Nuclear Yes-associated Protein Research Articles

Prognostic significance of nuclear Yes-associated protein 1 in patients with nonsmall cell lung cancer: A systematic review and meta-analysis.

Background:Nuclear Yes-associated protein 1 (YAP1) has often been regarded as an adverse prognostic indicator in various tumors. Recent studies have associated YAP1 with unfavorable prognosis in nonsmall cell lung cancer (NSCLC). However, due to small sample sizes, the prognostic value of nuclear YAP1 in NSCLC patients is not well understood. In the present study, we evaluated the prognostic role of nuclear YAP1 in NSCLC patients via a systematic review and meta-analysis.Methods:We searched the PubMed, EMBASE, Cochrane, Web of Science, China National Knowledge Infrastructure (CNKI), and Wanfang Databases for papers investigating the prognostic significance of nuclear YAP1 expression in NSCLC patients. Hazard ratios (HRs) and the corresponding 95% confidence intervals (CIs) were calculated with reference to overall survival (OS) and progression-free survival (PFS) of NSCLC patients to provide synthesized estimates of the effects of nuclear YAP1 expression.Results:Among 414 cases, higher nuclear YAP1 expression presented as a predictive factor of poorer OS (HR = 1.52; 95% CI: 1.11–2.08; P = .01; I2 = 0.0%) and decreased PFS (HR = 2.11; 95% CI: 1.52–2.93; P < .001; I2 = 44.2%) in NSCLC patients. Subgroup analysis revealed shortened OS (HR = 1.63; 95% CI: 1.14–2.34; P = .007; I2 = 0.0%) and worse PFS (HR = 2.25; 95% CI: 1.53–3.30; P < .001; I2 = 0.0%) in patients from Asia with higher nuclear YAP1 expression. Prognosis was also worse in patients with III–IV stage cancer (PFSHR = 2.09; 95% CI: 1.45–3.01; P < .001; I2 = 58.1%) and in patients treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) (OS HR = 1.59; 95% CI: 1.00–2.51; P = .048; I2 = 15.5%, and PFS HR = 2.35, 95% CI: 1.62–3.42; P < .001; I2 = 0.0%).Conclusion:High expression of nuclear YAP1 was associated with shorter survival outcome in patients with NSCLC.

Low shear stress induces ERK nuclear localization and YAP activation to control the proliferation of breast cancer cells

Micro-environmental regulation of cancer cell malignancy is one of the most basic cancer life phenomena. However, the study of cellular response to microenvironment has been long focused on signal processes mediated by various chemical factors and their receptors, the study of mechanical forces, another key environmental factor, has been less studied. In recent years, more and more attention has been paid to the physiological and pathological significance of mechanical microenvironment. However, it is still not clear how cells perceive environmental changes and the signal pathways that regulate cell physiological activities. In this study, we identified that low shear stress (LSS) significantly promoted breast cancer cell proliferation. The proliferation was closely associated with mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) and Yes-associated protein (YAP). Inhibition of ERK or YAP both abolished the LSS-induced proliferation activity of cancer cells. LSS induced ERK phosphorylation and YAP activations, which suggested the involvement of ERK and YAP under LSS treatment. Under LSS, ERK inhibitor U0126 decreased both active YAP and ERK expressions, while YAP inhibitor verteporfin failed to decrease ERK phosphorylation. Further study confirmed that ERK translocated to nucleus which showed an active state of ERK in LSS-treated group. LSS with verteporfin group showed no differences with LSS-treated group which confirmed ERK and YAP an upstream-downstream cascade. The above results demonstrated that LSS can promote breast cancer cell proliferation through ERK-YAP activation. These results not only highlight a new means of understanding mechanical transmission to cytoplasm mechanisms but also serve as a new basis for developing drug delivery systems for breast cancer treatment.

Hippo Deficiency Leads to Cardiac Dysfunction Accompanied by Cardiomyocyte Dedifferentiation During Pressure Overload.

The Hippo pathway plays an important role in determining organ size through regulation of cell proliferation and apoptosis. Hippo inactivation and consequent activation of YAP (Yes-associated protein), a transcription cofactor, have been proposed as a strategy to promote myocardial regeneration after myocardial infarction. However, the long-term effects of Hippo deficiency on cardiac function under stress remain unknown. We investigated the long-term effect of Hippo deficiency on cardiac function in the presence of pressure overload (PO). We used mice with cardiac-specific homozygous knockout of WW45 (WW45cKO), in which activation of Mst1 (Mammalian sterile 20-like 1) and Lats2 (large tumor suppressor kinase 2), the upstream kinases of the Hippo pathway, is effectively suppressed because of the absence of the scaffolding protein. We used male mice at 3 to 4 month of age in all animal experiments. We subjected WW45cKO mice to transverse aortic constriction for up to 12 weeks. WW45cKO mice exhibited higher levels of nuclear YAP in cardiomyocytes during PO. Unexpectedly, the progression of cardiac dysfunction induced by PO was exacerbated in WW45cKO mice, despite decreased apoptosis and activated cardiomyocyte cell cycle reentry. WW45cKO mice exhibited cardiomyocyte sarcomere disarray and upregulation of TEAD1 (transcriptional enhancer factor) target genes involved in cardiomyocyte dedifferentiation during PO. Genetic and pharmacological inactivation of the YAP-TEAD1 pathway reduced the PO-induced cardiac dysfunction in WW45cKO mice and attenuated cardiomyocyte dedifferentiation. Furthermore, the YAP-TEAD1 pathway upregulated OSM (oncostatin M) and OSM receptors, which played an essential role in mediating cardiomyocyte dedifferentiation. OSM also upregulated YAP and TEAD1 and promoted cardiomyocyte dedifferentiation, indicating the existence of a positive feedback mechanism consisting of YAP, TEAD1, and OSM. Although activation of YAP promotes cardiomyocyte regeneration after cardiac injury, it induces cardiomyocyte dedifferentiation and heart failure in the long-term in the presence of PO through activation of the YAP-TEAD1-OSM positive feedback mechanism.

Yes-associated protein expression in paired primary and local recurrent breast cancer and its clinical significance

Yes-associated protein (YAP) protein acts as tumorigenic factor in many solid tumors, but the situation in breast cancer is under debate. Here, we would analyze its status in breast cancer. YAP expression in the 110 primary breast cancer and their paired local recurrent tumors was investigated. Clinicopathologic data for age, histologic grading, hormone status, lymph nodes and HER2 status were also gathered and analyzed. 46.4% (51/110) primary breast cancer tissues were positive for total YAP expression which was significantly higher than that in the recurrent tissues (10.9%; P < 0.05). The expression of total YAP protein in the primary breast cancer tissues was positively associated with the tumor size, especially in triple negative breast cancer (TNBC) subtype (P < 0.05). Higher total or nuclear YAP expression in the primary tumor was correlated with poor disease-free survival among patients with TNBC (P < 0.05). In the multivariate models, nuclear YAP expression was an independently prognostic factor in TNBC. High total or nuclear YAP expression predicts poor prognosis among patients with TNBC. It might be a therapeutic target for TNBC in the future.

Blockade of YAP alleviates hepatic fibrosis through accelerating apoptosis and reversion of activated hepatic stellate cells

Yes-associated protein (YAP) is a significant downstream protein in the Hippo signaling pathway with important functions in cell proliferation, apoptosis, invasion and migration. YAP also plays a role in the progression and development of various liver diseases. In hepatic fibrosis development and reversion, the proliferation and apoptosis of activated hepatic stellate cells (HSCs) play a critical role. However, the contribution of YAP to hepatic fibrosis progression and reversion and the underlying mechanism have not been investigated. Here we investigated the expression and function of YAP in the proliferation and apoptosis of activated HSCs. We found that YAP expression was increased in liver fibrosis tissues from CCl4-induced model mice and restored to normal level after stopping CCl4 injection and 6 weeks of spontaneously recovery. YAP expression was elevated in HSC-T6 cells treated with TGF-β1 and recovered after MDI treatment. Silencing of YAP inhibited the activation and proliferation of HSC-T6 cells stimulated by TGF-β1. In addition, the apoptosis of activated HSC-T6 cells silenced for YAP was slightly enhanced. Furthermore, over-expression of YAP repressed the reversion of activated HSC-T6 cells mediated by MDI reversal. We found that HSC-T6 cells activated by TGF-β1 showed higher levels of nuclear YAP compared with MDI-treated cells, indicating that YAP was activated in HSC-T6 cells treated by TGF-β1. We also found that loss of YAP attenuated Wnt/β-catenin pathway activity in activated HSC-T6 cells. Treatment of VP, an inhibitor of the YAP-TEAD complex, reduced both activation and proliferation of HSC-T6 cells and increased apoptosis. Together these results indicated that reduced expression of YAP contributes to acquisition of the quiescent phenotype in HSCs. Our results suggest that YAP may be a useful target in HSCs activation and reversion.

Stiffness heterogeneity-induced double-edged sword behaviors of carcinoma-associated fibroblasts in antitumor therapy

Carcinoma-associated fibroblasts (CAFs) function as a double-edged sword in tumor progression. However, factors affecting the transition between tumor promotion and inhibition remain to be investigated. Here, we found that the transition was determined by stiffness heterogeneity of the tumor stroma in which tumor cells and CAFs were grown. When tumor cells were grown on a rigid plastic substrate, supernatants from CAFs inhibited the cytotoxic effects of 5-fluorouracil. In contrast, when tumor cells were grown on a soft substrate (5.3 kPa), supernatants from CAFs grown on a soft substrate increased the cytotoxicity of 5-fluorouracil. The diverse effects of CAFs were mediated by mechanotransduction factors, including stroma stiffness-induced cytokine expression in CAFs and signal transduction associated with stress fiber formation of CAFs. Moreover, we found that the cytokine expression in CAFs was regulated by nuclear Yes-associated protein, which changed according to cell stiffness, as characterized by atomic force microscopy. Overall, these findings suggested that modulating the mechanotransduction of the stroma together with CAFs might be important for increasing the efficacy of chemotherapy.

Distinct immunological properties of the two histological subtypes of adenocarcinoma of the ampulla of Vater.

Adenocarcinoma of the ampulla of Vater (AOV) is classified into intestinal type (IT) and pancreatobiliary type (PB); however, the immunological properties of these subtypes remain to be characterized. Here, we evaluated the clinical implications of PD-L1 expression and CD8+ T lymphocyte density in adenocarcinomas of the AOV and their potential association with Yes-associated protein (YAP). We analyzed 123 adenocarcinoma-of-the-AOV patients who underwent surgical resection, and tumors were classified into IT or PB type. Tumor or inflammatory cell PD-L1 expression, CD8+ T lymphocyte density in the cancer cell nest (intratumoral) or in the adjacent stroma, and YAP localization and intensity were analyzed using immunohistochemical staining. PB-type tumors showed higher tumoral PD-L1 expression than IT-type tumors, and tumoral PD-L1 expression was associated with a shorter disease-free survival (DFS) [hazard ratio (HR), 1.77; p = 0.045] and overall survival (OS) (HR 1.99; p = 0.030). Intratumoral CD8+ T lymphocyte density was higher in IT type than in PB type and was associated with a favorable DFS (HR 0.47; p = 0.022). The nuclear staining pattern of YAP in tumor cells, compared to non-nuclear staining patterns, was more frequently associated with PB type and increased tumoral PD-L1 expression. Nuclear YAP staining was a significant prognostic factor for OS (HR 2.21; p = 0.022). These results show that the two subtypes of adenocarcinoma of the AOV exhibit significant differences in tumoral PD-L1 expression and intratumoral CD8+ T lymphocyte density, which might contribute to their distinct clinical features.

Loss of Yes-associated Protein Represents an Aggressive Subtype of Colorectal Cancer.

Background: Yes-associated protein (YAP) is a downstream effecter of Hippo signaling pathway, and has been linked to the initiation and development of colorectal cancer (CRC). However, the clinical significance of YAP in CRC remains controversial. This study was designed to investigate the clinical significance of YAP in CRC.Methods: We selected 206 eligible patients diagnosed with CRC from 2003 to 2007. Tissue microarray (TMA) blocks were made from 206 formalin-fixed paraffin-embedded CRC tissues and 158 corresponding normal colonic tissues. Using the TMA blocks, we performed immunohistochemical staining of YAP and assessed its expression status in different subcellular locations. The patients were divided into four groups according to the expression status of YAP in the cytoplasm and nucleus. Statistical analysis was performed to explore the correlation between YAP expression and clinicopathological features and overall survival (OS) in CRC patients.Results: Our results showed that both cytoplasmic YAP and nuclear YAP were overexpressed in CRC tissues compared to normal colonic tissues. Complete loss of YAP expression in CRC was significantly correlated with larger tumor size (p=0.023), proximal tumor location (p=0.038), higher tumor grade (p=0.022) and worse OS (p<0.001). Univariate and multivariate Cox regression analyses revealed that complete loss of YAP expression was an independent indicator of poor prognosis in CRC (p<0.001).Conclusions: Loss of YAP expression correlates with poor prognosis and may represent a subgroup with more aggressive biological features in CRC.

Unbalanced YAP\u2013SOX9 circuit drives stemness and malignant progression in esophageal squamous cell carcinoma

Yes-associated protein (YAP) has been identified as a key regulator of tissue homeostasis. However, the precise role and regulatory mechanism of YAP in esophageal squamous cell carcinoma (ESCC) remains unclear. Here we report that the genetic or pharmacological inhibition of YAP repressed cancer stem cell (CSC)-like properties, including tumorsphere-forming potential, cell motility, and chemoresistance in vitro, and was sufficient to attenuate tumor growth and CSC marker expression in ESCC xenografts. Mechanistically, YAP transcriptionally activated its downstream target SOX9 via TEAD1-mediated binding. We also observed a positive correlation between YAP signaling and SOX9 expression in two independent clinical cohorts. Intriguingly, YAP-targeting microRNAs, including miR-506-3p, which were induced by SOX9, post-transcriptionally repressed YAP expression, contributing to a negative feedback mechanism. Dual inhibition of YAP and SOX9 robustly suppressed malignant phenotypes. Notably, ESCC samples from The Cancer Genome Atlas (TCGA) dataset had frequent (44%) instances of YAP gene amplification and genetic inactivation of Hippo pathway regulators. Nuclear YAP expression was elevated in 197 ESCC tissues from a Chinese cohort. Together, our findings provide evidence that genetic hyperactivation of YAP unbalances the YAP–SOX9 feedback loop and confers CSC-like features in ESCC, suggesting that this YAP–SOX9 circuit represents a potential therapeutic target.

Invited commentary

Neointimal hyperplasia (NIH) is the proliferation and migration of vascular smooth muscle cells primarily in the intima, resulting in the thickening of the arterial wall and decreased arterial lumen. NIH is a significant problem after revascularization that can lead to significant graft and stent failures. Novel research investigating pathways that are responsible for NIH may provide insight into the pathophysiologic process and effective treatment options. There are currently drug-eluting stents based on paclitaxel and sirolimus, which are designed to inhibit smooth muscle cell proliferation. Although they are effective at reducing NIH, in-stent restenosis and thrombosis continue to occur. This may be due to the nonspecific effects of the drug to the target pathways/cells or incomplete inhibition of the pathways responsible for NIH. In this very important study, the authors investigate the inhibition of Rho GTPase, specifically RhoA and its effect on Hippo pathway-related genes and YAP transcription coactivator. YAP (Yes-associated protein) is a protein that acts as a transcriptional regulator by activating the transcription of genes involved in cell proliferation and suppressing apoptotic genes. Initially, the study demonstrated that active RhoA expression affects the smooth muscle cell synthetic phenotype and decreases the contractile phenotype. Inhibition of RhoA by rhosin decreased cell proliferation but had no effect on apoptosis. The inhibition of RhoA-guanosine triphosphate involved the knockdown of cyclin D and affected the majority of cells in the S phase of the cell cycle. Next, the authors demonstrated that YAP plays an important role in the regulation of smooth muscle cell phenotypic modulation and is affected by RhoA, whereby RhoA inhibition reduces the expression of both cytoplasmic and nuclear YAP. Finally, the authors tested whether inhibition of RhoA in a carotid rabbit model would decrease NIH, using rhosin jelly-coated stents to inhibit RhoA. Compared with bare-metal stents, the rhosin-coated stents had less NIH, and cellular analysis demonstrated that the smooth muscle cell contractile phenotype was induced and the synthetic phenotype was suppressed. These effects were mitigated when active YAP lentivirus was instilled into the RhoA-inhibited stent carotid section for 30 minutes, indicating the involvement of YAP in switching the smooth muscle cell phenotype from contractile to synthetic, with increased NIH. In summary, the data demonstrated that the RhoA inhibitor-eluting stent attenuated in-stent restenosis through the YAP signaling pathway evaluated at 6 months. These novel mechanistic discoveries of RhoA and YAP involved in NIH in the context of arterial stents offer an exciting and promising area for clinical trials using RhoA inhibitor drug-eluting stents. Further work is necessary to validate the in vitro and in vivo animal studies from this study, to translate the basic scientific research of RhoA inhibition and decreased NIH, and to determine the effectiveness and durability in human trials. The opinions or views expressed in this commentary are those of the author and do not necessarily reflect the opinions or recommendations of the Journal of Vascular Surgery or the Society for Vascular Surgery. RhoA inhibitor-eluting stent attenuates restenosis by inhibiting YAP signalingJournal of Vascular SurgeryVol. 69Issue 5PreviewCurrent drug-eluting stent (DES) treatment is promising, but it still has the drawback of in-stent restenosis, which remains a clinically relevant problem. Efforts should be made to discover new signaling molecules and novel potential targets for the prevention of arterial restenosis. In this study, we fabricated a novel DES targeting the RhoA pathway and further examined this promising strategy in vitro and in a rabbit carotid model. Full-Text PDF Open Archive

Discovery of a Small Molecule to Increase Cardiomyocytes and Protect the Heart After Ischemic Injury

Accumulating data suggest that new cardiomyocytes in adults are generated from existing cardiomyocytes throughout life. To enhance the endogenous cardiac regeneration, we performed chemical screenings to identify compounds that activate pro-proliferative YES-associated protein and transcriptional enhancer factor domain activities in cardiomyocytes. We synthesized a novel fluorine-containing TT-10 (C11H10FN3OS2) from the biologically hit compound. TT-10 promoted cardiomyocyte proliferation and simultaneously exerted antioxidant and antiapoptotic effects invitro. TT-10 treatment in mice ameliorated myocardial infarction-induced cardiac dysfunction at least in part via enhancing clonal expansion of existing cardiomyocytes with nuclear YES-associated protein expression. Stimulating cardiomyocyte proliferation and/or protection with TT-10 might complement current therapies for myocardial infarction.

Distinct Mechanosensing of Human Neural Stem Cells on Extremely Limited Anisotropic Cellular Contact.

Human neural stem cells (hNSCs) can alter their fate choice in response to the biophysical cues provided during development. In particular, it has been reported that the differentiation of neural stem cells (NSCs) is enhanced by anisotropic contact, which facilitates focal adhesion (FA) formation and cytoskeletal organization. However, a biomolecular mechanism governing how the cells process the biophysical cues from these anisotropic geometries to their fate commitment is still poorly understood due to the limited availability of geometrical diversities (contact width above 50 nm) applicable to cell studies. Here, we firstly demonstrate that the biomolecular mechanism for enhanced neurogenesis on an anisotropic nanostructure is critically dependent on the resolution of a contact feature. We observed a totally different cellular response to anisotropic geometries by first utilizing a high-resolution nanogroove (HRN) structure with an extremely narrow contact width (15 nm). The width scale is sufficiently low to suppress the integrin clustering and enable us to elucidate how the contact area influences the neurogenesis of hNSCs at an aligned state. Both the HRN and control nanogroove (CN) pattern with a contact width of 1 μm induced the spontaneous topographic alignment of hNSCs. However, intriguingly, the focal adhesion (FA) formation and cytoskeletal reorganization were substantially limited on the HRN, although the cells on the CN showed enhanced FA formation compared with flat surfaces. In particular, the hNSCs on the HRN surface exhibited a strikingly lower fraction of nuclear yes-associated protein (YAP) than on the CN surface, which was turned out to be regulated by Rho GTPase in the same way as the cells sense the mechanical properties of the environment. Considering the previously reported role of YAP on neurogenesis, our finding newly substantiates that YAP and Rho GTPase also can be transducers of hNSCs to process topographical alternation to fate decision. Furthermore, this study with the unprecedented high-resolution nanostructure suggests a novel geometry sensing model where the functional crosstalk between YAP signaling and Rho GTPase integrally regulate the fate commitment of the hNSCs.

Nuclear YAP localization as a key regulator of podocyte function

Podocytes are crucial for the establishment of the blood-urine filtration barrier in the glomeruli of the kidney. These cells are mainly affected during glomerulopathies causing proteinuria and kidney function impairment. Ongoing podocyte injury leads to podocyte loss, finally followed by end-stage kidney disease. Podocytes display a predominant nuclear localization of YAP (Yes-associated protein), one effector protein of the Hippo pathway, which regulates the balance between proliferation, differentiation, and apoptosis in cells. Nuclear active YAP seems to be critical for podocyte survival in vivo and in vitro. We can show here that different treatments leading to sequestration of YAP into the cytoplasm in podocytes, like decreased rigidity of the substrate, incubation with dasatinib, or overexpression of Hippo pathway members result in the induction of apoptosis. A RNA sequencing analysis of large tumor suppressor kinase 2 (LATS2) overexpressing podocytes confirmed a significant upregulation of apoptotic genes. The downregulation of Hippo pathway components suggests a feedback mechanism in podocytes. Noteworthy was the regulation of genes involved in cell–cell junction, the composition of the extracellular space, and cell migration. This suggests an influence of Hippo pathway activity on podocyte integrity. As focal segmental glomerulopathy (FSGS) goes along with an activation of the Hippo pathway in podocytes, a comparison of our data with two independent studies of transcriptional regulation in human FSGS glomeruli obtained from the Nephroseq database was performed. This comparison affirmed a multitude of consistent transcriptional changes concerning the regulation of genes influencing apoptosis and the Hippo signaling pathway as well as cell junction formation and cell migration. The link between Hippo pathway activation in podocytes and the regulation of junction and migration processes in vivo might be a fundamental mechanism of glomerular sclerosis and loss of renal function.

Hippo Signaling Pathway Dysregulation in Human Huntington\u2019s Disease Brain and Neuronal Stem Cells

The Hippo signaling pathway is involved in organ size regulation and tumor suppression. Although inhibition of Hippo leads to tumorigenesis, activation of Hippo may play a role in neurodegeneration. Specifically, activation of the upstream regulator, mammalian sterile 20 (STE20)-like kinase 1 (MST1), reduces activity of the transcriptional co-activator Yes-Associated Protein (YAP), thereby mediating oxidative stress-induced neuronal death. Here, we investigated the possible role of this pathway in Huntington’s disease (HD) pathogenesis. Our results demonstrate a significant increase in phosphorylated MST1, the active form, in post-mortem HD cortex and in the brains of CAG knock-in HdhQ111/Q111 mice. YAP nuclear localization was also decreased in HD post-mortem cortex and in neuronal stem cells derived from HD patients. Moreover, there was a significant increase in phosphorylated YAP, the inactive form, in HD post-mortem cortex and in HdhQ111/Q111 brain. In addition, YAP was found to interact with huntingtin (Htt) and the chaperone 14-3-3, however this interaction was not altered in the presence of mutant Htt. Lastly, YAP/TEAD interactions and expression of Hippo pathway genes were altered in HD. Together, these results demonstrate that activation of MST1 together with a decrease in nuclear YAP could significantly contribute to transcriptional dysregulation in HD.

Abstract 3089: YAP and TAZ induce MCM protein expression to facilitate tumor-supporting properties in liver cancer

Abstract Overexpression and nuclear enrichment of the Hippo pathway-regulated transcriptional co-activator Yes-Associated Protein (YAP) is frequently detected in human hepatocellular carcinoma (HCC) and correlates with poor clinical outcome of cancer patients1. In mice, inducible overexpression of the constitutively active isoform YAPS127A induces hepatocellular proliferation leading to hepatomegaly followed by tumor formation within 12-15 weeks. How oncogenic YAP and its paralogue TAZ (syn. WWTR1, WW Domain Containing Transcription Regulator 1) induce uncontrolled proliferation in liver cells is not known, yet. Analysis of expression profiling data, derived from human liver cancer cells after siRNA-mediated inhibition of endogenous YAP, revealed that the Minichromosome Maintenance (MCMs) family members MCM2-7 were positively regulated. MCMs are key components of the pre-replication complex and involved in the formation of the replication fork, which is essential for efficient DNA duplication followed by mitosis2. We confirmed that silencing of YAP and TAZ by independent siRNAs reduced the mRNA and protein expression of MCM2-7 in liver cancer cell lines. In vitro analyses revealed that transcription factors of the TEAD family were the most relevant YAP binding partners needed for the transcriptional regulation of MCMs. Using chromatin immunoprecipitation and Dual Luciferase Reporter Assay, we showed that YAP, TAZ and TEAD4 directly bound the MCM promoter. First in vitro data suggested that viability of liver cancer cells, subjected to drug induced replication stress, was exclusively reduced in cells with reduced YAP, TAZ or MCM protein expression. Administration of the YAP/TAZ/TEAD inhibitor Verteporfin reduced the expression of all MCMs in liver cancer cells. Vice versa, the overexpression of YAPS127A in transgenic mice showed significantly increased levels of the proliferation marker Ki67 and MCM2-7. Expression data from 242 HCC patients illustrated an association between YAP and MCM mRNA levels, with elevated MCMs significantly correlating with worse overall survival and early cancer recurrence3. Immunohistochemical stains of HCC tissue micro-arrays revealed a highly significant correlation between Ki67 expression, nuclear YAP overexpression and nuclear MCM2-7 enrichment. In summary, our results strongly suggest that YAP and TAZ facilitate their tumor-supporting properties through the regulation of MCM2-6, a complete protein complex. We conclude that combined inhibition of YAP and TAZ or perturbation of MCM activity might represent an efficient therapeutic approach for the treatment of a subgroup of HCC patients. 1Tschaharganeh D, et al., Gastroenterol. 2013; 2Deegan TD, et al., Curr Opin Struct Biol. 2016; 3Roessler S, et al., Cancer Res. 2010. Citation Format: Maria Knaub, Sofia Weiler, Stefan Thomann, Peter Schirmacher, Kai Breuhahn. YAP and TAZ induce MCM protein expression to facilitate tumor-supporting properties in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3089.

Abstract 5039: The relationship between interstitial fluid pressure, collective invasion and YAP activation in engineered human breast tumors

Abstract The tumor microenvironment exhibits increased interstitial fluid pressure (IFP) due to combined effects of vascular leakage and inefficient drainage. Elevated IFP affects the transition of metastatic cells from the tumor to the healthy tissue. However, the complex architecture of the heterogeneous tumor tissues has made it difficult to correlate IFP with chemical and physical tumor responses that regulate invasion and metastasis. To overcome these limitations, we have engineered 3D breast tumor models for the quantitative characterization of biophysical and biochemical features that delineate its transition from a passive non-invasive to a metastatic state. In brief, we embed MDA-MB-231 human breast cancer cells in a collagen type I cavity molded within polydimethylsiloxane (PDMS) channels. The multicellular aggregates are subject to gradients of hydrostatic pressure through opposing reservoirs of culture media that are located at the base (Pbase) and the tip (Ptip) of the tumor. The pressure differential |Pbase – Ptip| defines the IFP gradient across the tumor. We found that the morphology of the engineered breast tumor models varies in response to the differential pressure gradients. For Pbase = Ptip and Pbase &amp;gt; Ptip, the engineered tumors displayed limited invadopodia formation. In contrast, for Pbase &amp;lt; Ptip, the engineered tumors displayed enhanced invadopodia formation. Moreover, we found that IFP affected cellular motility and invasion. Tumor cells demonstrated the highest average speed and directionality and were most invasive for Pbase &amp;lt; Ptip. Furthermore, we found that IFP-induced collective invasion coincides with the elevated expression of epithelial-mesenchymal transition (EMT) markers (vimentin, E-cadherin, Snail and keratin-8) in the engineered breast tumor models. In contrast with the previously demonstrated tumor suppressive activity of E-cadherin, engineered tumors that overexpressed E-cadherin developed a more invasive phenotype. The IFP-induced invasion through EMT in our engineered breast tumors motivated an investigation into the role of Yes-associated protein (YAP). YAP activation is implicated in EMT and regulated by fluid shear stress. Immunofluorescence staining for nuclear YAP suggests that YAP activation modulates the invasion and cell motility responses to IFP. Traction force microscopy will be used to further examine the relationship between IFP, force transduction into the surrounding microenvironment, and YAP activation. Citation Format: Andreas P. Kourouklis, Allison K. Simi, Alexandra Piotrowski-Daspit, Joe Tien, Celeste M. Nelson. The relationship between interstitial fluid pressure, collective invasion and YAP activation in engineered human breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5039.

Deregulation of the Hippo Pathway Promotes Tumor Cell Proliferation Through YAP Activity in Human Sporadic Vestibular Schwannoma

Vestibular schwannomas (VSs) can cause serious neurological defects including hearing loss and facial paralysis. The aim of this study is to identify whether Hippo signaling could be a potential targetable pathway for clinical treatment in VSs. Gene expression profiling was performed in 10 sporadic VSs and 4 normal nerves to identify aberrant genes expression of the Hippo pathway. Western blotting and immunohistochemical staining were used to examine the expression of Hippo core components in 20 VS samples. Neurofibromatosis type 2 (NF2) gene sequencing was also performed in all tumors using sanger sequencing. Verteporfin, inhibitor of yes-associated protein (YAP)-TEA domain family member, was used to assess the effect ofproliferation inhibition in human primary VS cells and RT4-D6P2T cell line. We found 51 differentially expressed genes of the Hippo pathway between VSs and healthy controls. Unsupervised analysis identified the 2 molecular variants that significantly related with distinct NF2 mutation status. The phosphorylation levels of large tumor suppressor 1 and YAP were significantly decreased in NF2-mutated VSs compared with wild-type VSs and normal nerves. Immunohistochemical staining showed that increased nuclear YAP expression in VSs was positively correlated with high Ki-67 index and low Merlin expression. Verteporfin reduced viability of primary VS cells and RT4-D6P2T cells. Our findings implicate that deregulation of the Hippo pathway as a molecular mechanism of pathogenesis in human VSs, and suggest inhibition of this pathway as a potential treatment strategy.

Role of YAP Activation in Nuclear Receptor CAR-Mediated Proliferation of Mouse Hepatocytes.

Constitutive androstane receptor (CAR) is a xenobiotic-responsive nuclear receptor that is highly expressed in the liver. CAR activation induces hepatocyte proliferation and hepatocarcinogenesis in rodents, but the mechanisms remain unclear. In this study, we investigated the association of CAR-dependent cell proliferation with Yes-associated protein (YAP), which is a transcriptional cofactor controlling organ size and cell growth through the interaction with various transcriptional factors including TEA domain family member (TEAD). In mouse livers, 1,4-bis-(2-[3,5-dichloropyridyloxy])benzene (TCPOBOP) (a mouse CAR [mCAR] activator) treatment increased the nuclear YAP accumulation and mRNA levels of YAP target genes as well as cell-cycle related genes along with liver hypertrophy and verteporfin (an inhibitor of YAP/TEAD interaction) cotreatment tended to attenuate them. Furthermore, in cell-based reporter gene assays, CAR activation enhanced the YAP/TEAD-dependent transcription. To investigate the role of YAP/TEAD activation in the CAR-dependent hepatocyte proliferation, we sought to establish an in vitro system completely reproducing CAR-dependent cell proliferation. Since CAR was only slightly expressed in cultured mouse primary hepatocytes compared with mouse livers and no proliferation was observed after treatment with TCPOBOP, we overexpressed CAR using mCAR expressing adenovirus (Ad-mCAR-V5) in mouse primary hepatocytes. Ad-mCAR-V5 infection and TCPOBOP treatment induced hepatocyte proliferation. Similar results were obtained with immortalized normal mouse hepatocytes as well. In the established in vitro system, CAR-dependent proliferation was strongly inhibited by Yap knockdown and completely abolished by verteporfin treatment. Our present results obtained in in vivo and in vitro experiments suggest that YAP/TEAD activation plays key roles in CAR-dependent proliferation of murine hepatocytes.

Tumorigenic role of YAP in hepatocellular carcinogenesis is involved in SHP2 whose function is different in vitro and in vivo

Yes-associated protein (YAP) is a nuclear effector of the cell-density sensing Hippo pathway and interacts with Src homology phosphotyrosine phosphatase 2 (SHP2), which controls cell proliferation and survival. The tumor promoting/suppressing activities of YAP and SHP2 during liver tumorigenesis remain controversial. This study aimed to investigate the tumorigenic roles of YAP and SHP2 in hepatocellular carcinogenesis. Cell density associated subcellular distributions of YAP and SHP2 in normal human hepatocytes (THLE-2) and hepatocellular carcinoma (HCC) cells (SK-Hep1, SNU-182) were investigated by Western blotting and cell block immunohistochemistry. The effects of YAP knockdown on proliferation, migration and invasion were studied using YAP-specific siRNAs. The prognostic significance of YAP and SHP2 expressions was investigated immunohistochemically using a tissue microarray (TMA) from 50 HCC cases. High-cell density decreased the nuclear expression of YAP and SHP2 in normal hepatocytes as compared with low-cell density. However, in HCC cells, nuclear YAP and SHP2 were observed regardless of cell density. Nuclear YAP influenced SHP2 expression and cell proliferation. In particular, YAP knockdown impacted nuclear levels of SHP2 protein in SK-Hep1 cells. In HCC tissues, nuclear YAP expression was elevated and cytoplasmic SHP2 expression was diminished as compared with adjacent non-tumor tissues. Notably, these expressions were found to be significantly associated with poor recurrence-free and overall survival rate in patients with HCC. Consequently, the tumor promoting role of YAP is involved in SHP2 which functions as a tumor promoter in vitro but as a tumor suppressor in vivo. YAP and SHP2 can be unfavorable prognostic markers in HCC.

Dishevelled has YAP nuclear export function in a tumour suppressor context dependent manner

The Hippo signaling is an evolutionary conserved pathway that inhibits cell proliferation by contact inhibition, and whose loss leads to organ growth and cancer development. The Yes‐associated protein (YAP) transcription co‐activator is a key regulator of the Hippo pathway. Phosphorylation‐dependent YAP translocation is a well‐known intracellular mechanism of the Hippo pathway, however the responsible molecular effectors by which the YAP leads cytosolic translocation remain undefined. Recent findings indicate that oncogenic YAP paradoxically suppresses canonical Wnt activity.We found that DVL, a scaffolding protein of the Wnt pathway as well as a key regulator of Wnt‐independent epithelial polarity, is a molecular effector for nuclear‐cytoplasmic shuttling of YAP in a YAP‐phosphorylation dependent manner. Mutational inactivation of the nuclear export signal in DVL leads to nuclear YAP retention, with increases TEAD transcriptional activity. DVL is also required for YAP intracellular dynamics induced by E‐cadherin, α‐catenin, or AMPK activation. Importantly, the nuclear‐cytoplasmic trafficking is largely dependent on the p53‐Lats2 or LKB1‐AMPK tumor suppressor axes, which determine YAP phosphorylation status. Conversely, loss of p53 or LKB1 relieves DVL‐linked reciprocal inhibition between the canonical Wnt pathway and nuclear YAP activity. Our observations provide novel mechanistic insights into controlled proliferation coupled with epithelial polarity during development and human cancer.Support or Funding InformationNFR‐2017R1A2B3002241 funded by the Korean government (MSIP) andBK 21 PLUS Project, Yonsei University College of Dentistry, Seoul, Korea.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.