Yes-associated Protein Protein Research Articles

The CGRP receptor component RAMP1 links sensory innervation with YAP activity in the regenerating liver.

The effectiveness of liver regeneration limits surgical therapies of hepatic disorders and determines patient outcome. Here, we investigated the role of the neuropeptide calcitonin gene-related peptide (CGRP) for liver regeneration after acute or chronic injury. Mice deficient for the CGRP receptor component receptor activity-modifying protein 1 (RAMP1) were subjected to a 70% partial hepatectomy or repeated intraperitoneal injections of carbon tetrachloride. RAMP1 deficiency severely impaired recovery of organ mass and hepatocyte proliferation after both acute and chronic liver injury. Mechanistically, protein expression of the transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) was decreased in regenerating livers of RAMP1-deficient mice. Lack of RAMP1 was associated with hyperphosphorylation of YAP on Ser127 and Ser397, which regulates YAP functional activity and protein levels. Consequently, expression of various YAP-controlled cell cycle regulators and hepatocyte proliferation were severely reduced in the absence of RAMP1. In vitro, CGRP treatment caused increased YAP protein expression and a concomitant decline of YAP phosphorylation in liver tissue slice cultures of mouse and human origin and in primary human hepatocytes. Thus, our results indicate that sensory nerves represent a crucial control element of liver regeneration after acute and chronic injury acting through the CGRP-RAMP1 pathway, which stimulates YAP/TAZ expression and activity.

Deletion of Cardiomyocyte YAP Potentiates Inflammatory Gene Expression in the Heart

Cardiac inflammation and recruitment of inflammatory cells to the heart are elicited by both ischemic and non‐ischemic injury and precede cardiac fibrosis. Both cardiac and systemic inflammation increases the risk of progression to heart failure. A role for RhoA activation in cardiac fibrosis following non‐ischemic cardiac injury has been demonstrated previously. Our lab and others have documented the importance of transcriptional responses to RhoA activation in the heart, and have shown that this pathway occurs in part through the transcriptional co‐activator Yes‐associated Protein (YAP). Here we tested the hypothesis that YAP and the genes it regulates are involved in generation of inflammatory signals in the cardiomyocyte, which subsequently contribute to recruitment of pro‐inflammatory immune cells. YAP was deleted in the cardiomyocyte compartment by injection of cardiomyocyte‐specific adeno‐associated virus serotype 9 (AAV9) expressing Cre recombinase into adult floxed YAP mice. After 21 days cardiac YAP protein levels were decreased by approximately 50%, with no concomitant decrease of YAP expression in the lung or liver, and without a compensatory increase in expression of the YAP homolog TAZ. Wildtype and YAP knockout mice were then infused with angiotensin‐II (AngII), a GPCR agonist which rapidly induces cardiac inflammation and fibrosis. Hearts were assessed for mRNA levels of various genes after one day of AngII infusion. YAP knockout hearts had attenuated expression of genes associated with cardiac hypertrophy, consistent with previously reported observations. Pro‐inflammatory cytokines and chemokines, including CCL2, IL‐6, and IL‐1β were upregulated in hearts from wildtype mice infused with AngII. Surprisingly these transcriptional responses, when assessed after one day of AngII infusion, were enhanced in hearts from YAP knockouts. We previously reported that pro‐inflammatory gene expression is increased in the heart after three hours of AngII infusion, a time at which macrophage recruitment to the heart has not yet occurred. Interestingly, in contrast to what we observed at one day of AngII, we determined that inflammatory gene expression was not potentiated in YAP knockouts at three hours of AngII. These data suggest that loss of cardiomyocyte YAP may enhance inflammation by increasing recruitment or activation of pro‐inflammatory immune cells, rather than directly affecting inflammatory gene expression in cardiomyocytes. We are currently investigating the relative contribution of cardiomyocytes and non‐cardiomyocytes to inflammatory gene expression by using isolated cell fractions. In summary, we propose that YAP dependent signaling in the cardiomyocyte suppresses inflammatory gene expression in response to AngII through release of factors that decrease macrophage recruitment or activation. Future studies will investigate YAP dependent transcriptional targets through which cardiomyocytes signal to regulate inflammatory responses.Support or Funding InformationThis work is supported by AHA fellowship 19POST34430051 (CSB), and NIH grant R37HL028143 (JHB).

Estradiol-17β regulates proliferation and apoptosis of sheep endometrial epithelial cells by regulating the relative abundance of YAP1

Yes-associated protein 1 (YAP1) transcription regulator of the Hippo protein kinase pathway, serves as a key regulator of tissue growth and organ size by regulating cell proliferation and apoptosis. Effects of YAP1 on proliferation and apoptosis of sheep endometrial epithelial cells (EEC) as a result of estradiol-17β (E2) treatment, however, remain unclear. In the present study, the abundance of YAP1 protein in the uterine horn was greater than that in the uterine body or cervix. The YAP1 protein was primarily localized in the endometrial luminal and glandular epithelial cells of the uterine horn of ewes on day 2 of the estrous cycle. Compared with control samples, there was a lesser abundance of YAP1 mRNA transcript that was associated with a lesser proliferation and greater apoptosis of EEC. There were also lesser concentrations of epidermal growth factor and insulin-like growth factor 1 in the spent culture medium when there was a lesser abundance of YAP1 mRNA in EEC compared with those in the control group. When there was a greater abundance of YAP1 mRNA transcript, there were greater concentrations of epidermal growth factor and insulin-like growth factor 1 in the spent media. Furthermore, with estradiol-17β treatment the abundance of YAP1 mRNA transcript was similar to that of the control samples. Taken together, estradiol-17β may function as an essential regulator of EEC proliferation and apoptosis by modulation of concentrations of YAP1 protein in the sheep uterus. These results indicate there are molecular mechanisms of estradiol-17β and YAP1 in EEC proliferation and apoptosis of ewes.

ANKHD1 promotes proliferation and invasion of non‑small‑cell lung cancer cells via regulating YAP oncoprotein expression and inactivating the Hippo pathway.

The ankyrin repeat and KH domain-containing 1 (ANKHD1) protein was recently reported to be a potential member of the Hippo signaling pathway. However, its role in human non-small-cell lung cancer (NSCLC) has not been extensively investigated. The aim of the present study was to examine the expression of ANKHD1 in primary human tissues and cells and determine whether it is correlated with the clinical characteristics of tumor growth. The biological functions of ANKHD1 were evaluated in vitro and in vivo. Yes-associated protein (YAP) expression and phosphorylation induced by ANKHD1 were evaluated by western blotting and immunoprecipitation. Marked upregulation of ANKHD1 protein expression was observed in NSCLC cells and tissues, which was associated with advanced pathological tumor-node-metastasis stage, lymph node metastasis and poor prognosis in patients with NSCLC. ANKHD1 overexpression also promoted the proliferation and invasion of NSCLC cells. ANKHD1 upregulation inactivated Hippo signaling via increasing YAP protein levels, as well as inhibiting YAP protein phosphorylation, whereas depletion of YAP abolished the effects of ANKHD1 on cell proliferation and invasion. Therefore, ANKHD1 may play an important role in NSCLC through regulating the YAP-dependent Hippo signaling pathway.

YAP1 is an independent prognostic marker in pancreatic cancer and associated with extracellular matrix remodeling

BackgroundPancreatic cancer is a major cause of cancer-related mortality. The identification of effective biomarkers is essential in order to improve management of the disease. Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway, a signal transduction system implicated in tissue repair and regeneration, as well as tumorigenesis. Here we evaluate the biomarker potential of YAP1 in pancreatic cancer tissue.MethodsYAP1 was selected as a possible biomarker for pancreatic cancer from global protein sequencing of fresh frozen pancreatic cancer tissue samples and normal pancreas controls. The prognostic utility of YAP1 was evaluated using mRNA expression data from 176 pancreatic cancer patients in The Cancer Genome Atlas (TCGA), as well as protein expression data from immunohistochemistry analysis of a local tissue microarray (TMA) cohort comprising 140 pancreatic cancer patients. Ingenuity Pathway Analysis was applied to outline the interaction network for YAP1 in connection to the pancreatic tumor microenvironment. The expression of YAP1 target gene products was evaluated after treatment of the pancreatic cancer cell line Panc-1 with three substances interrupting YAP–TEAD interaction, including Super-TDU, Verteporfin and CA3.ResultsMass spectrometry based proteomics showed that YAP1 is the top upregulated protein in pancreatic cancer tissue when compared to normal controls (log2 fold change 6.4; p = 5E−06). Prognostic analysis of YAP1 demonstrated a significant correlation between mRNA expression level data and reduced overall survival (p = 0.001). In addition, TMA and immunohistochemistry analysis suggested that YAP1 protein expression is an independent predictor of poor overall survival [hazard ratio (HR) 1.870, 95% confidence interval (CI) 1.224–2.855, p = 0.004], as well as reduced disease-free survival (HR 1.950, 95% CI 1.299–2.927, p = 0.001). Bioinformatic analyses coupled with in vitro assays indicated that YAP1 is involved in the transcriptional control of target genes, associated with extracellular matrix remodeling, which could be modified by selected substances disrupting the YAP1-TEAD interaction.ConclusionsOur findings indicate that YAP1 is an important prognostic biomarker for pancreatic cancer and may play a regulatory role in the remodeling of the extracellular matrix.

Geometry of the Carotid Artery and Its Association With Pathologic Changes in a Chinese Population.

ObjectivesCarotid artery geometry influences blood flow disturbances and is thus an important risk factor for carotid atherosclerosis. Extracellular matrix (ECM) and yes-associated protein (YAP) expression may play essential roles in the pathophysiology of carotid artery stenosis, but the effect of blood flow disturbances of carotid bifurcation location on the ECM is unknown. We hypothesized that carotid artery anatomy and geometry are independently associated with the ECM and YAP expression.MethodsIn this cross-sectional study, 193 patients were divided into two groups: an asymptomatic group (n = 111) and a symptomatic group (n = 82), symptomatic patients presenting with ischemic attack, amaurosis fugax, or minor non-disabling stroke. For all subjects before surgery, carotid bifurcation angle and internal artery angle were measured with computed tomography angiography (CTA), and laminar shear stress was measured with ultrasonography. After surgery, pathology of all plaque specimens was analyzed using hematoxylin and eosin (HE) staining and Movat special staining. Immunohistochemistry was performed to detect expression of YAP in a subset of 30 specimens.ResultsSymptomatic patients had increased carotid bifurcation angle and laminar shear stress compared to asymptomatic patients (P < 0.05), although asymptomatic patients had increased internal carotid angle compared to symptomatic patients (P < 0.001). Relative higher bifurcation angles were correlated with increased carotid bifurcation, decreased internal angle, and decreased laminar shear stress. For each change in intervertebral space or one-third of vertebral body height, carotid bifurcation angle changed 4.76°, internal carotid angle changed 6.91°, and laminar shear stress changed 0.57 dynes/cm2. Pathology showed that average fibrous cap thickness and average narrowest fibrous cap thickness were greater in asymptomatic patients than symptomatic patients (P < 0.05). Expression of proteoglycan and YAP protein in symptomatic patients was higher than in asymptomatic patients (P < 0.001), while collagen expression was lower in symptomatic patients than asymptomatic patients (P < 0.05).ConclusionGeometry of the carotid artery and position relative to cervical spine might be associated with ECM and YAP protein expression, which could contribute to carotid artery stenosis.

Gli1 interacts with YAP1 to promote tumorigenesis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the predominant esophageal cancer type in China. The aberrant activation of glioma-associated oncogene homolog1 (Gli1), a key factor in Hedgehog (Hh) signaling pathway, has been found in esophageal carcinoma. Moreover, Yes-associated protein 1 (YAP1), the major mediator of Hippo signaling pathway, has been linked to esophageal carcinoma progression. However, the precise roles and the underlying mechanism of both Gli1 and YAP1 in ESCC are unclear. Here, we found that Gli1 and YAP1 are overexpressed in ESCC and are associated with poor prognosis. In addition, we confirmed that knockdown of Gli1 or YAP1 suppresses ESCC cell growth, migration, and invasion in ESCC TE1 and EC109 cells. Significantly, Gli1 interacts with YAP1 in ESCC cells. Both Gli1 and YAP1 proteins are closely correlated with each other in human ESCC samples. Mechanistically, Gli1 upregulates YAP1 in a LATS1-independent manner. Conversely, YAP1 induces Gli1 by regulating phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Most importantly, we demonstrated that the interaction between Gli1 and YAP1 promotes ESCC tumor growth in vitro and in vivo. Our findings established a novel signaling mechanism by which the interaction between Gli1 and YAP1 promotes ESCC cell growth. This signaling regulation of the tumorigenesis provides a new therapeutic strategy for highly lethal ESCC.

MiR-195-5p Inhibits Malignant Progression of Cervical Cancer by Targeting YAP1.

PurposeOur previous studies have shown that miR-195 is reduced in cervical cancer tissues, and that upregulation of miR-195 suppressed cervical cancer cell growth and induced a cell cycle block. In this study, we aimed to further elucidate the mechanism of action between miR-195-5p and Yes-associated protein 1 (YAP1) in the malignant progression of cervical cancer.MethodsMiR-195-5p and YAP1 were detected using qRT-PCR in cervical cancer cells transfected with miR-195-5p mimics or inhibitor. Cell proliferation, migration, and invasion ability were detected using MTT, wound healing, and transwell invasion assays. Dual luciferase reporter assay, qRT-PCR, and Western blot analysis were used to demonstrate that YAP1 was a target of miR-195-5p.ResultsOur results showed that miR-195-5p is negatively correlated with YAP1 protein levels but not with mRNA expression. Moreover, upregulation of miR-195-5p by transient transfection with miR-195-5p mimics in HeLa and SiHa cells inhibited cell proliferation, migration ability, invasiveness, and the EMT. Conversely, miR-195-5p downregulation produced opposite results. In addition, multiple miRNA target prediction sites showed that YAP1 was a potential target gene; this was confirmed by dual luciferase assay. Rescue experiments further confirmed that YAP1 is involved in miR-195-5p-mediated inhibition of proliferation, migration ability, invasiveness, and the EMT of cervical cancer cells.ConclusionTaken together, our data suggest that miR-195-5p may act as a tumor suppressor which could provide a theoretical basis for cervical cancer patient targeted therapy.

Involvement of Yes-associated protein 1 (YAP1) in doxorubicin-induced cytotoxicity in H9c2 cardiac cells.

Cardiac cell death is one of the major events implicated in doxorubicin-induced cardiotoxicity, which leads to heart failure. We recently reported that Yes-associated protein 1 (YAP1) regulates cell survival and apoptosis. However, it is unclear whether YAP1 regulates doxorubicin-induced cell death in cardiomyocytes. We investigated whether YAP1 is involved in doxorubicin-induced cell death using H9c2 cardiac cells and mouse heart. In an in vivo study, YAP1 protein expression was significantly decreased in hearts of doxorubicin-treated mice with increased caspase-3 activation. Doxorubicin also caused cell death by increasing caspase-3 activation in H9c2 cells. Doxorubicin reduced YAP1 protein expression and messenger RNA expression accompanied by increased phosphorylation of YAP1 at Ser127. Doxorubicin further increased cell death with increased caspase-3/7 activation in the absence of YAP1 when compared with doxorubicin or siYAP1 treatment alone. Overexpression of constitutively active YAP1 (YAP1-5SA) using an adenovirus gene transfer technique significantly reversed doxorubicin-induced cell death by decreasing caspase-3/7 activation in H9c2 cells. Akt, a potential prosurvival factor, decreased in doxorubicin- and YAP1 short interfering RNA (siRNA)-treated cells. Doxorubicin further significantly decreased Akt protein expression when YAP1 was silenced. Overexpression of YAP1 canceled decreased Akt protein expression induced by doxorubicin treatment in H9c2 cells. In conclusion, these results suggest that doxorubicin-induced cardiac cell death is mediated in part by down-regulation of YAP1 and YAP1-targeted gene, Akt. Modulating YAP1 and its related Hippo pathway on local cardiomyocytes may be a promising therapeutic approach for doxorubicin-induced cardiotoxicity.

Maternal Yes-Associated Protein Participates in Porcine Blastocyst Development via Modulation of Trophectoderm Epithelium Barrier Function.

The establishment of a functional trophectoderm (TE) epithelium is an essential prerequisite for blastocyst formation and placentation. Transcription coactivator yes-associated protein (YAP), a downstream effector of the hippo signaling pathway, is required for specification of both the TE and epiblast lineages in mice. However, the biological role of YAP in porcine blastocyst development is not known. Here, we report that maternally derived YAP protein is localized to both the cytoplasm and nuclei prior to the morula stage and is then predominantly localized to the TE nuclei in blastocysts. Functionally, maternal YAP knockdown severely impeded blastocyst formation and perturbed the allocation of the first two lineages. The treatment of embryos with verteporfin, a pharmacological inhibitor of YAP, faithfully recapitulated the phenotype observed in YAP deleted embryos. Mechanistically, we found that maternal YAP regulates multiple genes which are important for lineage commitment, tight junction assembly, and fluid accumulation. Consistent with the effects on tight junction gene expression, a permeability assay revealed that paracellular sealing was defective in the trophectoderm epithelium. Lastly, YAP knockdown in a single blastomere at the 2-cell stage revealed that the cellular progeny of the YAP+ blastomere were sufficient to sustain blastocyst formation via direct complementation of the defective trophectoderm epithelium. In summary, these findings demonstrate that maternal YAP facilitates porcine blastocyst development through transcriptional regulation of key genes that are essential for lineage commitment, tight junction assembly, and fluid accumulation.

Interaction of YAP1 and mTOR promotes bladder cancer progression.

Yes-associated protein 1 (YAP1) and mammalian target of rapamycin (mTOR) signaling pathways have been found to be deregulated in bladder cancer and accelerate the malignant progression of bladder cancer. However, the cross-talk between YAP1 and mTOR and its role in bladder cancer progression remains unclear. The aim of the present study was to investigate this crosstalk and the results revealed that the expression of YAP1 and mTOR was elevated in bladder cancer tissues compared with that in adjacent normal tissues. Knockdown of either mTOR or YAP1 with siRNA transfection significantly repressed the proliferation ability and induced apoptosis of HT-1376 and J82 bladder cancer cells, particularly when YAP1 and mTOR were downregulated simultaneously. Upregulation of mTOR increased the mRNA and protein levels of YAP1 and enhanced its nuclear accumulation. In turn, YAP1 upregulation increased mTOR expression, reduced its protein degradation and increased its stability. In addition, immuno-fluorescence and Duolink assays demonstrated that YAP1 and mTOR were co-localized in the nucleus. Immunoprecipitation assay demonstrated that the YAP1 protein was able to bind to the mTOR protein. Moreover, YAP1 combined with S-phase kinase-associated protein 2 (SKP2) and positively regulated its expression. Furthermore, the promotion of cell growth and inhibition of cell apoptosis induced by YAP1 overexpression were abolished when SKP2 was downregulated in HT-1376 and J82 cells. Taken together, the findings of the present study indicated that the crosstalk between YAP1 and mTOR plays a pivotal role in accelerating the progression of bladder cancer, which may provide new insights into the role of the YAP1/mTOR axis in the occurrence and development of bladder cancer.

Inhibition of cyclin-dependent kinase 7 down-regulates yes-associated protein expression in mesothelioma cells.

Cyclin‐dependent kinase 7 (CDK7) is a protein kinase that plays a major role in transcription initiation. Yes‐associated protein (YAP) is a main effector of the Hippo/YAP signalling pathway. Here, we investigated the role of CDK7 on YAP regulation in human malignant pleural mesothelioma (MPM). We found that in microarray samples of human MPM tissue, immunohistochemistry staining showed correlation between the expression level of CDK7 and YAP (n = 70, r = .513). In MPM cells, CDK7 expression level was significantly correlated with GTIIC reporter activity (r = .886, P = .019). Inhibition of CDK7 by siRNA decreased the YAP protein level and the GTIIC reporter activity in the MPM cell lines 211H, H290 and H2052. Degradation of the YAP protein was accelerated after CDK7 knockdown in 211H, H290 and H2052 cells. Inhibition of CDK7 reduced tumour cell migration and invasion, as well as tumorsphere formation ability. Restoration of the CDK7 gene rescued the YAP protein level and GTIIC reporter activity after siRNA knockdown in 211H and H2052 cells. Finally, we performed a co‐immunoprecipitation analysis using an anti‐YAP antibody and captured the CDK7 protein in 211H cells. Our results suggest that CDK7 inhibition reduces the YAP protein level by promoting its degradation and suppresses the migration and invasion of MPM cells. Cyclin‐dependent kinase 7 may be a promising therapeutic target for MPM.

LINC01559 accelerates pancreatic cancer cell proliferation and migration through YAP-mediated pathway.

Pancreatic cancer (PC) is one of the top two most fatal cancers, with the poorest survival rate among all human malignancies. Increasing evidence suggests the involvement of long noncoding RNAs (lncRNAs) in the initiation and progression of various cancers. Herein, we investigated the role of lncRNA LINC01559 in PC. Several online databases indicated that LINC01559 was at a low expression in normal pancreatic tissues but was obviously upregulated in PAAD tissues. Further, our results showed that LINC01559 was stimulated in PC cell lines relative to normal controls. Furthermore, we validated that LINC01559 facilitated PC cell proliferation and migration in vitro. Also, silencing LINC01559 obstructed PC cell growth in vivo. Besides, LINC01559 was revealed to be mainly in the cytoplasm of PC cells and therefore served as a ceRNA of Yes-associated protein (YAP) in PC cells via sponging miR-607. Surprisingly, we also proved that LINC01559 could interact with YAP protein, which might hinder YAP phosphorylation and enhance YAP transcriptional activity in PC cells. Furthermore, we demonstrated that YAP was the downstream effector in LINC01559-regulated PC development. Collectively, our findings unmasked that LINC01559 accelerates PC progression through relying on YAP, providing a new potential target for clinical treatment of patients with PC.

Autophagy promotes triple negative breast cancer metastasis via YAP nuclear localization

The triple-negative breast cancer (TNBC) subtype is the most aggressive form of invasive breast cancer. Although autophagy is critical to the progression of TNBC, the mechanism of autophagy in regulating the metastatic potential of TNBC still remains unclear. Recently, the effector of the Hippo signaling pathway yes-associated protein (YAP) was shown to promote autophagy. To investigate autophagy regulation in YAP signaling in the context of cancer metastasis, we performed profiling analysis of YAP signaling, YAP subcellular localization, autophagosome formation and cell invasiveness in TNBC cell lines (MDA-MB-231 and Hs 578T) versus estrogen receptor (ER) positive breast cancer cell line MCF7. Our results showed that YAP transcriptional and protein expression was significantly upregulated in TNBC. When we triggered autophagy response in TNBC, YAP translocated into the nucleus and the expression of YAP target gene ankyrin repeat domain 1 (ANKRD1) increased remarkably. The correlation between autophagy response and YAP expression in TNBC was confirmed at the single-cell level. Furthermore, the inhibition of YAP nuclear entry greatly impeded the migration and invasion of TNBC cells while it did not affect the mobility of ER positive breast cancer cells. Therefore, this research established the autophagy-YAP-metastasis axis in TNBC and sheds light on the application of targeting YAP for TNBC therapeutics.

Fufang Xueshuantong protects retinal vascular endothelial cells from high glucose by targeting YAP

Fufang Xueshuantong (FXST), a Chinese patent medicine, is composed of Panax notoginseng, Salviae miltiorrhizae, Astragali Radix and Radix Scrophulariae and has been found to prevent diabetic retinopathy. Yes-associated protein (YAP) participates in the pathophysiology of retinal disease and promotes endothelial cell proliferation and angiogenesis. Although it is known that YAP activity is altered by FXST, the role of YAP in mediating the effect of FXST remains unclear. In high glucose-treated retinal vascular endothelial cells (RVECs), FXST significantly reduced cell viability, the number of migrating cells and tube length in the present study. Moreover, FXST decreased the levels of YAP mRNA and protein and inhibited the expression of vascular endothelial growth factor (VEGF). Transfection of sh-YAP into the cells decreased the ability of FXST to modulate cell migration and tube formation. The effect of FXST on VEGF expression was also decreased. Similar results were obtained when the cells were stimulated with a YAP inhibitor in combination with FXST. Thus, FXST is shown to protect high glucose-injured RVECs via YAP-mediated effects.

EphA2-to-YAP pathway drives gastric cancer growth and therapy resistance.

Yes-associated protein (YAP) is a transcriptional coactivator that promotes cell proliferation, stem cell maintenance and tissue homeostasis. The YAP activity is primarily regulated through an inhibitory phosphorylation by the serine/threonine kinases of Hippo pathway. Here, we show that receptor tyrosine kinase (RTK) erythropoietin-producing hepatocellular receptor A2 (EphA2) interacts with and phosphorylates YAP protein, leading to stabilization, nuclear translocation and activation of YAP in gastric cancer (GC) cells. EphA2 induces chemotherapy-resistance by increasing YAP stability and nuclear YAP protein. Knockdown of YAP blocks EphA2-induced tumor growth in GC xenograft mouse models. Importantly, the coactivation of EphA2 and YAP is manifested in clinical human GC, and is related to GC recurrence. Thus, our results establish a novel EphA2-to-YAP pathway that drives GC growth, progression and therapy-resistance, targeting this pathway would be an efficient way for the treatment of GC, particularly chemotherapy-resistant GC.

Mono (2-ethylhexyl) phthalate (MEHP) triggers the proliferation of hemangioma-derived endothelial cells via YAP signals

Hemangioma (HA) is tumor formed by hyper-proliferation of vascular endothelial cells. However, the potential effects of mono-(2-ethylhexyl) phthalate (MEHP) on the progression of HA are not well illustrated. Our present study revealed that MEHP exposure can significantly increase the in vitro proliferation of hemangioma-derived endothelial cells (HemECs). MEHP treatment can activate yes-associated protein (YAP), a key effector of Hippo pathway, by inhibiting its phosphorylation. The dephosphorylation of YAP induced by MEHP can promote the nuclear accumulation of YAP. Knockdown of YAP or its inhibitor can block MEHP triggered cell proliferation. MEHP can increase the levels of precursor and mature mRNA of YAP in HemECs. As well, MEHP extended the half-life of YAP protein. Mechanistically, MEHP can decrease the phosphorylation of YAP via suppressing the activity of large tumor suppressor kinase 1/2 (LATS1/2) to inhibit it induced degradation of YAP. Further, MEHP increased the expression of interferon regulatory factor 1 (IRF1), which can bind to the promoter of YAP to initiate its transcription. Collectively, we revealed that Hippo-YAP signal is involved in MEHP-induced proliferation of HA cells.

Abstract 4394: Molecular interplay between Tank-binding kinase (TBK1) and Yes-associated protein (YAP1) in KRAS mutant NSCLC

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality in the United States. Lung adenocarcinomas are highly correlated with smoking and are characterized by mutations in KRAS, EGFR, BRAF and other oncogenes. KRAS mutations are widespread in adenocarcinomas among smokers and known to be a key player in various downstream signaling pathways contributing to the tumorigenesis. Recently, a non-canonical IκB kinase, Tank Binding Kinase 1 (TBK1), has been found to contribute in KRAS mutant cancers. TBK1 has well documented functions in immune response, cell survival and in mitosis. While it has been suggested that TBK1-mediated regulation of Akt signaling might facilitate oncogenesis, the molecular mechanisms underlying TBK1 function downstream of KRAS is not fully elucidated. Yes associated protein 1 (YAP1) is an oncogenic component of the Hippo signaling cascade which could promote KRAS mediated oncogenesis and could substitute for the loss of Kras in mouse models of pancreatic cancer. In our present study, we demonstrate a unique and novel interplay between TBK1 and the oncogenic Hippo effector molecule, YAP1. YAP1 and its paralog, TAZ are known transcriptional co-activators that function to maintain organ size during development and is often activated in cancers. We find that TBK1 could physically interact with YAP1 and phosphorylate it at T110, T114, S128 and S131 residues in vitro. Knocking down (KD) or knock out (KO) of TBK1 resulted in a significant elevation of YAP1 expression at the protein level; surprisingly, without any effect at the mRNA level. Interestingly, the upregulation of YAP1 upon depletion of TBK1 was restricted to KRAS mutant NSCLC cell-lines and not in EGFR mutant cell lines. This elevation of YAP1 upon TBK1 KD was mainly observed in the nucleus; notably, there were only minimal changes in the levels of MST and LATS, raising the possibility that these changes occur independent of the classic hippo signaling pathway. Treatment with cycloheximide, an inhibitor of protein-translation, could not diminish the elevated level of YAP1 protein in the TBK1 depleted cells, indicating that the increased YAP1 level is due to enhanced protein stability, probably as a result of post-translational modification(s). Depletion of TBK1 also resulted in the induction of EMT-like features, promoting cell-migration in scratch assays and elevated the proportion of stem-like side-population cells, probably in a YAP1-dependent manner. An unbiased RNA-Seq analysis in A549 and H460 cells indicated that MAP kinase (MAPK) pathway is activated upon TBK1 KD, which might also be involved in the YAP1 protein regulation. Our recent experiments further support this argument. The in-depth molecular mechanism(s) by which TBK1 regulates YAP1 in KRAS mutant cells are under investigation, and we hypothesize that this regulatory event contributes to KRAS mediated oncogenesis in NSCLC. Citation Format: Biswarup Saha, Neha Jaiswal, Namrata Bora-Singhal, Srikumar Chellappan. Molecular interplay between Tank-binding kinase (TBK1) and Yes-associated protein (YAP1) in KRAS mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4394.

Abstract 2889: The Yes-Associated Protein plays a role in tumor formation, stemness, and therapy response in high risk neuroblastoma

Abstract Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood. Despite intensive multimodal therapy, &amp;gt;50% of high risk (HR) NB patients relapse with chemotherapy-resistant disease. RAS/RAF/MAPK pathway mutations and increased Yes-Associated Protein (YAP) transcriptional activity are more prevalent in relapsed NB compared to diagnostic tumors. YAP is a transcriptional co-activator, regulated by the Hippo pathway that binds with TEAD in the nucleus to activate genes promoting cell growth, self-renewal, and survival. YAP aberrant transcription has been implicated in many tumor types, yet the role for YAP in NB is unknown. YAP protein is heterogeneously expressed in HR NB cell lines. We used human derived NB cell lines stably transduced to express (NGPYAP) or inhibit (SK-N-ASshYAP, NLFshYAP) YAP to further characterize YAP’s role in NB. While TEAD expression increases with YAP genetic inhibition, expression of YAP paralog TAZ did not change. SK-N-ASshYAP xenografts in NSG mice showed significantly delayed time to tumor formation and slower growth compared to empty vector transduced xenografts. Unmodified NB cells grown as neurospheres in neural basal media showed increased YAP expression and stemness markers, OCT4 and SOX2, by RT-qPCR. OCT4 and SOX2 were dependent on YAP expression as they did not increase in NLFshYAP neurospheres. Furthermore, NLFshYAP had decreased number and size of neurospheres compared to control. Given YAP’s role in tumor stemness, we treated unmodified NB cells with 13-cis-retinoic acid (RA) to see if YAP-induced stemness would overcome RA-induced differentiation. Interestingly, YAP expression and downstream targets, OCT4 and SOX2, increased in response to RA treatment. This suggests that RA paradoxically induces YAP transcriptional activity and may be selecting for a more chemoresistant tumor initiating phenotype. Lastly, YAP knockdown sensitized NRAS- and NF-1-mutated NBs to the MEK inhibitor, Trametinib, and to chemotherapy in vitro. In adult cancers, YAP’s role in therapy resistance is due to its regulation of anti-apoptotic Bcl-2 family proteins. However, YAP knockdown or induced expression in NBs did not change Bcl-2 family protein levels, nor did it affect Bim binding patterns to Mcl-1 or Bcl-2. Thus, investigations are ongoing to understand how YAP negatively regulates Trametinib response. We treated NB cells with Verteporfin (VP), an FDA-approved drug used to treat macular degeneration that inhibits YAP-TEAD interactions in other cancers. While VP inhibited YAP expression in NBs, it also had significant off target effects, potently killing YAP null NBs via light-activated generation of reactive oxygen species. Given the relevance of YAP in relapsed NBs and its effects on NB tumor formation, stemness, and therapy resistance, the need for targeted and specific YAP inhibitors is critical to reinstating therapy sensitivity at relapse. Citation Format: Jenny Shim, Yoo Joo Lee, Kaitlyn Janssen, Alexa Fritz, Kelly C. Goldsmith. The Yes-Associated Protein plays a role in tumor formation, stemness, and therapy response in high risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2889.

Abstract 4388: A novel, reversible YAP1-HuR axis promotes pancreatic tumorigenesis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related death in the U.S. In roughly 95% of cases, gain-of-function mutations in KRAS combine with loss of tumor suppressors to progress pre-invasive neoplasms (PanINs) to late-stage ductal adenocarcinoma. Two important mediators of KRASfunction, Human Antigen R (HuR) and Yes-associated protein 1 (YAP1), are both highly overexpressed in PDAC. HuR is a RNA-binding protein that facilitates gene expression through the stabilization and increased translation of target pro-survival mRNAs upon stress. YAP1 is a transcriptional co-activator, which associates with a number of transcription factor families to sense and upregulate targets that lead to tumor growth and cellular cross-talk. While the functions of HuR and YAP1 are well known, the events that lead to their overexpression and regulation are poorly understood. YAP1 was first identified as a HuR target via ribonucleoprotein-immunoprecipitation assays in which HuR-bound mRNAs were run on a whole-transcriptome microarray. YAP1 mRNA was significantly bound to HuR as compared to the IgG isotype control (13.2-fold), and was in line with previously established targets (WEE1, 3.2-fold; PIM1, 13.9-fold). YAP1 mRNA bound to HuR is abolished when treated with a known HuR inhibitor, pyrvinium pamoate, even in the presence of an established HuR stressor (i.e., oxaliplatin). Actinomycin D chase assays demonstrated that YAP1 mRNA stability is significantly dependent on HuR proficiency. We validated that both YAP1 mRNA and protein expression levels are dependent on HuR via real-time quantitative PCR and Western blot analysis. Transcription of downstream YAP1 targets, CTGF and CYR61, was also significantly affected by knockdown of HuR. Surprisingly, we found that RNA silencing of YAP1 significantly reduced HuR mRNA and protein expression, as well as HuR targets, WEE1 and PIM1. Treatment with small-molecule inhibitors, Verteporfin and CA3, which target the interface of YAP1’s transcription-factor binding domain, recapitulated these effects in a dose and time-dependent manner. We are currently using chromatin immunoprecipitation sequencing to identify HuR’s promoter/enhancer domains for YAP1-mediated transcription. Our previous work has shown that overexpression of cytoplasmic HuR correlates strongly with tumor staging. Low-levels of HuR correspond to early PanINs with staining steadily increasing in late-stage PanIN lesions and gross overexpression in invasive adenocarcinoma. Conversely, YAP1 overexpression seems to be most critical for initial development and expansion of the tumor cells, while it converts to a maintenance role once PDAC is fully developed. Ongoing studies will address whether the temporal regulation of these proteins could explain their overexpression patterns in pancreatic pathologic stages as they relate to cooperating with KRAS activity. Citation Format: Samantha Z. Brown, Christopher W. Schultz, Tianyun Li, Aditi Jain, Raymond O'Neill, Wei Jiang, Charles P. Yeo, Jonathan R. Brody. A novel, reversible YAP1-HuR axis promotes pancreatic tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4388.