Yes-associated Protein Knockdown Research Articles

1231 Inhibition of YAP With Vertoporfin Makes Chordomas Sensitive to Radiation in Vitro and in Vivo: New Therapies to Combat Radioresistance

INTRODUCTION: Chordomas are locally invasive tumors of notochordal origin with a propensity to resist radiotherapy and with no FDA approved therapies. Verteporfin is a photosensitizer with the ability to inhibit the yes-associated-protein (YAP) pathway which is upregulated in chordomas and enables their resistance to radiotherapy. METHODS: We used five primary chordoma cell lines (JHC7, UCH1, UCH2, MUG-CHOR1, UM-CHOR1) to perform in vitro and in vivo experiments with 64 mice using a xenograft subcutaneous model of chordoma. We then knocked down the YAP gene in chordoma cells and performed experiments to confirm our results were secondary to verteporfin effects on YAP. RESULTS: Verteporfin has additive and synergistic effects on chordoma cells when combined with radiation. Chordoma cells treated with verteporfin have significantly more double-stranded DNA damage after radiation therapy (p < 0.0001) and verteporfin prevents repair of that DNA damage. Verteporfin significantly reduces DNA damage repair proteins and genes in chordoma cells (ATM: p = 0.0007, ATR: p < 0.0001, RADS1: p = 0.0033, BRCA1: p < 0.0001, BRCA2: p = 0.0012, PPP2R4: p < 0.0001). Verteporfin leads to significantly increased frequency of cells in G2/M phase (MUG-CHOR1: p < 0.0001, UCH2: p = 0.0021). We then performed all the above experiments using chordoma cells with the YAP gene knocked down and found that YAP knockdown led to enhanced radiation induced DNA damage (p<0.0001), diminished yH2AX response (p = 0.0264), and increased frequency of cells in G2/M phase (p < 0.0001). In vivo verteporfin treatment significantly inhibited chordoma tumor growth (p < 0.0001) and microparticles were most effective at radiosensitization. CONCLUSIONS: Inhibition of YAP using verteporfin radiosensitizes chordoma via 1) inhibition of the DNA damage repair cascade and 2) increasing frequency of the G2/M cell cycle phase when cells are most sensitive to radiation.

Targeting ROCK2 improves macromolecular permeability in a 3D fibrotic pancreatic cancer microenvironment model

Pancreatic cancer is characterized by a densely fibrotic stroma. The fibrotic stroma hinders the intratumoral penetration of nanomedicine and diminishes therapeutic efficacy. Fibrosis is characterized by an abnormal organization of extracellular matrix (ECM) components, namely the abnormal deposition and/or orientation of collagen and fibronectin. Abnormal ECM organization is chiefly driven by pathological signaling in pancreatic stellate cells (PSCs), the main cell type involved in fibrogenesis. However, whether targeting signaling pathways involved in abnormal ECM organization improves the intratumoral penetration of nanomedicines is unknown. Here, we show that targeting transforming growth factor-β (TGFβ)/Rho-associated kinase (ROCK) 1/2 signaling in PSCs normalizes ECM organization and concomitantly improves macromolecular permeability of the fibrotic stroma. Using a 3-dimensional cell culture model of the fibrotic pancreatic cancer microenvironment, we found that pharmacological inhibition of TGFβ or ROCK1/2 improves the permeation of various macromolecules. By using an isoform-specific pharmacological inhibitor and siRNAs, we show that targeting ROCK2, but not ROCK1, alone is sufficient to normalize ECM organization and improve macromolecular permeability. Moreover, we found that ROCK2 inhibition/knockdown attenuates Yes-associated protein (YAP) nuclear localization in fibroblasts co-cultured with pancreatic cancer cells in 3D. Finally, pharmacological inhibition or siRNA-mediated knockdown of YAP normalized ECM organization and improved macromolecular permeability. Our results together suggest that the TGFβ/ROCK2/YAP signaling axis may be therapeutically targeted to normalize ECM organization and improve macromolecular permeability to augment therapeutic efficacy of nanomedicines in pancreatic cancer.

USP40 promotes hepatocellular carcinoma progression through a YAP/USP40 positive feedback loop

Yes-associated protein (YAP) is an essential driver of hepatocellular carcinoma (HCC) progression and the ubiquitin-proteasome system controls its abundance. However, the role of ubiquitin-specific protease 40 (USP40) in YAP stability remains unclear. Here, USP40 was first identified as a novel regulator of YAP abundance and its target genes in HCC cells. USP40 interacted with YAP to remove the lysine 48 (K48)-linked polyubiquitination of YAP at K252 and K315 sites, thereby maintaining YAP stability. USP40 facilitated the proliferation, colony formation, migration and spheroid formation of HCC cells in vitro and promoted HCC growth in vivo in a YAP-dependent manner. In turn, YAP transcriptionally activated USP40 expression in HCC cells. RNA sequencing analysis showed that about 37% of USP40-regulated genes overlapped with YAP-regulated genes. Interestingly, stiffness-induced USP40 upregulation was abolished by YAP knockdown, and USP40 knockdown attenuated stiffness-induced YAP accumulation in HCC cells. Clinical data demonstrated that USP40 was positively associated with YAP expression in HCC tissues and its high expression indicated a poor prognosis. In conclusion, the USP40/YAP positive feedback loop contributes to HCC progression, suggesting that USP40 may be a promising drug target for anti-HCC.

Extracellular matrix stiffness modulates the mechanophenotypes and focal adhesions of colon cancer cells leading to their invasions via YAP1

Distal metastasis is the main cause of clinical treatment failure in patients with colon cancer. It is now known that the invasion and metastasis of cancer cells is precisely regulated by chemical and physical factors in vivo. However, the role of extracellular matrix (ECM) stiffness in colon cancer cell (CCCs) invasion and metastasis remains unclear. Here, bioinformatical analysis suggested that a high expression level of yes associated protein 1 (YAP1) was significantly associated with metastasis and poor prognosis in colon cancer patients. We further investigated the effects of polyacrylamide hydrogels with different stiffnesses (3, 20, and 38 ​kPa), which were simulated as ECM, on the mechanophenotype (F-actin cytoskeleton organization, electrophoretic rate, membrane fluidity, and Young's modulus) of CCCs. The results showed that a stiffer ECM could induce the maturation of focal adhesions and formation of stress fibers in CCCs, regulate their mechanophenotypes, and promote cell motility. We also demonstrated that the expression levels of YAP1 and paxillin were positively correlated in patients with colon cancer. YAP1 knockdown reduces paxillin clustering and cell motility and alters the cellular mechanophenotypes of CCCs. This is of great significance for an in-depth understanding of the invasion and metastatic mechanisms of colon cancer and for the optimization of clinical therapy from the perspective of mechanobiology.

Fluid shear stress induced-endothelial phenotypic transition contributes to cerebral ischemia-reperfusion injury and repair.

Long-term ischemia leads to insufficient cerebral microvascular perfusion and dysfunction. Reperfusion restores physiological fluid shear stress (FSS) but leads to serious injury. The mechanism underlying FSS-induced endothelial injury in ischemia-reperfusion injury (IRI) remains poorly understood. In this study, a rat model of middle cerebral artery occlusion was constructed to explore cerebrovascular endothelial function and inflammation in vivo. Additionally, the rat brain microvascular endothelial cells (rBMECs) were exposed to a laminar FSS of 0.5 dyn/cm2 for 6 h and subsequently restored to physiological fluid shear stress level (2 dyn/cm2) for 2 and 12 h, respectively. We found that reperfusion induced endothelial-to-mesenchymal transition (EndMT) in endothelial cells, leading to serious blood-brain barrier dysfunction and endothelial inflammation, accompanied by the nuclear accumulation of Yes-associated protein (YAP). During the later stage of reperfusion, cerebral endothelium was restored to the endothelial phenotype with a distinct change in mesenchymal-to-endothelial transition (MEndT), while YAP was translocated and phosphorylated in the cytoplasm. Knockdown of YAP or inhibition of actin polymerization markedly impaired the EndMT in rBMECs. These findings suggest that ischemia-reperfusion increased intensity of FSS triggered an EndMT process and, thus, led to endothelial inflammation and tissue injury, whereas continuous FSS induced a time-dependent reversal MEndT event contributing to the endothelial repair. This study provides valuable insight for therapeutic strategies targeting IRI.

YAP knockdown repressed autophagy in fibroblasts to accelerate wound healing through regulating En1/mTOR axis.

Wound repair dysfunction is becoming a major public health issue worldwide. Yes-associated protein (YAP) has previously been reported to be closely related to wound healing, while how YAP accelerates wound healing via regulating autophagy needs to be further probed. ICR male mice were involved in two independent animal experiments; the mice were randomly allocated into control, autophagy inhibitor (3-MA) (injection), and 3-MA (drip) group or control, si-NC, si-YAP group (8 mice for each). Full-thickness excisional wounds (8 mm) in mice were created by punch to construct an in vivo wound model to observe the effects of autophagy inhibitor (3-MA) (by injection and drip) and si-YAP by electrotransfection. Firstly, we found that the autophagy inhibitor (3-MA) accelerated wound closure in vivo. Loss-of-function experiments subsequently revealed that YAP knockdown led to increased proliferation and migration of fibroblasts as well as reduced autophagy, resulting in accelerated wound healing. In addition, our results revealed that YAP could positively regulate Engrailed-1 (En1) expression in fibroblasts. En1 knockdown also promoted the proliferation and migration of fibroblasts, meanwhile resulting in increased mammalian target of rapamycin (mTOR) levels and reduced autophagy in fibroblasts. YAP knockdown repressed autophagy in fibroblasts to accelerate wound closure by regulating the En1/mTOR axis.

YAP/TAZ Signaling Enhances Angiogenesis of Retinal Microvascular Endothelial Cells in a High-Glucose Environment

Purpose Diabetic retinopathy (DR) is a major cause of irreversible blindness in the working-age population. Neovascularization is an important hallmark of advanced DR. There is evidence that Yes-associated protein (YAP)/transcriptional co-activator with a PDZ binding domain (TAZ) plays an important role in angiogenesis and that its activity is regulated by vascular endothelial growth factor (VEGF). Therefore, the aim of this study was to investigate the effect of YAP/TAZ-VEGF crosstalk on the angiogenic capacity of human retinal microvascular endothelial cells (hRECs) in a high-glucose environment. Methods The expression of YAP and TAZ of hRECs under normal conditions, hypertonic conditions and high glucose were observed. YAP overexpression (OE-YAP), YAP silencing (sh-YAP), VEGF overexpression (OE-VEGF) and VEGF silencing (sh-VEGF) plasmids were constructed. Cell counting kit-8 assay was performed to detect cells proliferation ability, transwell assay to detect cells migration ability, and tube formation assay to detect tube formation ability. The protein expression of YAP, TAZ, VEGF, matrix metalloproteinase (MMP)-8, MMP-13, vessel endothelium (VE)-cadherin and alpha smooth muscle actin (α-SMA) was measured by western blot. Results The proliferation of hRECs was significantly higher in the high glucose group compared with the normal group, as well as the protein expression of YAP and TAZ (p < 0.01). YAP and VEGF promoted the proliferation, migration and tube formation of hRECs in the high glucose environment (p < 0.01), and increased the expression of TAZ, VEGF, MMP-8, MMP-13 and α-SMA while reducing the expression of VE-cadherin (p < 0.01). Knockdown of YAP effectively reversed the above promoting effects of OE-VEGF (p < 0.01) and overexpression of YAP significantly reversed the inhibition effects of sh-VEGF on above cell function (p < 0.01). Conclusion In a high-glucose environment, YAP/TAZ can significantly promote the proliferation, migration and tube formation ability of hRECs, and the mechanism may be related to the regulation of VEGF expression.

HDAC11 Regulates Palmitate-induced NLRP3 Inflammasome Activation by Inducing YAP Expression in THP-1 Cells and PBMCs.

Histone deacetylase 11 (HDAC11) has been implicated in the pathogenesis of metabolic diseases characterized by chronic low-grade inflammation, such as obesity. However, the influence of HDAC11 on inflammation and the specific effect of HDAC11 on the palmitic acid (PA)-induced NLR family pyrin domain containing 3 (NLRP3) inflammasome activation are poorly understood. The effect of PA treatment on HDAC11 activity and the NLRP3 inflammasome was investigated in human peripheral blood mononuclear cells and THP-1 cells. The PA-induced responses of key markers of NLRP3 inflammasome activation, including NLRP3 gene expression, caspase-1 p10 activation, cleaved IL-1β production, and extracellular IL-1β release, were assessed as well. The role of HDAC11 was explored using a specific inhibitor of HDAC11 and by knockdown using small interfering (si)HDAC11 RNA. The relationship between HDAC11 and yes-associated protein (YAP) in the PA-induced NLRP3 inflammasome was investigated in THP-1 cells with HDAC11 or YAP knockdown. Following PA treatment, HDAC11 activity and protein levels increased significantly, concomitant with activation of the NLRP3 inflammasome. Notably, PA-induced the upregulation of NLRP3, caspase-1 p10 activation, the production of cleaved IL-1β, and the release of IL-1β into the extracellular space, all of which were attenuated by FT895 treatment and by HDAC11 knockdown. In THP-1 cells, PA induced the expression of YAP and its interaction with NLRP3, resulting in NLRP3 inflammasome activation, whereas both were inhibited by FT895 and siHDAC11 RNA. These findings demonstrate a pivotal role for HDAC11 in the PA-induced activation of the NLRP3 inflammasome. HDAC11 inhibition thus represents a promising therapeutic strategy for mitigating NLRP3 inflammasome-related inflammation in the context of obesity.

Verteporfin induces lipid peroxidation and ferroptosis in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) has extremely poor prognosis, with a 5-year survival rate of approximately 11 %. Yes-associated protein (YAP) is a major downstream effector of the Hippo-YAP pathway and plays a pivotal role in regulation of cell proliferation and organ regeneration and tumorigenesis. Activation of YAP signaling has been associated with PDAC progression and drug resistance. Verteporfin (VP) is a photosensitizer used for photodynamic therapy and previous work showed that it can function as a YAP inhibitor. The efficacy of VP on human cancer are being tested in several trials. In this study, we examined the effect of VP on reactive oxygen species (ROS) and lipid peroxidation in pancreatic cancer cells, by using fluorescent molecular probes and by measuring the levels of malondialdehyde, a metabolic byproduct and marker of lipid peroxidation. We found that VP causes rapid increase of both overall ROS and lipid peroxide levels, independent of light activation. These effects were not dependent on YAP, as knockdown of YAP did not cause ROS or lipid peroxidation or enhance VP-induced ROS production. Temoporfin, another photodynamic drug, did not show similar activities. In addition, VP treatment led to loss of cell membrane integrity and reduction of viability. Notably, the activity of VP to induce lipid peroxidation was neutralized by ferroptosis inhibitors ferrostatin-1 or liproxstatin-1. VP treatment also reduced the levels of glutathione peroxidase 4 (GPX4), an enzyme that protects against lipid peroxidation. These results indicate that VP can induce lipid peroxidation and ferroptosis in the absence of light activation. Our findings reveal a novel mechanism by which VP inhibits tumor growth and provide insights into development of new therapeutic strategies for the treatment of pancreatic cancer.

Exosomes derived from mesenchymal stromal cells exert a therapeutic effect on hypoxia-induced pulmonary hypertension by modulating the YAP1/SPP1 signaling pathway

ObjectiveHypoxic pulmonary hypertension (HPH) is a progressive and life-threatening disease characterized by perivascular inflammation, pulmonary vascular remodeling, and occlusion. Mesenchymal stromal cell-derived exosomes (MSC-exo) have emerged as potential therapeutic agents due to their role in cell communication and the transportation of bioactive molecules. In this study, we aimed to investigate the therapeutic effects of MSC-exo against HPH and elucidate the underlying molecular mechanism. MethodsExosomes were isolated from conditioned media of human bone mesenchymal stromal cells using ultracentrifugation and characterized through western blotting, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). An HPH animal model was established in male SD rats, and MSC-exo or phosphate-buffered saline (PBS) were administered via the tail vein for three weeks. Subsequently, right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary vascular remodeling were evaluated. Lung tissues from HPH rats and normal rats underwent high-throughput sequencing and transcriptomic analysis. Gene Ontology (GO) analysis was employed to identify upregulated differentially expressed genes. Additionally, rat pulmonary artery smooth muscle cells (PASMC) exposed to platelet-derived growth factor-BB (PDGF-BB) were used to simulate HPH-related pathological behavior. In vitro cellular models were established to examine the molecular mechanism of MSC-exo in HPH. ResultsMSC-exo administration protected rats from hypoxia-induced increases in RVSP, RVHI, and pulmonary vascular remodeling. Additionally, MSC-exo alleviated PDGF-BB-induced proliferation and migration of PASMC. Transcriptomic analysis revealed 267 upregulated genes in lung tissues of HPH rats compared to control rats. Gene Ontology analysis indicated significant differences in pathways associated with Yes Associated Protein 1 (YAP1), a key regulator of cell proliferation and organ size. RT-qPCR and western blot analysis confirmed significantly increased expression of YAP1 in HPH lung tissues and PASMC, which was inhibited by MSC-exo treatment. Furthermore, analysis of datasets demonstrated that Secreted Phosphoprotein 1 (SPP1), also known as Osteopontin (OPN), is a downstream binding protein of YAP1 and can be upregulated by PDGF-BB. MSC-exo treatment reduced the expression of both YAP1 and SPP1. Lentivirus-mediated knockdown of YAP1 inhibited PDGF-BB-induced PASMC proliferation, migration, and SPP1 protein levels. ConclusionOur findings demonstrate that MSC-exo exert a therapeutic effect against hypoxia-induced pulmonary hypertension by modulating the YAP1/SPP1 signaling pathway. The inhibition of YAP1 and downstream SPP1 expression by MSC-exo may contribute to the attenuation of pulmonary vascular remodeling and PASMC proliferation and migration. These results suggest that MSC-exo could serve as a potential therapeutic strategy for the treatment of HPH. Further investigations are warranted to explore the clinical applicability of MSC-exo-based therapies in HPH patients.

YAP Facilitates NEDD4L-Mediated Ubiquitination and Degradation of ACSL4 to Alleviate Ferroptosis in Myocardial Ischemia-Reperfusion Injury.

Ferroptosis is a novel iron-dependent type of cell death that takes part in the progression of myocardial ischemia/reperfusion injury (MIRI). However, the detailed mechanism of ferroptosis underlying MIRI remains unclear. This study aimed to investigate the regulatory role of yes-associated protein (YAP) in ferroptosis during MIRI. The invivo and invitro MIRI models were established in the Sprague-Dawley (SD) rats and H9C2 cardiomyocytes. The infarct volume, pathologic changes, cardiac function, serum levels of lactate dehydrogenase (LDH) and creatine kinase (CK)-MB were detected. Western blotting and immunohistochemistry were performed to measure the expression of YAP, neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) and ferroptosis-related proteins. Ferroptosis was evaluated by Fe2+, malondialdehyde (MDA), LDH, glutathione (GSH), and lipid reactive oxygen species (ROS) levels. Molecular mechanism was analyzed by co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assay. YAP and NEDD4L were remarkably low expressed in MIRI models. YAP overexpression reduced myocardial infarct volume and improved cardiac function. In addition, YAP inhibited MIRI-induced ferroptosis as confirmed by reducing levels of Fe2+, MDA, LDH, lipid ROS, and ferroptosis-related protein ACSL4, and enhancing GSH level and cell viability. Mechanistically, YAP facilitated NEDD4L transcription that consequently caused ubiquitination and degradation of ACSL4, thereby restraining ferroptosis in MIRI. YAP knockdown aggravated MIRI-induced ferroptosis, which was counteracted by NEDD4L overexpression. YAP represses MIRI-induced cardiomyocyte ferroptosis via promoting NEDD4L transcription and subsequent ubiquitination and degradation of ACSL4. YAP-mediated ferroptosis inhibition might be a novel therapeutic strategy for MIRI.

Androgen receptor promotes cell stemness via interacting with co-factor YAP1 in gastric cancer

Cancer stem cells (CSCs) have been proposed to explain tumor relapse and chemoresistance in various types of cancers, and androgen receptor (AR) has been emerged as a potential regulator of stemness in cancers. However, the underlying mechanism of AR-regulated CSCs properties and chemoresistance in gastric cancer (GC) remains unknown. Here, we shown that AR is upregulated in GC tissues and correlates with poor survival rate and CSCs phenotypes of GC patients. According to our experimental data, overexpression of AR upregulated the expression of CSCs markers and this was consistent with the result concluded from data analysis that the expression of AR was positively correlated with CD44 in GC patients. In addition, AR overexpression obviously enhanced the tumor sphere formation ability and chemoresistance of GC cells in vitro. Whereas these effects were attenuated by inhibition of AR. These results were further validated in vivo that MGC-803 cells overexpressing AR had stronger properties to initiate gastric tumorigenesis than the control cells, and inhibition of AR increased the chemosensitivity of GC cells. Mechanically, AR upregulated CD44 expression by directly binding to its promoter region and Yes-associated protein 1 (YAP1) served as the co-factor of AR, which was demonstrated by the fact that the promoting effects of AR on GC cells stemness were partially counteracted by YAP1 knockdown. Thus, this study revealed that AR facilitates CSCs properties and chemoresistance of GC cells via forming complex with YAP1and indicates a potential therapeutic approach to GC patients.

Astrocytic expression of Yes-associated protein (YAP) regulates retinal neovascularization in a mouse model of oxygen-induced retinopathy

Pathological neovascularization is the hallmark of many vascular oculopathies. There is still a great deal of uncertainty surrounding retinal neovascularization research. A working hypothesis that astrocytic Yes-associated protein (YAP) act as a key factor in retinal neovascularization was proposed. And our study was conducted to verified this hypothesis. In vivo, we successfully generated mice deficient in YAP in astrocytes (YAPf/f GFAP-Cre mice) and set up oxygen-induced retinopathy (OIR) model. Pathological neovascularization was evaluated by immunofluorescence staining and western blotting. In vitro, cultured retinal astrocytes were transfected with YAP siRNA. Enzyme-linked immunosorbent assay (ELISA) and western blot were used to determine the proteins in the supernatants and cells. The results showed that YAP was upregulated and activated in the OIR mice retinas. Conditional ablation of YAP aggravated pathological neovascularization, along with the upregulation of vascular endothelial growth factor A (VEGF-A) and monocyte chemoattractant protein-1 (MCP-1). Studies in vitro confirmed that the knockdown of YAP in astrocytes lead to increases in VEGF-A and MCP-1 levels, thus enhancing pro-angiogenic capability of YAP-deficit astrocytes. In conclusion, astrocytic YAP alleviates retinal pathological angiogenesis by inhibiting the over-activation of astrocytes, which suppresses excessive VEGF-A production and neuroinflammation.

YAP mediates resistance to EGF-induced apoptosis in EGFR-mutated non-small cell lung cancer cells

Mechanisms underlying the growth and survival of non-small cell lung cancer (NSCLC) cells positive for activating mutations of the epidermal growth factor receptor gene (EGFR) have remained unclear. We here examined the functional relation between such mutant forms of EGFR and Yes-associated protein (YAP), a transcriptional coactivator of the Hippo signaling pathway that regulates cell proliferation and survival. Under the condition of serum deprivation, epidermal growth factor (EGF) induced activation of YAP in NSCLC cell lines positive for mutated EGFR but not in those wild type (WT) for EGFR. Similar EGF-induced activation of YAP was apparent in A549 lung cancer cells forcibly expressing mutant EGFR but not in those overexpressing the WT receptor. Furthermore, EGF induced apoptotic cell death in serum-deprived A549 cells overexpressing the WT form of EGFR but not in those expressing mutant EGFR, and knockdown of YAP rendered the latter cells sensitive to this effect of EGF. Our results thus suggest that activation of YAP mediates resistance of EGFR-mutated NSCLC cells to EGF-induced apoptosis and thereby contributes specifically to the survival of such cells.

Syndecan-2 modulates the YAP pathway in epithelial-to-mesenchymal transition-related migration, invasion, and drug resistance in colorectal cancer

Epithelial-to-mesenchymal transition (EMT) is associated with an invasive phenotype in colorectal cancer (CRC). Here, we examined the roles of YES-associated protein (YAP) and syndecan-2 (SDC2) in EMT-related progression, invasion, metastasis, and drug resistance in CRC. The expression levels of YAP and SDC2 in CRC patient tumor tissue were quantified by PCR and western blotting. EMT-associated characteristics were assessed using Transwell assays and immunohistochemistry. Co-immunoprecipitation, glutathione S-transferase pull-down, and luciferase reporter assays were used to assess interactions between YAP and SDC2. YAP was found to be highly expressed in tumor tissue from 13/16 CRC patients, while SDC2 was highly expressed in the tumor tissue of 12/16 CRC patients. Overexpression of YAP in colon cancer cells led to increased cell viability, invasion, migration, and oxaliplatin resistance demonstrating that YAP plays a role in EMT. In addition, overexpression of YAP led to decreased expression of the large tumor suppressor kinase 1 (LATS1) and mammalian sterile 20-like kinases (MST1/2). Decreased LATS1 expression was associated with increased levels of cell proliferation. Knockdown of YAP by shRNA interference led to decreased cell invasion, migration, and drug resistance in colon cancer cells and reduced tumorigenesis in a mouse xenograft model. Finally, we established that YAP interacted with SDC2, and demonstrated that SDC2 mediated the YAP pathway through the EMT-related factors BMP4, CTGF and FOXM1.

Transcriptomic analysis of hepatocytes reveals the association between ubiquitin-specific peptidase 1 and yes-associated protein 1 during liver regeneration

ObjectivesThe liver has an excellent ability to regenerate, and disrupted liver regeneration after various injuries leads to an unfavorable prognosis for patients. In this study, we sought to identify novel therapeutic hallmarks that are associated with yes-associated protein 1 (YAP1)-mediated hepatocyte proliferation during the process of liver regeneration. MethodsPartial hepatectomy was conducted to induce liver regeneration in rats. Primary hepatocytes were isolated and cultured. Hepatocyte proliferation was assessed using immunohistochemistry staining, and expression of YAP1 was detected. RNA sequencing and bioinformatics analysis were used to search for potential regulators of YAP1. The association between ubiquitin-specific peptidase 1 (USP1) and YAP1 was validated using in vivo and in vitro experiments. ResultsYAP1 was significantly elevated in regenerative hepatocytes, especially in the nucleus. Knockdown of YAP1 using small interfering RNA or pharmacological inhibition using verteporfin significantly attenuated the proliferation of hepatocytes. The bioinformatics analysis results revealed that USP1 was associated with YAP1-mediated hepatocyte proliferation during liver regeneration. ML-323, a specific inhibitor of USP1-USP1 associated factor 1 (UAF1), significantly decreased the expression of YAP1, Cyclin D1, and proliferating cell nuclear antigen, while these decreased expressions could be rescued by YAP1 overexpression. Furthermore, ML-323 treatment significantly inhibited liver regeneration following partial hepatectomy. ConclusionsIn conclusion, we identified USP1 as a novel biomarker that is associated with YAP1-mediated hepatocyte proliferation in liver regeneration. Pharmacological inhibition of USP1 by ML-323 substantially impairs hepatocyte proliferation during liver regeneration.

Hippo (YAP)-autophagy axis protects against hepatic ischemia-reperfusion injury through JNK signaling.

Hepatic ischemia-reperfusion injury (HIRI) remains a common complication during liver transplantation (LT) in patients. As a key downstream effector of the Hippo pathway, Yes-associated protein (YAP) has been reported to be involved in various physiological and pathological processes. However, it remains elusive whether and how YAP may control autophagy activation during ischemia-reperfusion. Human liver tissues from patients who had undergone LT were obtained to evaluate the correlation between YAP and autophagy activation. Both an in vitro hepatocyte cell line and in vivo liver-specific YAP knockdown mice were used to establish the hepatic ischemia-reperfusion models to determine the role of YAP in the activation of autophagy and the mechanism of regulation. Autophagy was activated in the post-perfusion liver grafts during LT in patients, and the expression of YAP positively correlated with the autophagic level of hepatocytes. Liver-specific knockdown of YAP inhibited hepatocytes autophagy upon hypoxia-reoxygenation and HIRI ( P <0.05). YAP deficiency aggravated HIRI by promoting the apoptosis of hepatocytes both in the in vitro and in vivo models ( P <0.05). Attenuated HIRI by overexpression of YAP was diminished after the inhibition of autophagy with 3-methyladenine. In addition, inhibiting autophagy activation by YAP knockdown exacerbated mitochondrial damage through increasing reactive oxygen species ( P <0.05). Moreover, the regulation of autophagy by YAP during HIRI was mediated by AP1 (c-Jun) N-terminal kinase (JNK) signaling through binding to the transcriptional enhanced associate domain (TEAD). YAP protects against HIRI by inducing autophagy via JNK signaling that suppresses the apoptosis of hepatocytes. Targeting Hippo (YAP)-JNK-autophagy axis may provide a novel strategy for the prevention and treatment of HIRI.

Epstein–Barr virus LMP1 protein promotes proliferation and inhibits differentiation of epithelial cells via activation of YAP and TAZ

Latent Epstein-Barr virus (EBV) infection promotes undifferentiated nasopharyngeal carcinomas (NPCs) in humans, but the mechanism(s) for this effect has been difficult to study because EBV cannot transform normal epithelial cells invitro and the EBV genome is often lost when NPC cells are grown in culture. Here we show that the latent EBV protein, LMP1 (Latent membrane protein 1), induces cellular proliferation and inhibits spontaneous differentiation of telomerase-immortalized normal oral keratinocytes (NOKs) in growth factor-deficient conditions by increasing the activity of the Hippo pathway effectors, YAP (Yes-associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif). We demonstrate that LMP1 enhances YAP and TAZ activity in NOKs both by decreasing Hippo pathway-mediated serine phosphorylation of YAP and TAZ and increasing Src kinase-mediated Y357 phosphorylation of YAP. Furthermore, knockdown of YAP and TAZ is sufficient to reduce proliferation and promote differentiation in EBV-infected NOKs. We find that YAP and TAZ are also required for LMP1-induced epithelial-to-mesenchymal transition. Importantly, we demonstrate that ibrutinib (an FDA-approved BTK inhibitor that blocks YAP and TAZ activity through an off-target effect) restores spontaneous differentiation and inhibits proliferation of EBV-infected NOKs at clinically relevant doses. These results suggest that LMP1-induced YAP and TAZ activity contributes to the development of NPC.

LXA4 inhibits TGF-β1-induced airway smooth muscle cells proliferation and migration by suppressing the Smad/YAP pathway

The aims of the present study were to examine the signaling mechanisms for transforming growth factor-β1 (TGF-β1)-induced rat airway smooth muscle cells (ASMCs) proliferation and migration and to determine the effect of lipoxin A4 (LXA4) on TGF-β1-induced rat ASMCs proliferation and migration and its underlying mechanisms. TGF-β1 upregulated transcriptional coactivator Yes-associated protein (YAP) expression by activating Smad2/3 and then upregulated cyclin D1, leading to rat ASMCs proliferation and migration. This effect was reversed after treatment with the TGF-β1 receptor inhibitor SB431542. YAP is a critical mediator of TGF-β1-induced ASMCs proliferation and migration. Knockdown of YAP disrupted the pro-airway remodeling function of TGF-β1. Preincubation of rat ASMCs with LXA4 blocked TGF-β1-induced activation of Smad2/3 and changed its downstream targets, YAP and cyclin D1, resulting in the inhibition of rat ASMCs proliferation and migration. Our study suggests that LXA4 suppresses Smad/YAP signaling to inhibit rat ASMCs proliferation and migration and therefore has potential value in the prevention and treatment of asthma by negatively modulating airway remodeling.

Yes-associated protein nuclear translocation promotes anabolic activity in human articular chondrocytes

Yes-associated protein (YAP) has been widely studied as a mechanotransducer in many cell types, but its function in cartilage is controversial. The aim of this study was to identify the effect of YAP phosphorylation and nuclear translocation on the chondrocyte response to stimuli relevant to osteoarthritis (OA). Cultured normal human articular chondrocytes from 81 donors were treated with increased osmolarity media as an in vitro model of mechanical stimulation, fibronectin fragments (FN-f) or IL-1β as catabolic stimuli, and IGF-1 as an anabolic stimulus. YAP function was assessed with gene knockdown and inhibition by verteporfin. Nuclear translocation of YAP and its transcriptional co-activator TAZ and site-specific YAP phosphorylation were determined by immunoblotting. Immunohistochemistry and immunofluorescence to detect YAP were performed on normal and OA human cartilage with different degrees of damage. Chondrocyte YAP/TAZ nuclear translocation increased under physiological osmolarity (400mOsm) and IGF-1 stimulation, which was associated with YAP phosphorylation at Ser128. In contrast, catabolic stimulation decreased the levels of nuclear YAP/TAZ through YAP phosphorylation at Ser127. Following YAP inhibition, anabolic gene expression and transcriptional activity decreased. Additionally, YAP knockdown reduced proteoglycan staining and levels of type II collagen. Total YAP immunostaining was greater in OA cartilage, but YAP was sequestered in the cytosol in cartilage areas with more severe damage. YAP chondrocyte nuclear translocation is regulated by differential phosphorylation in response to anabolic and catabolic stimuli. Decreased nuclear YAP in OA chondrocytes may contribute to reduced anabolic activity and promotion of further cartilage loss.