination with Crizotinib (E) or PHA665752 (F). * p < 0.05, ** p < 0.01, *** p < 0.005. The uncropped blots are shown in Figure S4.

Figure 2

MET inhibition diminishes IFN-γ-induced PD-L1 expression by reducing STAT3 activation. Analysis of MET, AKT, ERK, FAK, GSK3β, and STAT3 activation/expression together with PD-L1 expression in lysates obtained from four melanoma cells treated for 48 h with IFN-γ (25 ng/mL) alone or in combination with the Crizotinib (Crizo) (A,B). Densitometric quantification for PD-L1 (C) and phosphorylated-STAT3 expression (D) were quantified using Image Studio^TM Lite v 5.2 (LI-COR, USA) software and quantitative data are presented as mean ± SD based on 3 independent experiments. * p < 0.05, ** p < 0.01. The uncropped blots are shown in Figure S5A,B.

Figure 3

Co-immunoprecipitation of MET and PD-L1 from lysates of melanoma cells. Extracts were prepared from each cell line after treatment with HGF (25 ng/mL) at the indicated times and immunoprecipitated using c-MET (A,C) or PD-L1 (B,D) antibodies. Each immunoprecipitant was probed for the presence of MET, pMET (Tyr 1234/35), pMET (Tyr 1349), and PD-L1. Immunoblotting of whole cell lysates (WCL) was performed with antibodies against c-MET and PD-L1 to examine total levels of MET and PD-L1. WCL immunoblotting with actin was used as the loading control. Co-localization analysis of MET and PD-L1 expression by immunofluorescence analysis. Colocalization of PD-L1 (Alexa Fluor 488) with MET (Alexa Fluor 647) in the plasma membrane (WGA, Alexa Fluor 555) as analyzed by confocal laser scanning microscopy in SH-4 (E), RPMI-7951 (F), SK-MEL-28 (G), and WM-35 (H) melanoma cell lines. Pearson’s correlation between PD-L1 and MET expression in melanoma cells is shown in the colocalized PD-L1 and MET column in each cell line. Scale bars: 10 μm. The uncropped blots are shown in Figure S6A–D.

Figure 4

Schematic diagram of the proposed working model for the inhibition of PD-L1 by MET inhibitors in melanoma cell lines. MET inhibitors inhibit IFN-γ-induced STAT3 phosphorylation, leading to reduced PD-L1 expression and membrane presentation. IFN-γ, interferon-gamma (Created with BioRender.com: OD25HC9TFN).