Yes-associated protein (YAP) is a main mediator of the Hippo pathway, which promotes cancer development. Here we show that YAP promotes resistance to erlotinib in human non-small cell lung cancer (NSCLC) cells. We found that forced YAP overexpression through YAP plasmid transfection promotes erlotinib resistance in HCC827 (exon 19 deletion) cells. In YAP plasmid-transfected HCC827 cells, GTIIC reporter activity and Hippo downstream gene expression of AREG and CTGF increased significantly (P<0.05), as did ERBB3 mRNA expression (P<0.05). GTIIC reporter activity, ERBB3 protein and mRNA expression all increased in HCC827 erlotinib-resistance (ER) cells compared to parental HCC827 cells. Inhibition of YAP by small interfering RNA (siRNA) increased the cytotoxicity of erlotinib to H1975 (L858R+T790M) cells. In YAP siRNA-transfected H1975 cells, GTIIC reporter activity and downstream gene expression of AREG and CTGF decreased significantly (P<0.05). Verteporfin, YAP inhibitor had an effect similar to that of YAP siRNA; it increased sensitivity of H1975 cells to erlotinib and in combination with erlotinib, synergistically reduced migration, invasion and tumor sphere formation abilities in H1975 cells. Our results indicate that YAP promotes erlotinib resistance in the erlotinib-sensitive NSCLC cell line HCC827. Inhibition of YAP by siRNA increases sensitivity of erlotinib-resistant NSCLC cell line H1975 to erlotinib.

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.

Alterations of the EGFR/ERK and Hippo/YAP pathway have been found in non-small cell lung cancer (NSCLC). Herein, we show that ERK1 and ERK2 have an effect on the Hippo/YAP pathway in human NSCLC cells. Firstly, inhibition of ERK1/2 by siRNA or small-molecular inhibitors decreased the YAP protein level, the reporter activity of the Hippo pathway, and the mRNA levels of the Hippo downstream genes, CTGF, Gli2, and BIRC5. Secondly, degradation of YAP protein was accelerated after ERK1/2 depletion in NSCLC cell lines, in which YAP mRNA level was not decreased. Thirdly, forced over-expression of the ERK2 gene rescued the YAP protein level and Hippo reporter activity after siRNA knockdown targeting 3′UTR of the ERK2 gene in NSCLC cells. Fourthly, depletion of ERK1/2 reduced the migration and invasion of NSCLC cells. Combined depletion of ERK1/2 had a greater effect on cell migration than depletion of either one separately. Finally, the MEK1/2 inhibitor Trametinib decreased YAP protein level and transcriptional activity of the Hippo pathway in NSCLC cell lines. Our results suggest that ERK1/2 inhibition participates in reducing YAP protein level, which in turn down-regulates expression of the downstream genes of the Hippo pathway to suppress migration and invasion of NSCLC cells.

Malignant mesothelioma is an aggressive cancer that is resistant to current therapy. The poor prognosis of mesothelioma has been associated with elevated Yes‐associated protein (YAP) activity. In this study, we evaluated the effect of targeting YAP in mesothelioma. First, we comprehensively studied YAP activity in five mesothelioma cell lines (211H, H2052, H290, MS‐1 and H2452) and one normal mesothelial cell line (LP9). We found decreased phospho‐YAP to YAP protein ratio and consistently increased GTIIC reporter activity in 211H, H2052 and H290 compared to LP9. The same three cell lines (IC50s < 1 μM) were more sensitive than LP9 (IC50 = 3.5 μM) to the YAP/TEAD inhibitor verteporfin. We also found that verteporfin significantly reduced YAP protein level, mRNA levels of YAP downstream genes and GTIIC reporter activity in the same three cell lines, indicating inhibition of YAP signaling by verteporfin. Verteporfin also impaired invasion and tumoursphere formation ability of H2052 and H290. To validate the effect of specific targeting YAP in mesothelioma cells, we down‐regulated YAP by siRNA. We found siYAP significantly decreased YAP transcriptional activity and impaired invasion and tumoursphere formation ability of H2052 and H290. Furthermore, forced overexpression of YAP rescued GTIIC reporter activity and cell viability after siYAP targeting 3′UTR of YAP. Finally, we found concurrent immunohistochemistry staining of ROCK2 and YAP (P < 0.05). Inhibition of ROCK2 decreased GTIIC reporter activity in H2052 and 211H suggesting that Rho/ROCK signaling also contributed to YAP activation in mesothelioma cells. Our results indicate that YAP may be a potential therapeutic target in mesothelioma.

Rationale: Glucagon is involved in hepatic gluconeogenesis, playing a key role in type 2 diabetes. Ginsenosides are reported to have antidiabetic activities. Ginsenoside Rg1 is a major propanaxatriol-type saponin in ginseng. This study aims to investigate the regulatory effects of Rg1 on glucagon-induced hepatic glucose production.Methods: The effects of Rg1 were investigated in high-fat-diet (HFD)-fed mice and glucagon-challenged C57BL/6J mice. Glucose metabolism was evaluated by oral glucose tolerance test and pyruvate tolerance test. The role of Rg1 on the regulation of Akt-FoxO1 interaction was performed using immunofluorescence, immunoprecipitation, siRNA silencing, pharmacological inhibitor and active-site mutant in primary hepatocytes or HepG2 cells.Results: Abnormally elevated fasting glucagon levels were observed in HFD-fed mice, contributing significantly to increased fasting plasma glucose levels. Inappropriate fasting glucagon secretion inactivated Akt and promoted hepatic glucose production via upregulation of FoxO1 activity. Rg1 preserved glucagon-impaired Akt activation partly by binding to Akt at Ser473 site. Rg1 also promoted Akt binding to FoxO1 and inactivated FoxO1 by phosphorylation. Consequently, Rg1 decreased the hepatic glucose production through a decrease in transcription of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase). Both siRNA silencing of Akt and Akt inhibitor triciribine attenuated the effects of Rg1 in response to fasting hormone glucagon.Conclusion: Akt phosphorylation at Ser473 by ginsenoside Rg1 is critical for its gluconeogenesis-lowering effect, suggesting a potential for pharmaceutical intervention in response to fasting hormone glucagon.

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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.

BackgroundCasein kinase 2 (CK2) is involved in various cellular events such as proliferation, apoptosis, and the cell cycle. CK2 overexpression is associated with multiple human cancers and may therefore be a promising target for cancer therapy. To identity novel classes of inhibitors for CK2, we screened a natural product library obtained from National Cancer Institute.MethodsThe quantitative luminescent kinase assay ADP-Glo™ was used to screen CK2 inhibitors from the natural product library. The same assay was used to determine cell-free dose-dependent response of CK2 inhibitors and conduct a kinetic study. Docking was performed to predict the binding patterns of selected CK2 inhibitors. Western blot analysis was used to evaluate Akt phosphorylation specific to CK2 and apoptosis effect. The cell viability assay CellTiter-Glo® was used to evaluate the inhibition effects of CK2 inhibitors on cancer cells.ResultsWe identified coumestrol as a novel reversible ATP competitive CK2 inhibitor with an IC50 value of 228 nM. Coumestrol is a plant-derived compound that belongs to the class of phytoestrogens, natural compounds that mimic the biological activity of estrogens. In our study, coumestrol showed high selectivity among 13 kinases. The hydrogen bonds formed between coumestrol and the amino acids in the ATP binding site were first reviewed by a molecular docking study that suggested a possible interaction of coumestrol with the hinge region of ATP site of CK2. In addition, coumestrol inhibited cancer cell growth partially through down-regulation of CK2-specific Akt phosphorylation. Finally, coumestrol exerted strong inhibition effects on the growth of three cancer cell lines.ConclusionOur study shows that coumestrol, a novel ATP competitive and cell permeable CK2 inhibitor with submicromolar IC50, had inhibition effects on the growth of three cancer cell lines and may represent a promising class of CK2 inhibitors.