PD-L1 expression is regulated by both DNA methylation and NF-kB during EMT signaling in non-small cell lung carcinoma

Abstract

Tumor cells, which undergo Epithelial-mesenchymal transition (EMT) acquire increased capacities of proliferation, invasion and have the ability to generate metastases by escaping the immune system during their systemic migration. To escape the immune system, cancer cells may induce tolerance or resist elimination by immune effectors via multiple mechanisms and we hypothesized that EMT may control the expression of immune checkpoint inhibitors, then promoting immune evasion. PD-L1 (programmed cell death ligand 1) but not PD-L2 nor Galectin 9 or Death receptor (DR4, DR5 and Fas) and ligands (FasL and TRAIL) expression was up-regulated during cytokine-driven EMT in a reversible manner. Moreover PD-L1 is overexpressed in VIMENTIN positive NSCLC tissues. We also demonstrated that the expression of PD-L1 required both TNFα and TGFβ1. Indeed, TGFβ1 decreased DNMT1 content and that resulted in PD-L1 promoter demethylation whereas TNFα induced the NF-κB pathway that promoted expression of demethylated PD-L1 promoter.

Keywords: DNA methylation; NF-kB; PD-L1; death receptors; epithelial-mesenchymal transition; lung cancer, cancer.

Figure 1.

TNF-α and TGF-β1 combination induced A549 EMT in a reversible manner. (A) Pipeline of EMT induction and reversion (MET) in A549 cells. Cells were treated for 5 days with TNFα/TGF-β1 and then the cytokines were removed during the 5 next days (arrows) (B) Lung cancer cells A549 were observed on the fifth day of adherence with or without TNFα/TGF-β1 treatment under the microscope (40X magnification). To observe the cells undergoing MET (mesenchymal epithelial transition), the cytokines TNF-α and TGF-β1 were removed during the next 5 days. (C) Expression of epithelial marker E-CADHERIN and mesenchymal marker VIMENTIN in A549 after treatment with TNF-α and TGF-β1 were measured by Western-Blotting. One or two weeks after the removal of cytokines, expressions of E- CADHERIN and VIMENTIN were tested again. (D) EPCAM staining measured by FACS after treatment with TNF-α and TGF-β1, as well as, after the removal of cytokines during the next five days. The experiments in panels B, C and D were realized 4 times with similar results. (E) Representative staining of F-Actin using Rhodamine Phalloidin in A549 treated or not with TNF-α/TGF-β1. Nuclei were stained with DAPI. (F) The change in the invasive capacity of A549 with or without TNFα/TGF-β1 treatment was measured using Matrigel system. This experiment was performed in duplicate, and repeated 3 times (left: quantification; right: representative pictures).

Figure 2.

TNF-α and TGF-β1 cooperate to promote PD-L1 expression during EMT (A) The expression of immune-checkpoint inhibitors as Galectin-9, CEACAM1, PD-L1, PD-L2, Vista before and after induction of EMT on A549 cells was measured by FACS and measurements were independently repeated at least three times. (B) By FACS the changes in the expression of PD-L1 and EPCAM were also confirmed with 5 days of treatment and after the removal of cytokines during the next five days. (C) At last, the changes in the expression of PD-L1 with TNF-α or TGF-β1 alone, as well as with the combination of these cytokines were measured by FACS. (D and E) Expression of PD-L1 protein and mRNA were measured following TNFα/TGF-β1 treatment by ELISA or qRT-PCR. These experiments were repeated three times with similar results.

Figure 3.

PD-L1 expression is not regulated by conventional EMT transcriptional factors in A549. (A) Expression of several transcription factors of EMT as: ZEB-1, SNAIL-1, SLUG, GLI1, GLI2, TWIST in protein levels was measured by Western Blotting after 5 days of treatment of A549 cells with TNF-α and TGFβ. This experiment was realized 3 times. (B) qRT-PCR analysis was realized to test the expression of different transcription factors of EMT mRNA levels after treatment with cytokines. GAPDH was used as a housekeeping gene. (C) The expression of VIMENTIN and E-CADHERIN were measured using Western-blotting in A549 cells after transfection with SNAIL-1 or control vector. (D) expression of EPCAM and PD-L1 was measured by FACS analysis during treatment with TNF-α and TGFβ on A549 cancer cells transfected with SNAIL-1 or control vector. Results from Fig. 3A and 3 C and 3D are representative examples from at least 3 independent experiments.

Figure 4.

PD-L1 expression blockade by E-CADHERIN overexpression during EMT in A549 cells. (A) Expression of E-CADHERIN was controlled by western blotting in A549 transfected by a pcDNA3.1 void vector or expressing E-CADHERIN. (B) reversion of mesenchymal to epithelial phenotype was measured using immunofluorescence in A549 transfected by a pcDNA3.1 void vector or expressing E-CADHERIN. Nuclei were stained by DAPI (blue), beta CATENIN by FITC (green) and E-CADHERIN by TexasRed (Red). (C) expression of PD-L1 mRNA was measured by qRT-PCR in A549 control or A549 cells transfected with a plasmid expressing E-CADHERIN (D) Expression of PD-L1 was measured by cytometry in A549 transfected by a pcDNA3.1 void vector or expressing E-CADHERIN treated or not with TNFα/TGFβ1.

Figure 5.

PD-L1 expression during EMT is dependent from NF-kB in signaling in A549 cells. (A) Activity of NF-kB was measured in A549 cells treated or not with TNF-α and TGFβ1. (B) The expression of NF-kB subunits was measured by Western Blotting after treatment with NF-kB inhibitor. (C) The expression of PD-L1 was measured using cytometry following NF-kB inhibition with JSH-23. (D) Expression of EPCAM and PD-L1 was measured using cytometry in A549 treated or not with TNF-α and TGFβ1 and transfected or not with si IKKε. These experiments were repeated tree times with similar results.

Figure 6.

PD-L1 expression is regulated by DNA methylation during EMT in A549 cells. Quantification of 5meC and 5hmeC content using ELISA in A549 cells treated or not with TNF-α and TGFβ1. (B) Quantification of GADD45 A, SMUG, AICA, DNMT1, DNMT3 A, DNMT3B, TET1, TET2 and TET3 gene expression using qRT-PCR in A549 treated or not with TNF-α and TGFβ1. (C) Expression of DNMT1 using Western blotting in cytosolic or nuclear fractions of A549 cells treated or not with TNF-α and TGFβ1. (D) Methylation of PD-L1 promoter was quantified in A549 cells treated or not with TNF-α and/or TGFβ1. (E and F) Expression of PD-L1 mRNA using qRT-PCR and methylation of PD-L1 promoter were quantified in A549 cells treated or not with TNF-α and TGFβ1 in presence or not of BAY11, or cells treated with procalamide or treated with procalamide, TNF-α and BAY11. (G and H) Recruitment of NF-kB and recruitment of DNMT1 were quantified by ChIP in in A549 cells treated or not with TNF-α and TGFβ1.

Figure 7.

PD-L1 expression is correlated to EMT-like phenotype. PD-L1 and VIMENTIN expression was quantified using IHC in a cohort of 40 non small cell lung carcinoma. (A) Examples of anti-VIMENTIN and anti-PD-L1 immunostainings in EMT- and EMT+ tumors. (B) Quantification of PD-L1 staining in EMT- and EMT+ tumors.