YAP regulates PD-L1 expression in human NSCLC cells

Abstract

Programmed death-ligand 1 (PD-L1) is a membrane protein on tumor cells that binds to the PD-1 receptor expressed on immune cells, leading to the immune escape of tumor cells. Yes-associated protein (YAP) is a main effector of the Hippo/YAP signaling pathway, which plays important roles in cancer development. Here we show that YAP regulates PD-L1 expression in human non-small cell lung cancer (NSCLC) cells. First, we investigated YAP and PD-L1 expression at the protein level in 142 NSCLC samples and 15 normal lung samples. In tumor tissue, immunohistochemistry showed positive staining for YAP and PD-L1, which correlated significantly (n = 142, r = 0.514, P < 0.001). Second, in cell lines that express high levels of PD-L1 (H460, SKLU-1, and H1299), the ratio of p-YAP/YAP was lower and GTIIC reporter activity of the Hippo pathway was higher than those in three cell lines expressing low levels of PD-L1 (A549, H2030, and PC9) (P < 0.05). Third, in the same three cell lines, inhibition of YAP by two small interfering RNAs (siRNAs) decreased the mRNA and protein level of PD-L1 (P < 0.05). Fourth, forced overexpression of the YAP gene rescued the PD-L1 mRNA and protein level after siRNA knockdown targeting 3'UTR of the endogenous YAP gene. Finally, chromatin immunoprecipitation (ChIP) assays using a YAP-specific monoclonal antibody resulted in the precipitation of PD-L1 enhancer region encompassing two putative TEAD binding sites. Our results indicate that YAP regulates the transcription of PD-L1 in NSCLC.

Keywords: hippo pathway; non-small cell lung cancer; programmed death-ligand 1; yes-associated protein.

Figure 1. Immunohistochemistry of YAP and PD-L1 in human NSCLC tissues

Representative image showing expression of YAP protein (A) and PD-L1 protein (B) in human NSCLC tissues and normal lung tissues analyzed by immunohistochemistry. (A:1) and (B:1) are normal lung tissues. (A:2–7) and (B: 2–7) are NSCLC tissues. (A:5–7) Staining of YAP was localized in nuclei (arrow) and (B:5–7) staining of PD-L1 was localized in membrane (arrow), under a 20× objective lens. – and + mean negative; ++ and +++ mean positive.

Figure 2. Expression of YAP and PD-L1 in NSCLC cell lines

(A–B) The mRNA levels of YAP and PD-L1 in NSCLC cell lines were measured using qRT-PCR, and LP-9 cell line was used as control (F = 174.10 P < 0.001; F = 635.77 P < 0.001). VS LP-9: ^*P < 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001. (C): GTIIC reporter activity of the Hippo pathway in NSCLC cell lines, and LP-9 cell line was used as control (F = 311.39; P < 0.001). VS LP-9: ^*P < 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001. (D) pYAP/YAP ratio in NSCLC cell lines based on the value of Western blot. (E) Western blot was used to detect levels of YAP, pYAP and PD-L1 in NSCLC cell lines. GAPDH was detected as a loading control. Band intensity was analyzed with ImageJ software and normalized with the intensity of GAPDH band.

Figure 3. YAP and PD-L1 expression in H460, SKLU-1 and H1299 cell lines after knockdown of YAP

(A) YAP mRNA level in H460, SKLU-1 and H1299 cells after YAP inhibition by siRNA1,2 was measured using qRT-PCR. (B) PD-L1 mRNA level in H460, SKLU-1 and H1299 cells after YAP inhibition by siRNA1,2 was measured using qRT-PCR. (C) Western Blot analysis in H460, SKLU-1 and H1299 cell lines transfected with YAP siRNA1,2 or control siRNA. GAPDH was detected as a loading control. Band intensity was analyzed with ImageJ software and normalized with the intensity of GAPDH band. YAP-knockdown reduced PD-L1 expression significantly. ^*P < 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001.

Figure 4. Expression of PD-L1 and YAP after YAP forced over-expression in siRNA-YAP H460 cells

(A–B) qRT-PCR analysis of mRNA levels of YAP and PD-L1 after YAP silencing by siRNA and/or forced over-expression of the YAP gene in H460 cells. (C) Western blot analysis of YAP, PD-L1 after YAP silencing by siRNA and/or forced over-expression of the YAP gene in H460 cells. GAPDH was detected as a loading control. Band intensity was analyzed with ImageJ software and normalized with the intensity of GAPDH band. ^*P < 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001.

Figure 5. YAP regulates PD-L1 at the transcriptional level through binding to PD-L1 enhancer

(A) Schematic of the PD-L1 promoter region. Sequence analysis revealed a putative YAP1-TEAD co-binding site between −7911 to −7941 nucleotides upstream of the transcription start site. (B) ChIP assays were performed with SKLU-1 and H1299 cells in two regions (203 bp and 210 bp), which were shown by gel bands of RT-PCR products with 36 cycles. (C) ChIP assays were performed with A549, H1975, H1299 and SKLU-1 cells in two regions (203 bp and 210 bp), which were shown by gel bands of RT-PCR products with 30 cycles.