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.

Loss of VGLL 4 suppresses tumor PD ‐L1 expression and immune evasion

“…Indeed, the expression of miR-130a mimic or miR-130a sponge suppressed or enhanced VGLL4 and PD-L1 levels, respectively, in A549 cells (Fig 6A-C). Consistent with previous reports Lee et al, 2017;Miao et al, 2017;Janse van Rensburg et al, 2018;Kim et al, 2018), we observed an increased PD-L1 mRNA level when YAP5SA, a transcriptionally active and Hippo pathway-resistant YAP mutant, was expressed in A549 cells (Fig EV5B). Consistent with previous reports Lee et al, 2017;Miao et al, 2017;Janse van Rensburg et al, 2018;Kim et al, 2018), we observed an increased PD-L1 mRNA level when YAP5SA, a transcriptionally active and Hippo pathway-resistant YAP mutant, was expressed in A549 cells (Fig EV5B).…”

“…Furthermore, expression of YAP target genes CTGF and CYR61 was slightly decreased by inhibition of miR-130a ( Fig 6D), while knockdown of VGLL4 slightly increased miR-130a expression ( Fig EV5A), confirming the positive feedback loop of YAP-miR-130a-VGLL4 axis. Consistent with previous reports Lee et al, 2017;Miao et al, 2017;Janse van Rensburg et al, 2018;Kim et al, 2018), we observed an increased PD-L1 mRNA level when YAP5SA, a transcriptionally active and Hippo pathway-resistant YAP mutant, was expressed in A549 cells ( Fig EV5B). However, we detected a strong inhibition on ◀ Figure 3.…”

“…Recently, several studies suggest that YAP/TAZ promote PD-L1 expression through TEAD-mediated transcriptional regulation Lee et al, 2017;Miao et al, 2017;Janse van Rensburg et al, 2018;Kim et al, 2018). Interestingly, one study showed that VGLL4 expression is repressed by YAP/TEAD transcriptional complex through miR-130a , which promotes us to test whether miR-130a and YAP could suppress IFNc-inducible PD-L1 expression.…”

“…Many studies reported that the dysregulation of several oncogenic or tumor-suppressive pathways constitutively activates the expression of PD-L1, suggesting that this is a general mechanism of tumorigenesis. PTEN deletions, PI3K/AKT mutations (Parsa et al, 2007;Lastwika et al, 2016), EGFR mutations (Akbay et al, 2013), MYC overexpression (Casey et al, 2016), CDK5 disruption (Dorand et al, 2016), and YAP/TAZ activation Lee et al, 2017;Miao et al, 2017;Janse van Rensburg et al, 2018;Kim et al, 2018) represent a rapidly growing list of genetic mechanisms of constitutive PD-L1 expression. Expression of PD-L1 can be induced by many cytokines, of which interferon-gamma (IFNc) is the most potent via JAK1/2-STAT1/2/3-IRF1 signaling axis, with IRF1 binding to PD-L1 promoter (Dong et al, 2002;Loke & Allison, 2003;Lee et al, 2006;Garcia-Diaz et al, 2017).…”

Transforming growth factor beta induces fibroblasts to express and release the immunomodulatory protein PD‐L1 into extracellular vesicles

“…As shown in Figure5B, YAP/ TAZ knockdown prevented TGFβ stimulated PD-L1 expression in both human and murine fibroblasts, consistent with previous reports that PD-L1 is a direct transcriptional target of YAP/TAZ in several tumor models. [47][48][49]…”