Yes-associated Protein Protein Research Articles

The Hippo effector YAP1 biochemically and functionally interacts with the nuclear factor‐kappa B/RELA transcription factor

The transcriptional co-activator YAP1 (yes-associated protein 1) is a critical nuclear effector of the Hippo pathway. The Hippo pathway regulates cell growth, cell motility, cell migration, and carcinogenesis, but poorly defined mechanism. We investigated biochemical and functional interactions between YAP1 and the nuclear factor (NF)-kappa B/RELA subunit in prostate cancer cell models. We demonstrated that endogenous YAP1 and RELA form protein complexes in the cell, as revealed by co-immunoprecipitation and western blotting. Compared with control, we found that combined treatment of cells with androgen and SDF-1a (stromal cell-derived factor-1 alpha) or RANKL (receptor activator of NF-kappa B ligand) enhanced the protein-protein interaction between YAP1 and RELA, as showed by proximity ligation assay. Our confocal microscopy experiment further showed that combined SDF-1a and androgen treatment promoted the YAP1 and RELA colocalization instead of single-agent treatment. Moreover, our promoter-reporter and RNAi experiments showed that knockdown of YAP1 or TEAD, a key mediator of the YAP transcription, significantly reduced the NF-Kappa B promoter-reporter gene activity. Also, disruption of YAP1 activity attenuated the TEAD-RELA interaction. Furthermore, controlled expression of MST1/STK4, a potent inhibitor of YAP1, attenuated the NF-Kappa B-promoter reporter activity. Additionally, our unbiased bioinformatics analysis of the exiting ChIP-seq (chromatin immunoprecipitation-sequencing) data sets identified several genes that are likely co-regulated by the YAP1/TEAD and NF-Kappa B transcription factor. These findings suggest that cooperative androgen and cytokine signaling regulates Hippo/YAP and NF-Kappa B interaction. The YAP1/TEAD and NF-Kappa B interaction may have critical roles in cellular biology and human diseases.

Selective Targeting of Vascular Endothelial YAP Activity Blocks EndMT and Ameliorates Unilateral Ureteral Obstruction-Induced Kidney Fibrosis.

Kidney fibrosis is accompanied by vascular dysfunction. Discovering new ways to ameliorate dysfunctional angiogenesis may bypass kidney fibrosis. YAP (Yes-associated protein) plays a multifaceted role during angiogenesis. Here, we found that selectively targeting YAP signaling in the endothelium ameliorates unilateral ureteral obstruction (UUO)-induced kidney fibrosis. Genetic deletion of Yap1, encoding YAP protein, in VE-cadherin+ endothelial cells inhibited endothelial-to-mesenchymal transition (EndMT) and dysfunctional angiogenesis and improved obstructive nephropathy and kidney fibrosis. Treatment with the systemic YAP inhibitor verteporfin worsened kidney fibrosis symptoms because of its lack of cell specificity. In an attempt to identify endothelial-specific YAP modulators, we found that G-protein-coupled receptor coagulation factor II receptor-like 1 (F2RL1) was highly expressed in vessels after UUO-induced kidney fibrosis. The F2RL1 peptide antagonist FSLLRY-NH2 selectively blocked YAP activity in endothelial cells and ameliorated kidney fibrosis. Thus, selective antagonization of endothelial YAP activity might bypass kidney fibrosis and provide new avenues for the design of antifibrotic therapies.

PVHL promotes lysosomal degradation of YAP in lung adenocarcinoma

Yes-associated protein (YAP) is a vital transcriptional co-activator that activates cell proliferation and evasion of apoptosis for the promotion of tumorigenesis. The von Hippel-Lindau tumor suppressor protein (pVHL), as a critical component of E3 ubiquitin ligase, targets various substrates to regulate tumor progression. However, the precise molecular mechanisms of pVHL during tumorigenesis remain largely unclear. Herein, we found that there was a significant negative correlation between pVHL and YAP at protein level in the TCGA-LUAD dataset and our cohort. Over-expression of pVHL decreased YAP protein expression and reduced its transcriptional activity. Further study indicated that pVHL did not affect YAP mRNA level but decreased YAP protein stability in a lysosome-dependent manner. In addition, the pVHL-mediated degradation of YAP inhibited cellular proliferation, migration, and enhanced chemosensitivity to cisplatin in lung adenocarcinoma cells. Interestingly, the pVHL-mediated YAP degradation was blocked by elevated O-GlcNAcylation. Collectively, our findings demonstrate that pVHL modulates the lysosomal degradation of YAP, and may provide more clues to better understanding the tumor suppressive effects of pVHL.

Low-Temperature Argon Plasma Regulates Skin Moisturizing and Melanogenesis-Regulating Markers through Yes-Associated Protein.

Extensive water loss and melanin hyperproduction can cause various skin disorders. Low-temperature argon plasma (LTAP) has shown the possibility of being used for the treatment of various skin diseases, such as atopic dermatitis and skin cancer. However, the role of LTAP in regulating skin moisturizing and melanogenesis has not been investigated. In this study, we aimed to determine the effect of LTAP on yes-associated protein (YAP), a major transcriptional coactivator in the Hippo signaling pathway that is involved in skin moisturizing and melanogenesis-regulating markers. In normal human epidermal keratinocytes (NHEKs), the human epidermal keratinocyte line HaCaT, and human dermal fibroblasts (HDFs), we found that LTAP exhibited increased expression levels of YAP protein. In addition, the expression levels of filaggrin (FLG), which is involved in natural moisturizing factors (NMFs), and hyaluronic acid synthase (HAS), transglutaminase (TGM), and involucrin (IVL), which regulate skin barrier and moisturizing, were also increased after exposure to LTAP. Furthermore, collagen type I alpha 1 and type III alpha 1 (COL1A1, COL3A1) were increased after LTAP exposure, but the expression level of matrix metalloproteinase-3 (MMP-3) was reduced. Moreover, LTAP was found to suppress alpha-melanocyte stimulating hormone (α-MSH)-induced melanogenesis in murine melanoma B16F10 cells and normal human melanocytes (NHEMs). LTAP regulates melanogenesis of the melanocytes through decreased YAP pathway activation in a melanocortin 1 receptor (MC1R)-dependent manner. Taken together, our data show that LTAP regulates skin moisturizing and melanogenesis through modulation of the YAP pathway, and the effect of LTAP on the expression level of YAP varies from cell to cell. Thus, LTAP might be developed as a treatment method to improve the skin barrier, moisture content, and wrinkle formation, and to reduce melanin generation.

YAP expression in endothelial cells prevents ventilator-induced lung injury.

Ventilator-induced lung injury is associated with an increase in mortality in patients with respiratory dysfunction, although mechanical ventilation is an essential intervention implemented in the intensive care unit. Intrinsic molecular mechanisms for minimizing lung inflammatory injury during mechanical ventilation remain poorly defined. We hypothesize that Yes-associated protein (YAP) expression in endothelial cells protects the lung against ventilator-induced injury. Wild-type and endothelial-specific YAP-deficient mice were subjected to a low (7 mL/kg) or high (21 mL/kg) tidal volume (VT) ventilation for 4 h. Infiltration of inflammatory cells into the lung, vascular permeability, lung histopathology, and the levels of inflammatory cytokines were measured. Here, we showed that mechanical ventilation with high VT upregulated YAP protein expression in pulmonary endothelial cells. Endothelial-specific YAP knockout mice following high VT ventilation exhibited increased neutrophil counts and protein content in bronchoalveolar lavage fluid, Evans blue leakage, and histological lung injury compared with wild-type littermate controls. Deletion of YAP in endothelial cells exaggerated vascular endothelial (VE)-cadherin phosphorylation, downregulation of vascular endothelial protein tyrosine phosphatase (VE-PTP), and dissociation of VE-cadherin and catenins following mechanical ventilation. Importantly, exogenous expression of wild-type VE-PTP in the pulmonary vasculature rescued YAP ablation-induced increases in neutrophil counts and protein content in bronchoalveolar lavage fluid, vascular leakage, and histological lung injury as well as VE-cadherin phosphorylation and dissociation from catenins following ventilation. These data demonstrate that YAP expression in endothelial cells suppresses lung inflammatory response and edema formation by modulating VE-PTP-mediated VE-cadherin phosphorylation and thus plays a protective role in ventilator-induced lung injury.

A novel isoform of Homeodomain-interacting protein kinase-2 promotes YAP/TEAD transcriptional activity in NSCLC cells.

Homeodomain-interacting protein kinase-2 (HIPK2) can either promote or inhibit transcription depending on cellular context. In this study, we show that a new HIPK2 isoform increases TEAD reporter activity in NSCLC cells. We detected HIPK2 copy number gain in 5/6 (83.3%) NSCLC cell lines. In NSCLC patients with high HIPK2 mRNA expression in the Human Protein Atlas, the five-year survival rate is significantly lower than in patients with low expression (38% vs 47%; p = 0.047). We also found that 70/78 (89.7%) of NSCLC tissues have moderate to strong expression of the N-terminal HIPK2 protein. We detected and cloned a novel HIPK2 isoform 3 and found that its forced overexpression promotes TEAD reporter activity in NSCLC cells. Expressing HIPK2 isoform 3_K228A kinase-dead plasmid failed to increase TEAD reporter activity in NSCLC cells. Next, we showed that two siRNAs targeting HIPK2 decreased HIPK2 isoform 3 and YAP protein levels in NSCLC cells. Degradation of the YAP protein was accelerated after HIPK2 knockdown in NSCLC cells. Inhibition of HIPK2 isoform 3 decreased the mRNA expression of YAP downstream gene CTGF. The specific HIPK2 kinase inhibitor TBID decreased TEAD reporter activity, reduced cancer side populations, and inhibited tumorsphere formation of NSCLC cells. In summary, this study indicates that HIPK2 isoform 3, the main HIPK2 isoform expressed in NSCLC, promotes YAP/TEAD transcriptional activity in NSCLC cells. Our results suggest that HIPK2 isoform 3 may be a potential therapeutic target for NSCLC.

Effects of miR-599 targeting YAP1 on proliferation, invasion and apoptosis of bladder urothelial carcinoma cells

The aim of this study was to investigate the effects of microRNA (miR)-599 targeting Yes-associated protein 1 (YAP1) on the proliferation, invasion and apoptosis of bladder urothelial carcinoma cell line J82. J82 cells cultured in vitro were divided into miR-nc group (transfected with analog negative control miR-NC), miR-599 group (transfected with miR-599 analog), miR-599 + pcDNA3.1 group (co transfected with miR-599 analog and pcDNA3.1 empty vector), miR-599 + pcDNA3.1-YAP1 group (co transfected with miR-599 analog and pcDNA3.1-YAP1 over expression vector), the targeting relationship between miR-599 and YAP1 was detected by double luciferase reporter gene assay, the expression level of miR-599 was detected by real-time fluorescence quantitative PCR, the expression of YAP1 was detected by Western blot, cell proliferation was detected by MTT method, cell invasion was detected by Transwell's cell experiment, and apoptosis was detected by flow cytometry. The miR-599 was able to bind to YAP1 targetingly; compared with those in miR-NC group, miR-599 expression level and apoptosis rate in miR-599 group were significantly higher, however, the expression level of YAP1 protein and the abilities of cell proliferation and invasion were significantly lower (P < 0.05); there was no significant difference in the above indexes between miR-599 group and miR-599 + pcDNA3.1 group (P > 0.05); compared with those in miR-599 + pcDNA3.1 group, the expression level of YAP1 protein and the abilities of cell proliferation and invasion in miR-599 + pcDNA3.1-YAP1 group were significantly higher, while the apoptotic rate was significantly lower (P < 0.05). MiR-599 may inhibit the proliferation, invasion and promote apoptosis of bladder urothelial carcinoma J82 cells by targetingly regulating YAP1.

Metformin inhibits mTOR and c-Myc by decreasing YAP protein expression in OSCC cells

Metformin is an antidiabetic drug that has been reported to have an inhibitory effect on different types of cancers, including oral squamous cell carcinoma(OSCC). However, few studies have explored the role of Yes‑associated protein(YAP), a vital factor contributing to OSCC biology, in metformin‑induced anticancer activity in OSCC cells. Thus, the purpose of the present study was to investigate the molecular relationship between metformin and YAP in OSCC cells. CAL27 and SCC25 cell lines were treated with various concentrations of metformin for 24h. Cell proliferation was detected by Cell Counting Kit‑8(CCK‑8) and flow cytometric assays. Cell apoptosis was analyzed using flow cytometry. The intracellular protein levels of target genes were determined by western blotting. It was demonstrated that metformin affected the cell cycle and apoptosis of CAL27 and SCC25 cells. Alteration of YAP protein expression affected metformin‑mediated changes in the cell cycle and apoptosis of CAL27 and SCC25 cells. In addition, compared to the control treatment, metformin treatment decreased the protein levels of YAP, mTOR, p‑mTOR and c‑Myc. The overexpression of YAP alleviated the inhibitory effect of metformin on the protein expression of mTOR, p‑mTOR and c‑Myc. The combination of metformin and verteporfin markedly enhanced the effects of metformin on the protein expression of mTOR, p‑mTOR and c‑Myc. Therefore, the results of the present study suggest that metformin suppresses OSCC by inhibiting YAP protein expression and by suppressing the YAP‑mediated effects of metformin on the protein expression of mTOR and c‑Myc.

The Antiasthma Medication Ciclesonide Suppresses Breast Cancer Stem Cells through Inhibition of the Glucocorticoid Receptor Signaling-Dependent YAP Pathway.

Ciclesonide is an FDA-approved glucocorticoid used to treat asthma and allergic rhinitis. However, whether it has anticancer and anti-cancer stem cell (CSC) effects is unknown. This study focused on investigating the effect of ciclesonide on breast cancer and CSCs and determining its underlying mechanism. Here, we showed that ciclesonide inhibits breast cancer and CSC formation. Similar glucocorticoids—dexamethasone and prednisone—did not inhibit CSC formation. Ciclesonide-induced glucocorticoid receptor (GR) degradation was dependent on ubiquitination. We showed via GR small interfering RNA (siRNA) that GR plays an important role in CSC formation. We showed via western blot and immunofluorescence assays that ciclesonide reduces the nuclear level of GR. The GR antagonist RU-486 also inhibited CSC formation. Ciclesonide reduced the protein level of the Hippo transducer Yes-associated protein (YAP). GR siRNA induced a decrease in YAP protein expression and inhibited mammosphere formation. The YAP inhibitor verteporfin inhibited CSC formation and transcription of the connective tissue growth factor and cysteine-rich protein 61 genes. The GR/YAP1 pathway regulated breast CSC formation. We showed that the GR/YAP signaling pathway regulates breast CSC formation and revealed a new approach for targeting GR and YAP to inhibit CSC formation.

Bone morphogenetic protein-2 enhances gonadotropin-independent follicular development via sphingosine kinase 1.

Pre-ovulatory mature follicles are not readily induced from gonadotropin (Gn)-independent early follicles in the poor ovarian response (POR) state, characterized by reduced number of retrieved oocytes. Bone morphogenetic protein (BMP), which is expressed in the ovary, contributes to early folliculogenesis, but its precise underlying mechanism remains unknown. The purpose of this study was to examine the effects of BMP-2 on granulosa cells (GCs) of Gn-independent early follicles. Sphingosine kinase 1 (SPHK1) localization, which produces sphingosine 1-phosphate (S1P), was examined in human early follicles by immunohistochemistry. SPHK1 mRNA levels were examined in Gn-independent bovine GCs (bGCs) and human nonluteinized granulosa cell line (HGrC1) cells. Phosphorylated Yes-associated protein (YAP) expression was evaluated by Western blot, and its localization was evaluated immunocytochemically in bGCs. Verteporfin, a selective YAP inhibitor, was used to explore the influence of YAP on BMP-2-induced bGCs proliferation. The expression of SPHK1 was observed in human GCs of primary and secondary follicles. BMP-2 significantly induced SPHK1 mRNA expression in bGCs and HGrC1 cells. Both BMP-2 and S1P decreased phosphorylated YAP protein levels and induced the nuclear translocation of YAP significantly, thereby increasing the number of bGCs by suppressing the Hippo pathway. This BMP-2-induced cell proliferation was completely blocked by verteporfin. This is a first report showing that BMP-2 up-regulated SPHK1 mRNA expression in GCs and promoted GCs proliferation through Hippo pathway suppression. Thus, BMP-2 contributes to Gn-independent folliculogenesis via SPHK1, suggesting a potential therapeutic strategy for the POR patients with follicular dysgenesis.

MiR-103a-3p promotes tumour glycolysis in colorectal cancer via hippo/YAP1/HIF1A axis

BackgroundGlycolysis plays an essential role in the growth and metastasis of solid cancer and has received increasing attention in recent years. However, the complex regulatory mechanisms of tumour glycolysis remain elusive. This study aimed to explore the molecular effect and mechanism of the noncoding RNA miR-103a-3p on glycolysis in colorectal cancer (CRC).MethodsWe explored the effects of miR-103a-3p on glycolysis and the biological functions of CRC cells in vitro and in vivo. Furthermore, we investigated whether miR-103a-3p regulates HIF1A expression through the Hippo/YAP1 pathway, and evaluated the role of the miR-103a-3p-LATS2/SAV1-YAP1-HIF1A axis in promoting glycolysis and angiogenesis in CRC cells and contributed to invasion and metastasis of CRC cells.ResultsWe found that miR-103a-3p was highly expressed in CRC tissues and cell lines compared with matched controls and the high expression of miR-103a-3p was associated with poor patient prognosis. Under hypoxic conditions, a high level of miR-103a-3p promoted the proliferation, invasion, migration, angiogenesis and glycolysis of CRC cells. Moreover, miR-103a-3p knockdown inhibited the growth, proliferation, and glycolysis of CRC cells and promoted the Hippo-YAP1 signalling pathway in nude mice in a xenograft model. Here, we demonstrated that miR-103a-3p could directly target LATS2 and SAV1. Subsequently, we verified that TEAD1, a transcriptional coactivator of Yes-associated protein 1 (YAP1), directly bound to the HIF1A promoter region and the YAP1 and TEAD1 proteins co-regulated the expression of HIF1A, thus promoting tumour glycolysis.ConclusionsMiR-103a-3p, which is highly expressed in CRC cells, promotes HIF1A expression by targeting the core molecules LATS2 and SAV1 of the Hippo/YAP1 pathway, contributing to enhanced proliferation, invasion, migration, glycolysis and angiogenesis in CRC. Our study revealed the functional mechanisms of miR-103a-3p/YAP1/HIF1A axis in CRC glycolysis, which would provide potential intervention targets for molecular targeted therapy of CRC.

Inhibition of yes-associated protein dephosphorylation prevents aggravated periodontitis with occlusal trauma.

Occlusal trauma can aggravate periodontitis, but the mechanism remains unclear. Yes-associated protein (YAP), a mechanical stressor protein, may play an important role in this process. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were applied to detect the expression of YAP and inflammatory factors in patients with periodontitis accompanied with or without occlusal trauma. Through local administration of Porphyromonas gingivalis and composite resin bonding on maxillary molars in mice, we established periodontitis and occlusal trauma models. Treatment with or without XAV939, to inhibit YAP activation, was performed in these models. Micro-computed tomography, immunofluorescence (IF), and qRT-PCR were used to explore the YAP pathway in periodontitis with occlusal trauma. Cyclic stress and lipopolysaccharide (LPS) stimuli were applied to the L929 mouse fibroblast cell line with or without XAV939. Western blot, IF, and qRT-PCR were used to verify the in vivo results. Activated dephosphorylated YAP and increased expression of inflammatory factors were observed in patients with periodontitis accompanied with occlusal trauma. In the mouse model of periodontitis with occlusal trauma, YAP transferred into the nucleus, resulting in Jun N-terminal kinases (JNK) related pro-inflammatory pathway up-regulation. L929 cell cyclic stress and LPS stimulation results confirmed the in vivo results. Application of XAV939 inhibited YAP protein dephosphorylation and reduced JNK pro-inflammatory pathway factor expression in vivo and in vitro. Occlusal trauma can activate YAP nuclear transfer, resulting in the up-regulation of the JNK pro-inflammatory pathway. This can be inhibited by the XAV939 YAP inhibitor.

Rational Design and Intramolecular Cyclization of Hotspot Peptide Segments at YAP-TEAD4 Complex Interface.

The Yes-Associated Protein (YAP) is a central regulator of Hippo pathway involved in carcinogenesis, which functions through interaction with TEA Domain (TEAD) transcription factors. Pharmacological disruption of YAP-TEAD4 complexes has been recognized as a potential therapeutic strategy against diverse cancers by suppressing the oncogenic activity of YAP. We systematically examine the crystal structure of YAP complex with TEAD4 and rationally identify two hotspot segments at the complex interface; they could be exploited as self-inhibitory peptides to target the complex interaction. Two peptides, termed PS-1 and PS-2 are split from the interfacial context of YAP protein. Dynamics simulations, energetics analyses and fluorescence polarizations are employed to characterize the intrinsic disorder as well as binding energy/affinity of the two YAP peptides to TEAD4 protein. The native conformation of PS-2 peptide is a cyclic loop, which is supposed to be constrained by adding a disulfide bond across the spatially vicinal residue pair Arg87-Phe96 or Met86- Phe95 at the peptide's two ends, consequently resulting in two intramolecular cyclized counterparts of linear PS-2 peptide, namely PS-2(cyc87,96) and PS-2(cyc86,95). The linear PS-2 peptide is determined as a weak binder of TEAD4 (Kd = 190 μM), while the two cyclic PS-2(cyc87,96) and PS-2(cyc86,95) peptides are measured to have moderate or high affinity towards TEAD4 (Kd = 21 and 45 μM, respectively). PS-1 and PS-2 peptides are highly flexible and cannot maintain in native active conformation when splitting from the interfacial context, and thus would incur a considerable entropy penalty upon rebinding to the interface. Cyclization does not influence the direct interaction between PS-2 peptide and TEAD4 protein, but can largely reduce the intrinsic disorder of PS-2 peptide in free state and considerably minimize indirect entropy effect upon the peptide binding.

Inhibition of Yes-Associated Protein-1 (YAP1) Enhances the Response of Invasive Breast Cancer Cells to the Standard Therapy.

PurposeThe deregulation of the Hippo pathway results in translocation ofYes-associated protein-1 (YAP1) to the nucleus to exert an oncogenic effect. This effect has been demonstrated in several malignancies, yet, in breast cancer (BC), it remains controversial. The present study aimed to investigate the significance of YAP1 expression in BC, its relation to cancer stem cells (CSCs), and the effect of its inhibition on tumor cell survival.Patients and MethodsWe evaluated the expression of YAP1 protein and gene using immunohistochemistry (IHC) and RT-qPCR in FFPE tissue from normal and breast cancer cases. We also studied its association with CSC expression (OCT4, NANOG, and SOX2) and with different clinicopathologic characteristics. Two BC cell lines (MCF7 and MDA-MB-231) were exposed to different concentrations of YAP1 inhibitor “verteporfin” and cell viability was subsequently assessed.ResultsYAP1 mRNA was higher in BC compared to the normal breast tissue (p-value=0.040) and was higher in luminal tumors compared to triple-negative breast cancer (TNBC) (p-value= 0.017). Its expression in tumors was significantly associated with the expression of pluripotency markers (OCT4 and NANOG) (p-value= 0.030 and 0.035, respectively) and its inhibition resulted in a significant reduction of CSC expression in both MCF-7 and MDA-MB-231 cells. YAP1 nuclear expression by IHC, which signifies its activation, was more evident in invasive carcinomas compared to normal breast tissue and in-situ foci where the expression was limited to the cytoplasm. The pretreatment of BC cells (MCF7 and MDA-MB-231) with YAP1 inhibitor “verteporfin” resulted in their sensitization to the effect of tamoxifen and doxorubicin, respectively, and significantly decreased tumor cell proliferation and survival.ConclusionOur results imply that YAP1 is highly expressed and activated in BC and its inhibition could represent a possible novel therapeutic strategy that should be further explored and investigated to improve the outcome of breast cancer patients.

Chronic GPER1 Activation Protects Against Oxidative Stress-Induced Cardiomyoblast Death via Preservation of Mitochondrial Integrity and Deactivation of Mammalian Sterile-20-Like Kinase/Yes-Associated Protein Pathway.

Introduction: Estrogen (17β-estradiol, E2) is well-known to induce cardioprotective effects against ischemia/reperfusion (I/R) injury. We recently reported that acute application of E2 at the onset of reperfusion in vivo induces cardioprotective effects against I/R injury via activation of its non-steroidal receptor, G protein-coupled estrogen receptor 1 (GPER1). Here, we investigated the impact and mechanism underlying chronic GPER1 activation in cultured H9c2 rat cardiomyoblasts.Methods: H9c2 rat cardiomyoblasts were cultured and pretreated with the cytotoxic agent H2O2 for 24 h and incubated in the presence of vehicle (control), GPER1 agonists E2 and G1, or GPER1 agonists supplemented with G15 (GPER1 antagonist) for 48 or 96 h. After treatment, cells were collected to measure the rate of cell death and viability using flow cytometry and Calcein AM assay or MTT assay, respectively. The resistance to opening of the mitochondrial permeability transition pore (mPTP), the mitochondrial membrane potential, and ATP production was assessed using fluorescence microscopy, and the mitochondrial structural integrity was observed with electron microscopy. The levels of the phosphorylation of mammalian sterile-20-like kinase (MST1) and yes-associated protein (YAP) were assessed by Western blot analysis in whole-cell lysate, while the expression levels of mitochondrial biogenesis genes, YAP target genes, and proapoptotic genes were measured by qRT-PCR.Results: We found that after H2O2 treatment, chronic E2/G1 treatment decreased cell death effect was associated with the prevention of the S phase of the cell cycle arrest compared to control. In the mitochondria, chronic E2/G1 activation treatment preserved the cristae morphology, and increased resistance to opening of mPTP, but with little change to mitochondrial fusion/fission. Additionally, chronic E2/G1 treatment predominantly reduced phosphorylation of MST1 and YAP, as well as increased MST1 and YAP protein levels. E2 treatment also upregulated the expression levels of TGF-β and PGC-1α mRNAs and downregulated PUMA and Bim mRNAs. Except for ATP production, all the E2 or G1 effects were prevented by the cotreatment with the GPER1 antagonist, G15.Conclusion: Together, these results indicate that chronic GPER1 activation with its agonists E2 or G1 treatment protects H9c2 cardiomyoblasts against oxidative stress-induced cell death and increases cell viability by preserving mitochondrial structure and function as well as delaying the opening of mPTP. These chronic GPER1 effects are associated with the deactivation of the non-canonical MST1/YAP mechanism that leads to genetic upregulation of cell growth genes (CTGF, CYR61, PGC-1α, and ANKRD1), and downregulation of proapoptotic genes (PUMA and Bim).

Yes-Associated Protein Contributes to Cell Proliferation and Migration of Gastric Cancer via Activation of Gli1.

ObjectiveIn the present study, we aimed to explore the potential oncogenic property and the internal mechanism of yes-associated protein (YAP) in gastric cancer (GC).Materials and MethodsYAP protein levels were evaluated in human GC tissues and paired normal tissues using immunohistochemistry (IHC). The role of YAP in regulating GC cell proliferation and migration was verified by genetic manipulation in vitro. Western blot analysis was used to determine the molecular signaling to explain the mechanism of the observed YAP effects in GC.ResultsNuclear YAP protein expression was upregulated in GC tissues, and high nuclear YAP level was significantly correlated with lymph node metastasis (LNM) and tumor node metastasis (TNM) stage in patients suffered from GC. YAP knockdown inhibited GC cell proliferation, migration and epithelial–mesenchymal transition (EMT) progress in vitro, whereas YAP elevation did the opposite. YAP regulated glioma-associated oncogene-1 (Gli1) expression independent of smoothened homolog (SMO). YAP modulated protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway in GC cells.ConclusionYAP enhanced GC cell proliferation and migration potentially via its regulation of Gli1 expression through the non-classical Hedgehog pathway, indicating suppression of YAP/Gli1 signaling axis may highlight a new entry point for combination therapy of GC.

&lt;p&gt;Rab14 Overexpression Promotes Proliferation and Invasion Through YAP Signaling in Non-Small Cell Lung Cancers&lt;/p&gt;

IntroductionSeveral reports have shown that Rab14 is dysregulated in human cancers suggesting that it is an oncogenic protein closely related to tumorigenesis. However, whether Rab14 plays a role in the development and progression of human non-small cell lung cancer (NSCLC) remains unclear.MethodsRab14 protein levels were examined in 115 cases of NSCLC tissues and 6 cancer cell lines. Rab14 knockdown was performed in H1299 and A549 cell lines. Rab14 plasmid transfection was performed in the LK2 cell line. The biological roles and mechanisms of Rab14 were examined using MTT, colony formation, Matrigel invasion assay, migration assay, cell cycle analysis, Western blotting, and RT-qPCR.ResultsWe found that Rab14 was upregulated in 65 of 115 lung cancer tissues. Rab14 high expression was significantly correlated with advanced TNM stage and nodal metastasis. Rab14 protein levels were higher in lung cancer cell lines than in normal bronchial cell line. Functionally, Rab14 overexpression increased growth rate, colony formation, invasion/migration ability and cell cycle progression, while Rab14 siRNA decreased the cell proliferation rate, colony numbers and inhibited invasion/migration ability and cell cycle progression. Rab14 upregulated cyclin D1, cyclin E, connective tissue growth factor (CTGF) and downregulated p27 protein and mRNA levels in both A549 and H1299 cell lines, while Rab14 siRNA produced the opposite effects. Further study showed that Rab14 overexpression increased luciferase reporter activity from transcriptional enhanced associate domain (TEAD) protein. Accordingly, Rab14 increased total Yes-associated protein (YAP) and nuclear YAP protein while decreased phosphorylated (p)-YAP and cytoplasmic YAP protein expression. Cycloheximide treatment showed that Rab14 downregulated the level of YAP degradation. Depletion of YAP using siRNA abolished the influence of Rab14 on cyclin D1, cyclin E, and CTGF. YAP knockdown also partly abolished the effects of Rab14 on cell proliferation and invasion.ConclusionIn summary, our data showed that Rab14 is overexpressed in human NSCLC. Rab14 facilitated proliferation and invasion, possibly through regulation of YAP signaling.

RGFP966 inactivation of the YAP pathway attenuates cardiac dysfunction induced by prolonged hypothermic preservation.

Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4-6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP-transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway.

YAP1 inhibits ovarian endometriosis stromal cell invasion through ESR2.

Endometriosis is an estrogen-dependent disease, and estrogen receptor 2 (ESR2) plays a critical role in the pathogenesis of ovarian endometriosis by promoting cell invasion. Yes-associated protein 1 (YAP1) plays suppressive roles in several types of tumors. However, the relationship between YAP1 and ESR2 is not fully understood. The aim of this study was to investigate the regulatory mechanism of YAP1 in terms of ESR2 and YAP1 regulation of endometriotic stromal cell (ECSC) invasion in ovarian endometriosis. Our results demonstrated that YAP1 mRNA and protein levels in eutopic endometrium (EU) tissues were higher than those in paired ectopic endometrium (EC) tissues. ECSCs transfected with siYAP1 exhibited a significant increase in both ESR2 mRNA levels and protein expression. Simultaneously, YAP1 overexpression in ECSCs yielded the opposite results. Co-IP assays demonstrated YAP1-NuRD complex formation by YAP1, CHD4 and MTA1 in ECSCs. YAP1 bound to two sites, (-539, -533) and (-158, -152), upstream of the ESR2 transcription initiation site. YAP1 binding to the two sites of the ESR2 promoter in ECSCs was significantly lower than that in eutopic endometrial stromal cells (EUSCs) from EU tissues. ECSCs transfected with siYAP1 exhibited increased invasion activity, while ECSCs transfected with siESR2 showed inhibition of invasion. However, transfection with siYAP1 and siESR2 together decreased the number of invading cells compared with transfection with siYAP1 alone. Therefore, we conclude that decreased levels of YAP1 in ovarian endometriomas enhance ESR2 expression via formation of a YAP1-NuRD complex, which further binds to the ESR2 promoters. Furthermore, YAP1 inhibits ECSCs invasion.

Corosolic Acid Inhibits Cancer Progress Through Inactivating YAP in Hepatocellular Carcinoma.

Chemotherapy is critical for the treatment of hepatocellular carcinoma (HCC). Despite the proapoptotic effects of corosolic acid (CA) treatment, its underlying mechanism is not completely clear. The aim of this study was to determine the molecular mechanism of CA in HCC treatment. MTT assay was used to determine the IC50 of CA. Immunoprecipitation and immunofluorescence were used to detect the interaction and subcellular localization of Yes-associated protein (YAP) and mouse double minute 2 (MDM2). In addition, in vivo xenotransplantation was performed to assess the effects of CA, YAP, and MDM2 on tumorigenesis. The IC50 of CA was about 40 μM in different HCC cell lines, and CA decreased YAP expression by reducing its stability and increasing its ubiquitination. CA treatment and MDM2 overexpression significantly decreased the crosstalk between YAP and cAMP-responsive element-binding protein (CREB), TEA domain transcription factor (TEAD), and Runt-related transcription factor 2 (Runx2). CA stimulation promoted the translocation of YAP and MDM2 from the nucleus to the cytoplasm and increased their binding. In addition, CA treatment obviously reduced tumorigenesis, whereas this effect was abolished when cells were transfected with sh-MDM2 or Vector-YAP. The present study uncovered that CA induced cancer progress repression through translocating YAP from the nucleus in HCC, which might provide a new therapeutic target for HCC.