Inhibition of yes-associated protein down-regulates PD-L1 (CD274) expression in human malignant pleural mesothelioma

Abstract

Although tumour PD-L1 (CD274) expression had been used as a predictive biomarker in checkpoint immunotherapy targeting the PD1/PD-L1 axis in various cancers, the regulation of PD-L1 (CD274) expression is unclear. Yes-associated protein (YAP), an important oncogenic protein in Hippo signalling pathway, reportedly promotes cancer development. We investigated whether inhibition of YAP down-regulates PD-L1 (CD274) in human malignant pleural mesothelioma (MPM). Western blotting showed that 2 human MPM cell lines (H2052 and 211H) had increased PD-L1 protein expression compared to H290, MS-1 and H28 cells. In H2052 and 211H cells, PD-L1 mRNA expression was significantly increased compared to other MPM cell lines; YAP knockdown by small interfering RNA decreased PD-L1 protein and mRNA expression. Forced overexpression of the YAP gene increased PD-L1 protein expression in H2452 cells. Chromatin immunoprecipitation (ChIP) assay showed the precipitation of PD-L1 enhancer region encompassing 2 putative YAP-TEAD-binding sites in H2052 cells. We found that, in human MPM tissue microarray samples, YAP and PD-L1 concurrently expressed in immunohistochemistry stain (n = 70, P < .05, chi-square). We conclude that PD-L1 is correlated with YAP expression, and inhibition of YAP down-regulates PD-L1 expression in human MPM. Further study of how YAP regulates PD-L1 in MPM is warranted.

Keywords: PD-L1; checkpoint immunotherapy; hippo signaling; malignant pleural mesothelioma; yes-associated protein.

Figure 1

PD‐L1 and yes‐associated protein (YAP) expression in human malignant pleural mesothelioma cell lines. (A) PD‐L1, YAP and p‐YAP (S127) protein expression and p‐YAP (S127)/YAP ratio in mesothelioma cell lines. (B) GTIIC reporter activity of human normal mesothelial cell line LP9 and mesothelioma cell lines. (C) PD‐L1 mRNA expression in human normal mesothelial cell line LP9 and mesothelioma cell lines. (D) YAP mRNA expression in human normal mesothelial cell line LP9 and mesothelioma cell lines. Error bars indicate standard deviations; ***P ≤ .001

Figure 2

Inhibition of yes‐associated protein (YAP) decreased PD‐L1 expression in mesothelioma H2052 and 211H cells. Western blotting was used to determine PD‐L1 protein expression after YAP knockdown (A). In both cell lines, after YAP knockdown, YAP mRNA expression significantly decreased (B, C), as did PD‐L1 mRNA expression (D, E), and GTIIC reporter activity (F, G). (H) Hippo/YAP downstream gene AREG and CTGF mRNA expression decreased after YAP knockdown in H2052 cells (H) and in 211H cells (I). Error bars indicate standard deviations; *P < .05; **P ≤ .01 ***P ≤ .001

Figure 3

Yes‐associated protein (YAP) forced overexpression rescued PD‐L1 expression during YAP inhibition. After YAP forced overexpression in H2052 and 211H cells treated with YAP 3′UTR siRNA, PD‐L1 protein expression increased in both cell lines, as shown by Western blotting (A), YAP mRNA expression increased significantly in H2052 cells (B) and in 211H cells (D), and PD‐L1 mRNA expression increased significantly in H2052 cells (C) and in 211H cells (E). Error bars indicate standard deviations; **P ≤ .01

Figure 4

Yes‐associated protein (YAP) binds the enhancers of PD‐L1 and regulates PD‐L1 at the transcriptional level. (A) Sequence analysis of PD‐L1 showed putative YAP binding sites between −7911 and −7946 nucleotides upstream of the transcription start site. (B) ChIP assays were performed with H2052 cells in 2 regions (203 and 210 bp), shown by gel bands of RT‐PCR products

Figure 5

The yes‐associated protein (YAP) inhibitor verteporfin decreased PD‐L1 expression in H2052 and 211H cells. After cells were treated 1.0 and 2.0 μmol/L verteporfin, (A, B) Western blotting showed that PD‐L1 protein expression decreased, (C, D) real‐time PCR showed that PD‐L1 mRNA expression significantly decreased, (E, F) GTIIC reporter activity significantly decreased, and (G, H) Hippo/YAP downstream genes AREG and CTGF significantly decreased. Error bars indicate standard deviations; *P < 0.05; **P ≤ .01 ***P ≤ .001

Figure 6

Immunohistochemistry of yes‐associated protein (YAP) and PD‐L1 staining in mesothelioma and normal pleura samples. (A, B, I, J) Normal pleura sample. (C‐H) Mesothelioma samples. (C, D, K, L) Weak PD‐L1 and YAP stain. (E, F, M, N) Moderate PD‐L1 and YAP stain. (G, H, O, P) Strong PD‐L1 and YAP stain. YAP staining localized at nuclei (arrows) of mesothelioma under a 40× objective lens. (Q) Cytoplasmic PD‐L1 staining was concurrent with nucleus YAP staining in mesothelioma samples (P < .05, chi‐square test)