Yes-associated Protein Activation Research Articles

Two faces of Hippo: activate or suppress the Hippo pathway in cancer.

The Hippo pathway has generated considerable interest in recent years because of its involvement in several key hallmarks of cancer progression and metastasis. Research on the Hippo signaling pathway in cancer has been used to determine the activity of yes-associated protein (YAP) in tumorigenesis and disease progression. Previous studies have shown that the Hippo pathway can be used as a target to inhibit YAP activity and is a viable treatment for cancer. However, more studies are required to further advance our understanding of the Hippo signaling pathway in cancer. It has been shown that knockout of serine/threonine-kinases LATS1/2 in the Hippo pathway suppresses cancer immunity in mice. In addition, suppression of the oncogene YAP could contribute toward cancer immune therapy. Therefore, regulation of Hippo signaling can be an attractive alternative strategy for cancer treatment. This review will provide a summary of currently known compounds that activate or suppress the Hippo pathway.

β1 integrin–dependent Rac/group I PAK signaling mediates YAP activation of Yes-associated protein 1 (YAP1) via NF2/merlin

Cell adhesion to the extracellular matrix or to surrounding cells plays a key role in cell proliferation and differentiation and is critical for proper tissue homeostasis. An important pathway in adhesion-dependent cell proliferation is the Hippo signaling cascade, which is coregulated by the transcription factors Yes-associated protein 1 (YAP1) and transcriptional coactivator with PDZ-binding motif (TAZ). However, how cells integrate extracellular information at the molecular level to regulate YAP1's nuclear localization is still puzzling. Herein, we investigated the role of β1 integrins in regulating this process. We found that β1 integrin-dependent cell adhesion is critical for supporting cell proliferation in mesenchymal cells both in vivo and in vitro β1 integrin-dependent cell adhesion relied on the relocation of YAP1 to the nucleus after the down-regulation of its phosphorylated state mediated by large tumor suppressor gene 1 and 2 (LATS1/2). We also found that this phenotype relies on β1 integrin-dependent local activation of the small GTPase RAC1 at the plasma membrane to control the activity of P21 (RAC1)-activated kinase (PAK) of group 1. We further report that the regulatory protein merlin (neurofibromin 2, NF2) interacts with both YAP1 and LATS1/2 via its C-terminal moiety and FERM domain, respectively. PAK1-mediated merlin phosphorylation on Ser-518 reduced merlin's interactions with both LATS1/2 and YAP1, resulting in YAP1 dephosphorylation and nuclear shuttling. Our results highlight RAC/PAK1 as major players in YAP1 regulation triggered by cell adhesion.

YAP1 inhibition radiosensitizes triple negative breast cancer cells by targeting the DNA damage response and cell survival pathways.

The Hippo pathway is an evolutionarily conserved signaling pathway that regulates proliferation and apoptosis to control organ size during developmental growth. Yes-associated protein 1 (YAP1), the terminal effector of the Hippo pathway, is a transcriptional co-activator and a potent growth promoter that has emerged as a critical oncogene. Overexpression of YAP1 has been implicated in promoting resistance to chemo-, radiation and targeted therapy in various cancers. However, the role of YAP1 in radioresistance in triple-negative breast cancer (TNBC) is currently unknown. We evaluated the role of YAP1 in radioresistance in TNBC in vitro, using two approaches to inhibit YAP1: 1) genetic inhibition by YAP1 specific shRNA or siRNA, and 2) pharmacological inhibition by using the small molecule inhibitor, verteporfin that prevents YAP1 transcriptional activity. Our findings demonstrate that both genetic and pharmacological inhibition of YAP1 sensitizes TNBC cells to radiation by inhibiting the EGFR/PI3K/AKT signaling axis and causing an increased accumulation of DNA damage. Our results reveal that YAP1 activation exerts a protective role for TNBC cells in radiotherapy and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of TNBC.

Butyrate stimulates the growth of human intestinal smooth muscle cells by activation of yes-associated protein.

Intestinal smooth muscle cells play a critical role in the remodeling of intestinal structure and functional adaptation after bowel resection. Recent studies have shown that supplementation of butyrate (Bu) contributes to the compensatory expansion of a muscular layer of the residual intestine in a rodent model of short-bowel syndrome (SBS). However, the underlying mechanism remains elusive. In this study, we found that the growth of human intestinal smooth muscle cells (HISMCs) was significantly stimulated by Bu via activation of Yes-Associated Protein (YAP). Incubation with 0.5 mM Bu induced a distinct proliferative effect on HISMCs, as indicated by the promotion of cell cycle progression and increased DNA replication. Notably, YAP silencing by RNA interference or its specific inhibitor significantly abolished the proliferative effect of Bu on HISMCs. Furthermore, Bu induced YAP expression and enhanced the translocation of YAP from the cytoplasm to the nucleus, which led to changes in the expression of mitogenesis genes, including TEAD1, TEAD4, CTGF, and Cyr61. These results provide evidence that Bu stimulates the growth of human intestinal muscle cells by activation of YAP, which may be a potential treatment for improving intestinal adaptation.

Biomechanical strain-induced modulation of proliferation coincides with an ERK1/2-independent nuclear YAP localization

Biomechanical strain induces activation of the transcriptional co-activator yes-associated protein (YAP) by nuclear re-distribution. Recent findings indicate that the mechanically responsive mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 1/2 is involved in the amount of nuclear YAP, reflecting its activation. In this context, we conducted experiments to detect how biomechanical strain acts on the subcellular localization of YAP in periodontal cells. To this end, cells were subjected to 2.5% static equiaxial strain for different time periods. Western blot and fluorescence imaging-based analyses revealed a clear modulation of nuclear YAP localization. This modulation fairly coincided with the altered course of the KI-67 protein amount in conjunction with the percentage of KI-67-positive and thus proliferating cells. The inhibition of the ERK1/2 activity via U0126 yielded an unchanged strain-related modulation of nuclear YAP localization, while YAP amount in whole cell extracts of strained cells was decreased. Administration of the YAP-inhibiting drug Verteporfin evoked a clear reduction of KI-67-positive and thus proliferating cells by approximately 65%, irrespective of strain. Our data reveal YAP as a regulator of strain-modulated proliferation which occurs in a MAPK-independent fashion.

Hypoxia-inducible factor 2α (HIF-2α) promotes colon cancer growth by potentiating Yes-associated protein 1 (YAP1) activity

Colorectal cancer (CRC) is the third-leading cause of cancer mortality in the United States and other industrialized countries. A hypoxic microenvironment is a hallmark for solid tumors. The hypoxia-induced signal transduction is transcriptionally mediated by hypoxia-inducible factor (HIF). Three major HIF isoforms, HIF-1α, HIF-2α, and HIF-3α, are present in the intestine. Our previous work demonstrates that HIF-2α is essential for CRC growth and progression. However, the mechanisms mediating cell proliferation after hypoxia or HIF-2α activation in CRC are unclear. Data mining of RNA-Seq experiments with mouse models of intestinal HIF-2α or Yes-associated protein 1 (YAP1) overexpression indicates a significant overlap of genes in these conditions. YAP1 is a transcriptional co-activator in the Hippo signaling pathway, and YAP1-induced transcriptional responses are essential in cancer cell proliferation. Here, we report that HIF-2α robustly increases YAP1 expression and activity in CRC-derived cell lines and in mouse models. The potentiation of YAP1 activity by HIF-2α was not via canonical signaling mechanisms such as Src (non-receptor tyrosine kinase), PI3K, ERK, or MAPK pathways. Moreover, we detected no direct interaction of HIF-2α with YAP1. Of note, YAP1 activation was critical for cancer cell growth under hypoxia. Our findings indicate that HIF-2α increases cancer cell growth by up-regulating YAP1 activity, suggesting that this pathway might be targeted in potential anti-cancer approaches for treating CRC patients.

Increased YAP Activation Is Associated With Hepatic Cyst Epithelial Cell Proliferation in ARPKD/CHF

Autosomal recessive polycystic kidney disease/congenital hepatic fibrosis (ARPKD/CHF) is a rare but fatal genetic disease characterized by progressive cyst development in the kidneys and liver. Liver cysts arise from aberrantly proliferative cholangiocytes accompanied by pericystic fibrosis and inflammation. Yes-associated protein (YAP), the downstream effector of the Hippo signaling pathway, is implicated in human hepatic malignancies such as hepatocellular carcinoma, cholangiocarcinoma, and hepatoblastoma, but its role in hepatic cystogenesis in ARPKD/CHF is unknown. We studied the role of the YAP in hepatic cyst development using polycystic kidney (PCK) rats, an orthologous model of ARPKD, and in human ARPKD/CHF patients. The liver cyst wall epithelial cells (CWECs) in PCK rats were highly proliferative and exhibited expression of YAP. There was increased expression of YAP target genes, Ccnd1 (cyclin D1) and Ctgf (connective tissue growth factor), in PCK rat livers. Extensive expression of YAP and its target genes was also detected in human ARPKD/CHF liver samples. Finally, pharmacological inhibition of YAP activity with verteporfin and short hairpin (sh) RNA-mediated knockdown of YAP expression in isolated liver CWECs significantly reduced their proliferation. These data indicate that increased YAP activity, possibly through dysregulation of the Hippo signaling pathway, is associated with hepatic cyst growth in ARPKD/CHF.

Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest

The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including β-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both β-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies.

Central role of mTORC1 downstream of YAP/TAZ in hepatoblastoma development

Hepatoblastoma (HB) is the most common type of liver malignancy in children. Recent studies suggest that activation of Yes-associated protein (YAP) is a major molecular event in HB development, as activated YAP synergizes with mutant β-catenin to promote HB formation in mice (YAP/β-catenin). However, how YAP regulates HB development remains poorly defined. Similarly, de-regulation of mammalian target of rapamycin complex 1 (mTORC1) signaling has been implicated in multiple tumor types, but its functional role in HB development is scarcely understood. In the present study, we found that mTORC1 is activated in human HB cells and YAP/β-catenin-induced mouse HB tumor tissues. mTOR inhibitor MLN0128 significantly inhibits human HB cell growth in vitro. Furthermore, ablation of Raptor, the unique subunit of mTORC1, strongly delayed YAP/β-catenin-induced HB development in mice. At the molecular level, we found that expression of the amino acid transporter SLC38A1 is induced in mouse HB tissues, and amino acid deprivation leads to mTORC1 suppression in HB cell lines. Silencing of YAP and/or its paralog, transcriptional co-activator with PDZ binding motif (TAZ), decreased SLC38A1 expression as well as mTORC1 activation in HB cells. Furthermore, a frequent and concomitant upregulation of mTORC1 and SLC38A1 was detected in a collection of human HB specimens. Altogether, our study demonstrates the key role of mTORC1 in HB development. YAP and TAZ promote HB development via inducing SLC38A1 expression, whose upregulation leads to mTORC1 activation. Targeting mTOR pathway or amino acid transporters may represent novel therapeutic strategies for the treatment of human HB.

Hydrogel-based microchannels to measure confinement- and stiffness-sensitive Yes-associated-protein activity in epithelial clusters

Abstract

Palmitic acid dysregulates the Hippo–YAP pathway and inhibits angiogenesis by inducing mitochondrial damage and activating the cytosolic DNA sensor cGAS–STING–IRF3 signaling mechanism

Impaired angiogenesis and wound healing carry significant morbidity and mortality in diabetic patients. Metabolic stress from hyperglycemia and elevated free fatty acids have been shown to inhibit endothelial angiogenesis. However, the underlying mechanisms remain poorly understood. In this study, we show that dysregulation of the Hippo-Yes-associated protein (YAP) pathway, an important signaling mechanism in regulating tissue repair and regeneration, underlies palmitic acid (PA)-induced inhibition of endothelial angiogenesis. PA inhibited endothelial cell proliferation, migration, and tube formation, which were associated with increased expression of mammalian Ste20-like kinases 1 (MST1), YAP phosphorylation/inactivation, and nuclear exclusion. Overexpression of YAP or knockdown of MST1 prevented PA-induced inhibition of angiogenesis. When searching upstream signaling mechanisms, we found that PA dysregulated the Hippo-YAP pathway by inducing mitochondrial damage. PA treatment induced mitochondrial DNA (mtDNA) release to cytosol, and activated cytosolic DNA sensor cGAS-STING-IRF3 signaling. Activated IRF3 bound to the MST1 gene promoter and induced MST1 expression, leading to MST1 up-regulation, YAP inactivation, and angiogenesis inhibition. Thus, mitochondrial damage and cytosolic DNA sensor cGAS-STING-IRF3 signaling are critically involved in PA-induced Hippo-YAP dysregulation and angiogenesis suppression. This mechanism may have implication in impairment of angiogenesis and wound healing in diabetes.

AB072. A feed forward loop between lncARSR and YAP activity promotes expansion of renal tumor-initiating cells

BackgroundRenal cell carcinoma (RCC) is the most common kidney cancer in adults prognosis. Increasing appreciation of cell heterogeneity and a challenging disease with poor within clear cell renal cell carcinoma (ccRCC) has focused attention on a distinct subpopulation of cells called tumour initiating cells (T-ICs) or cancer stem cells (CSCs) in ccRCC. T-ICs exhibit extended self-renewal potential and tumour initiating ability. Tumours that harbour an abundant T-IC population or have high expression of stemness-related genes may signal a poor clinical outcome in RCC patients. Therefore, identification of the underlying mechanisms governing renal T-ICs propagation may lead to the discovery of promising therapeutic strategies for RCC patients.MethodsLong non-coding RNA (lncRNA) is a subgroup of transcripts with more than 200 nt and limited coding potential. lncRNAs modulate biological process via diverse mechanisms, including mobilizing transcriptional co-regulators or chromatin-modifying complex at transcription level, and interacting with RNAs and protein complex or modifying signal proteins at post-transcription level. Several lncRNAs have been reported to regulate the self-renewal of T-ICs especially liver T-ICs. Nevertheless, the role of lncRNA in the regulation of renal T-ICs remains unknown. lncARSR (lncRNA Activated in RCC with Sunitinib Resistance, ENST00000424980) was a newly identified lncRNA to promote the sunitinib resistance of RCC in our previous study. Accumulating evidence indicated that T-ICs surviving from drug therapy and giving rise to tumour regrowth might be a major culprit for therapeutic resistance. Indeed, the expression signature of stem cell or targets of Nanog, Oct4, Sox2 and c-Myc (NOSM) in human ESCs were significantly enriched in our mRNA profile of sunitinib-resistant RCC cells (GSE69535), prompting us to explore the role of lncARSR in renal T-ICs.ResultsIn this study, we first find that lncARSR is highly expressed in primary renal T-ICs and predicts poor prognosis. Next, by using loss-of-function analysis in T-ICs and gain-of-function analysis in RCC cells, we demonstrate that lncARSR promotes the self-renewal capacity, tumorigenicity and metastasis of renal T-ICs. Further mechanism study reveals that lncARSR interacts with Yes-associated protein (YAP) to block its phosphorylation by LATS1, facilitating YAP nuclear translocation. Interestingly, we find that YAP in turn promotes the transcription of lncARSR, forming a feed-forward loop. Clinical investigation also confirms the correlation between lncARSR and YAP, and demonstrates the value of combining lncARSR and YAP to improve the prognostic accuracy for RCC patients.ConclusionsAltogether, we discover that lncARSR promotes the expansion of renal T-ICs via interacting with YAP.

The roles of YAP T425A mutation in the regulation of YAP activity.

The Hippo signaling pathway plays a critical role in body development and tissue growth. As the core effector of the Hippo signaling pathway, Yes-associated protein (YAP) has been reported to be involved in various kinds of human cancers. However, the mechanism for the regulation of YAP activity has not been completely understood. In this study, we constructed a YAP Thr425Ala mutant and found that this mutation decreased YAP transcriptional activity. Further, T425A retained YAP in the cytoplasm without affecting the phosphorylation of YAP S127. Moreover, we observed that the T425A mutation attenuated the ability of YAP in driving MCF10A cell migration. Our research indicates that T425 of YAP is important for the regulation of YAP localization and activity.

A forceful connection: mechanoregulation of oncogenic YAP.

The Yes‐associated protein (YAP) is an important transcriptional co‐activator that mediates the cellular response to mechanical and cytoskeletal cues. In two recent papers published in The EMBO Journal , Dae‐Sik Lim and colleagues show how YAP activity affects cancer formation and metastasis via a crosstalk with myocardin‐related transcription factors (MRTFs; Kim et al , 2017) and SKP2‐dependent cell cycle progression (Jang et al , 2017).

Down regulation of lincRNA-p21 contributes to gastric cancer development through Hippo-independent activation of YAP.

Long intergenic non-coding RNA p21 (lincRNA-p21), known as the direct transcriptional target of p53, was found down-regulated in several human solid tumors. However, little is known about the role of lincRNA-p21 in gastric cancer. The expression levels of lincRNA-p21 in tissue samples and cell lines were detected by qRT-PCR. MGC-803 and MKN-45 cells were transfected with siRNAs targeting lincRNA-p21 or control siRNAs to determine the effect of reduced lincRNA-p21 expression on tumorigenesis. We also overexpressed lincRNA-p21 in MGC-803 cells. Cell proliferation was measured by CCK-8 and Ethynyl-2-deoxyuridine (EdU) incorporation assays. Migration and invasion abilities of cells were measured by wound healing and transwell assay. We demonstrated that lincRNA-p21 was significantly reduced in gastric cancer tissues (p<0.001) compared with that in normal tissues and this lower level of lincRNA-p21 was significantly correlated with higher invasion depth grade (p=0.024), more distant metastasis (p=0.009) and advanced TNM stage (p=0.011). Further study revealed that knock down of lincRNA-p21 could promote malignant behavior of gastric cancer cells and induce epithelial to mesenchymal transition (EMT). Overexpressing lincRNA-p21 showed opposite effects. Moreover, knocking down lincRNA-p21 could elevate the expression of Yes associated protein (YAP), the core effector of Hippo signaling, by elevating mRNA levels and increasing its nucleus translocation instead of the canonical Hippo pathway. Overexpression experiments verified the regulation role of lincRNA-p21 in YAP expression. Collectively, these data suggest that lincRNA-p21 could serve as a potential biomarker and a vital therapeutic target in gastric cancer.

Insulin Receptor and GPCR Crosstalk Stimulates YAP via PI3K and PKD in Pancreatic Cancer Cells.

We examined the impact of crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling pathways on the regulation of Yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in the context of human pancreatic ductal adenocarcinoma (PDAC). Stimulation of PANC-1 or MiaPaCa-2 cells with insulin and neurotensin, a potent mitogenic combination of agonists for these cells, promoted striking YAP nuclear localization and decreased YAP phosphorylation at Ser127 and Ser397 Challenging PDAC cells with either insulin or neurotensin alone modestly induced the expression of YAP/TEAD-regulated genes, including connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61), and CXCL5, whereas the combination of neurotensin and insulin induced a marked increase in the level of expression of these genes. In addition, siRNA-mediated knockdown of YAP/TAZ prevented the increase in the expression of these genes. A small-molecule inhibitor (A66), selective for the p110α subunit of PI3K, abrogated the increase in phosphatidylinositol 3,4,5-trisphosphate production and the expression of CTGF, CYR61, and CXCL5 induced by neurotensin and insulin. Furthermore, treatment of PDAC cells with protein kinase D (PKD) family inhibitors (CRT0066101 or kb NB 142-70) or with siRNAs targeting the PKD family prevented the increase of CTGF, CYR61, and CXCL5 mRNA levels in response to insulin and neurotensin stimulation. Thus, PI3K and PKD mediate YAP activation in response to insulin and neurotensin in pancreatic cancer cells.Implications: Inhibitors of PI3K or PKD disrupt crosstalk between insulin receptor and GPCR signaling systems by blocking YAP/TEAD-regulated gene expression in pancreatic cancer cells. Mol Cancer Res; 15(7); 929-41. ©2017 AACR.

Abstract 2054: Cerivastatin overcomes the acquired resistance to crizotinib in EML4-ALK positive non-small cell lung cancer by controlling YAP activation

Abstract Crizotinib is highly effective in patients with non-small cell lung cancer (NSCLC) harboring the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion. However, its efficacy has been limited by the development of acquired resistance, and the mechanisms of such resistance remain largely unknown. Herein, we investigated a possible candidate for circumventing the acquired resistance to crizotinib. We established a model of acquired resistance to crizotinib (H3122-CR) by exposing EML4-ALK-positive H3122 lung cancer cells to increasing doses of crizotinib, and performed MTT screening using a library of FDA (Food and Drug Administration)-approved drugs composed of a collection of 640 clinically used compounds. Our MTT screening identified that cerivastatin, a drug targeting 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, significantly inhibited the cell growth in H3122-CR cells, which was completely restored by addition of geranylgeranyl pyrophosphate (GGPP), a key metabolite of mevalonate pathway. We also found that yes-associated protein (YAP), a major effector of the Hippo tumor suppressor pathway, was robustly accumulated in nucleus with a concomitant decrement of YAP phosphorylation in H3122-CR cells compared to parental cells. Importantly, inhibition of geranylgeranylation (GGylation) by GGTI-298 or cerivastatin markedly increased YAP phosphorylation, led to cytoplasmic translocation of YAP, and subsequently induced YAP inactivation. Moreover, the enrichment of EGFR signaling pathway and cell cycle signature including transcriptional targets of YAP was enhanced in H3122-CR cells, whose induction contributed YAP-mediated resistance to crizotinib. Inhibition of YAP function with siRNA or verteporfin as a YAP inhibitor greatly abrogated cell growth of H3122-CR cells by modulating cell cycle regulators and EGFR activation. Finally, we confirmed up-regulation of nuclear YAP expression in crizotinib-acquired resistant patient derived tumor xenograft (PDTX) models established from EML4-ALK positive NSCLC patients. Collectively, our findings define the GGylation-mediated YAP transcriptional activation as a new mechanism of resistance to crizotinib, providing a rationale for further exploring statins as a possible anticancer agent to overcome the acquired resistance to crizotinib in EML4-ALK positive NSCLC cells. Citation Format: Miran Yun, Hun Mi Choi, Kyoung Ho Pyo, Byoung Chul Cho. Cerivastatin overcomes the acquired resistance to crizotinib in EML4-ALK positive non-small cell lung cancer by controlling YAP activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2054. doi:10.1158/1538-7445.AM2017-2054

176 Serotonin receptor 2b (5-ht2b) modulates cardiomyocyte proliferation by regulating the hippo pathway

Heart failure is one of the leading causes of death worldwide. In part, this is due to the inadequate regenerative capacity of cardiomyocytes post-injury. Modulation of the Hippo signalling pathway...

Identification of a novel YAP-14-3-3ζ negative feedback loop in gastric cancer.

Growing evidence indicates that 14-3-3ζ and yes-associated protein (YAP) substantially promote tumorigenesis and tumor development. However, the regulatory mechanism underlying these two proteins remains unknown. Herein, we report a new regulatory role of 14-3-3ζ in the phosphorylation of YAP and the feedback inhibition of 14-3-3ζ by YAP. YAP and 14-3-3ζ expression exhibited a negative correlation in gastric cancer (GC) tissues. Moreover, patients with higher YAP and lower 14-3-3ζ expression had poor prognoses. Studies have revealed that 14-3-3ζ promotes cytoplasmic retention and suppresses the transcriptional activity of YAP by inducing its phosphorylation. Furthermore, we observed that the overexpression of YAP significantly reduced the expression of 14-3-3ζ by inducing its ubiquitination. YAP, 14-3-3ζ, and mouse double minute 2 homolog (MDM2) were colocalized, and the knockdown of MDM2 by siRNA attenuated the YAP-induced decrease of 14-3-3ζ. The binding of 14-3-3ζ and MDM2 was also restrained when the expression of YAP was interfered. Our results indicated the presence of a 14-3-3ζ–YAP negative regulatory feedback loop, which has a crucial role in cell proliferation and survival and is a potential target for the clinical treatment of GC.

Targeting YAP in malignant pleural mesothelioma.

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.