Yes-associated Protein Activation Research Articles

Statins improve endothelial function via suppression of epigenetic-driven EndMT.

The pleiotropic benefits of statins in cardiovascular diseases that are independent of their lipid-lowering effects have been well documented, but the underlying mechanisms remain elusive. Here we show that simvastatin significantly improves human induced pluripotent stem cell-derived endothelial cell functions in both baseline and diabetic conditions by reducing chromatin accessibility at transcriptional enhanced associate domain elements and ultimately at endothelial-to-mesenchymal transition (EndMT)-regulating genes in a yes-associated protein (YAP)-dependent manner. Inhibition of geranylgeranyltransferase (GGTase) I, a mevalonate pathway intermediate, repressed YAP nuclear translocation and YAP activity via RhoA signaling antagonism. We further identified a previously undescribed SOX9 enhancer downstream of statin-YAP signaling that promotes the EndMT process. Thus, inhibition of any component of the GGTase-RhoA-YAP-SRY box transcription factor 9 (SOX9) signaling axis was shown to rescue EndMT-associated endothelial dysfunction both in vitro and in vivo, especially under diabetic conditions. Overall, our study reveals an epigenetic modulatory role for simvastatin in repressing EndMT to confer protection against endothelial dysfunction.

Acyl-CoA thioesterase 12 suppresses YAP-mediated hepatocarcinogenesis by limiting glycerolipid biosynthesis

Cancer cells use acetate to support the higher demand for energy and lipid biosynthesis during uncontrolled cell proliferation, as well as for acetylation of regulatory proteins. Acyl-CoA thioesterase 12 (Acot12) is the enzyme that hydrolyzes acetyl-CoA to acetate in liver cytosol and is downregulated in hepatocellular carcinoma (HCC). A mechanistic role for Acot12 in hepatocarcinogenesis was assessed in mice in response to treatment with diethylnitrosamine(DEN)/carbon tetrachloride (CCl4) administration or prolonged feeding of a diet that promotes non-alcoholic steatohepatitis (NASH). Relative to controls, Acot12−/− mice exhibited accelerated liver tumor formation that was characterized by the hepatic accumulation of glycerolipids, including lysophosphatidic acid (LPA), and that was associated with reduced Hippo signaling and increased yes-associated protein (YAP)-mediated transcriptional activity. In Acot12−/− mice, restoration of hepatic Acot12 expression inhibited hepatocarcinogenesis and YAP activation, as did knockdown of hepatic YAP expression. Excess LPA produced due to deletion of Acot12 signaled through LPA receptors (LPARs) coupled to Gα12/13 subunits to suppress YAP phosphorylation, thereby promoting its nuclear localization and transcriptional activity. These findings identify a protective role for Acot12 in suppressing hepatocarcinogenesis by limiting biosynthesis of glycerolipids including LPA, which preserves Hippo signaling.

YAP promotes AP-1 expression in tubular epithelial cells in the kidney.

Chronic kidney disease (CKD) is a major health problem. Kidney fibrosis is a hallmark and final common pathway of CKD. The Hippo/yes-associated protein (YAP) pathway regulates organ size, inflammation, and tumorigenesis. Our previous study demonstrated tubular YAP activation by tubule-specific double knockout of mammalian STE20-like protein kinase 1/2 (Mst1/2) induced CKD in mice, but the underlying mechanisms remain to be fully elucidated. Activator protein (AP)-1 activation was found to promote tubular atrophy and tubulointerstitial fibrosis. Therefore, we studied whether YAP regulates AP-1 expression in the kidney. We found that expression of various AP-1 components was induced in kidneys subjected to unilateral ureteric obstruction and in Mst1/2 double knockout kidneys, and these inductions were blocked by deletion of Yap in tubular cells, with Fosl1 being most affected compared with other AP-1 genes. Inhibition of Yap also most highly suppressed Fosl1 expression among AP-1 genes in HK-2 and IMCD3 renal tubular cells. YAP bound to the Fosl1 promoter and promoted Fosl1 promoter-luciferase activity. Our results suggest that YAP controls AP-1 expression and that Fosl1 is the primary target of YAP in renal tubular cells.NEW & NOTEWORTHY Yes-associated protein (YAP) activation leads to tubular injury, renal inflammation, and fibrosis, but the underlying mechanisms are not fully understood. We now provide genetic evidence that YAP promotes activator protein-1 expression and that Fosl1 is the primary target of YAP in renal tubular cells.

Low-Intensity Pulsed Ultrasound Attenuates Periodontal Ligament Cells Apoptosis by Activating Yes-Associated Protein-Regulated Autophagy

The goal of the work described here was to determine if low-intensity pulsed ultrasound (LIPUS) has an anti-inflammatory effect on lipopolysaccharide (LPS)-induced inflammation in periodontal ligament cells (PDLCs). The mechanism underlying this effect remains to be explored and is likely related to PDLC apoptosis regulated by Yes-associated protein (YAP) and autophagy. To verify this hypothesis, we used a rat model of periodontitis and primary human PDLCs. We examined alveolar bone resorption in rats and apoptosis, autophagy and YAP activity in LPS-treated PDLCs with and without application of LIPUS by cellular immunofluorescence, transmission electron microscopy and Western blotting. Then, siRNA transfection was used to decrease YAP expression to confirm the regulatory role of YAP in the anti-apoptotic effect of LIPUS on PDLCs. We found that LIPUS attenuated alveolar bone resorption in rats and this was accompanied by YAP activation. LIPUS inhibited hPDLC apoptosis by YAP activation, and promoted autophagic degradation to help autophagy completion. These effects were reversed after YAP expression was blocked. LIPUS attenuates PDLC apoptosis by activating Yes-associated protein-regulated autophagy.

The biophysical property of the limbal niche maintains stemness through YAP

The cell fate decisions of stem cells (SCs) largely depend on signals from their microenvironment (niche). However, very little is known about how biochemical niche cues control cell behavior in vivo. To address this question, we focused on the corneal epithelial SC model in which the SC niche, known as the limbus, is spatially segregated from the differentiation compartment. We report that the unique biomechanical property of the limbus supports the nuclear localization and function of Yes-associated protein (YAP), a putative mediator of the mechanotransduction pathway. Perturbation of tissue stiffness or YAP activity affects SC function as well as tissue integrity under homeostasis and significantly inhibited the regeneration of the SC population following SC depletion. In vitro experiments revealed that substrates with the rigidity of the corneal differentiation compartment inhibit nuclear YAP localization and induce differentiation, a mechanism that is mediated by the TGFβ−SMAD2/3 pathway. Taken together, these results indicate that SC sense biomechanical niche signals and that manipulation of mechano-sensory machinery or its downstream biochemical output may bear fruits in SC expansion for regenerative therapy.

Synergistic Effect of QNZ, an Inhibitor of NF-κB Signaling, and Bone Morphogenetic Protein 2 on Osteogenic Differentiation in Mesenchymal Stem Cells through Fibroblast-Induced Yes-Associated Protein Activation.

Biomaterials carrying recombinant human bone morphogenetic protein 2 (BMP2) have been developed to enhance bone regeneration in the treatment of bone defects. However, various reports have shown that in the bone repair microenvironment, fibroblasts can inhibit BMP2-induced osteogenic differentiation in mesenchymal stem cells (MSCs). Thus, factors that can target fibroblasts and improve BMP2-mediated osteogenesis should be explored. In this project, we focused on whether or not an inhibitor of the NF-κB signaling pathway, QNZ (EVP4593), could play a synergistic role with BMP2 in osteogenesis by regulating the activity of fibroblasts. The roles of QNZ in regulating the proliferation and migration of fibroblasts were examined. In addition, the effect of QNZ combined with BMP2 on the osteogenic differentiation of MSCs was evaluated both in vitro and in vivo. Furthermore, the detailed mechanisms by which QNZ improved BMP2-mediated osteogenesis through the modulation of fibroblasts were analyzed and revealed. Interestingly, we found that QNZ inhibited the proliferation and migration of fibroblasts. Thus, QNZ could relieve the inhibitory effects of fibroblasts on the homing and osteogenic differentiation of mesenchymal stem cells. Furthermore, biomaterials carrying both QNZ and BMP2 showed better osteoinductivity than did those carrying BMP2 alone both in vitro and in vivo. It was found that the mechanism of QNZ involved reactivating YAP activity in mesenchymal stem cells, which was inhibited by fibroblasts. Taken together, our results suggest that QNZ may be a candidate factor for assisting BMP2 in inducing osteogenesis. The combined application of QNZ and BMP2 in biomaterials may be promising for the treatment of bone defects in the future.

Abstract CT006: First-in-class, first-in-human phase 1 trial of VT3989, an inhibitor of yes-associated protein (YAP)/transcriptional enhancer activator domain (TEAD), in patients (pts) with advanced solid tumors enriched for malignant mesothelioma and other tumors with neurofibromatosis 2 (NF2) mutations

Abstract Background: Inactivation of the Hippo pathway is a common finding in MM and other cancers, leading to constitutive YAP activation, but has thus far been undruggable. NF2 encodes Merlin that activates Hippo kinase resulting in inactivation of YAP, which is the major effector of Hippo signaling. NF2-deficient tumors, often due to inactivating NF2 mutations (NF2m), are dependent on YAP for growth. VT3989 is an oral, highly potent and selective inhibitor of TEAD palmitoylation, which blocks YAP function and has shown promising preclinical activity. Materials and Methods: This FIH trial used a 3 + 3 dose escalation design and evaluated VT3989 from 25 to 200 mg QD continuously and 50 to 200 mg intermittently. Pts with refractory solid tumors, ECOG PS 0-1, serum albumin > 2.5, urinary protein creatinine ratio ≤ 0.5 mg/mg and albumin creatinine ratio ≤ 100 mg/g were enrolled. Objectives were to determine the safety, tolerability and recommended Phase 2 dose (RP2D) of VT3989; to evaluate antitumor activity, pharmacokinetics (PK) and correlate NF2m and other alterations in tumor and ctDNA with response. Results: 67 pts have been enrolled. Median age 63.5y (21-83y), ECOG PS 0:1 13:54 pts. Median prior therapies 3 (range 0-8). Tumor types: 42 MM (29 pleural and 13 non-pleural) and 25 solid tumors including 9 meningioma and 4 sarcoma pts. 34 pts had NF2m (29 somatic and 5 germline). All MM pts had progressed on prior platinum/pemetrexed; all but 2 had prior immune checkpoint inhibitors. VT3989 is safe and well tolerated with no dose limiting toxicities. No MTD was defined up to 200 mg QD. The most common AEs are proteinuria, albuminuria and peripheral edema, mainly observed with the continuous schedule. There were 7 possibly related G3 AEs (fatigue, ALT, AST, dehydration, dyspnea, hypotension and peripheral edema) and 1 G4 cardiomyopathy. No pt developed decreased renal function or nephrotic syndrome. VT3989 has a 12-15 day half-life, dose proportional PK and reaches steady state within 2 weeks. Seven pts (6 refractory MM and 1 NF2m sarcoma) achieved RECIST v1.1 partial responses (PRs) with 4 confirmed, 3 unconfirmed (1 pending). 3 PRs in MM pts are ongoing up to 18+ months. The clinical benefit response rate (PR + SD > 8 weeks, per protocol) in MM pts is 57%. Of the 6 MM pts (1 pericardial, 1 pleural/peritoneal and 4 peritoneal) with PR, 2 have NF2m, 3 are wildtype, and 1 is unknown. Conclusions: VT3989 is safe and well tolerated with durable RECIST v1.1 antitumor responses in pts with advanced MM and NF2m cancers, providing the first early clinical proof-of-concept for effectively drugging the Hippo-YAP-TEAD pathway. RP2D optimization including further schedule evaluation and expansion are ongoing in pts with MM and NF2m tumors. Citation Format: Timothy A. Yap, David J. Kwiatkowski, Jayesh Desai, Ibiayi Dagogo-Jack, Michael Millward, Hedy L. Kindler, Anthony W. Tolcher, Sophia Frentzas, Archie W. Thurston, Len Post, F Andrew Dorr. First-in-class, first-in-human phase 1 trial of VT3989, an inhibitor of yes-associated protein (YAP)/transcriptional enhancer activator domain (TEAD), in patients (pts) with advanced solid tumors enriched for malignant mesothelioma and other tumors with neurofibromatosis 2 (NF2) mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT006.

Phase separation of YAP mediated by CC domain promotes hepatoblastoma proliferation via activation of transcription.

Yes-associated protein (YAP), a key transcriptional co-activator associated with cell fate and tumor progression, has been reported to be a powerful driver of hepatoblastoma (HB). In this study, we investigated the mechanism underlying oncogenic role of YAP in HB. The expression of YAP in HB tissues was measured through WB and qRT-PCR. The IHC and IF were performed to determine the distribution of YAP. The phase separation of YAP was proved by living cell imaging and FRAP experiment. The effect of YAP phase separation in HB cells in vitro an in vivo were tested using CCK8, flow cytometry and xenograft tumors. YAP was overexpressed and activated in HB. Nuclear YAP formed an active transcriptional site via LLPS to recruit the crucial transcription factor TEAD4. Thus, YAP phase separation facilitated transcription of oncogenic genes and subsequently mediated chemoresistance of HB. Mechanistically, the phase separation ability of YAP depends on the coiled-coil domain, which is a typical phase-separation domain. The electrostatic interactions and hydrophobic interactions within YAP are also vital to YAP phase separation. More importantly, YAP inhibitor Verteporfin is potential treatment for HB and combination with cisplatin enhanced therapeutic efficacy. Highly expressed and active YAP exerts an oncogenic effect in HB via phase separation and provides new insights for the treatment of HB.

Abstract 1448: The YAP1/TEAD and NF-Kappa B transcription factors may cooperate to mediate prostate cancer progression

Abstract Metastatic castration-resistant prostate cancer is morbid and lethal. The transcriptional coregulator YAP1 (Yes-associated protein 1) is a nuclear effector of the Hippo pathway. The Hippo pathway regulates cell growth, motility, and carcinogenesis. Here, we investigated biochemical and functional interactions between YAP1 and the nuclear factor (NF) kappa B/RELA subunit in prostate cancer cell models. We demonstrated that endogenous YAP1 and RELA form protein complexes in the cell, as revealed by co-immunoprecipitation and western blotting. Compared with control, we found that combined treatment of cells with androgen and SDF1a (stromal cell-derived factor-1 alpha) or RANKL (receptor activator of NF-kappa Β ligand) enhanced the protein-protein interaction between YAP1 and RELA, as showed by proximity ligation assay. Our confocal microscopy experiment further showed that combined SDF1a and androgen treatment augmented the YAP1 and RELA colocalization relative to single-agent. Moreover, our promoter-reporter and RNAi experiments showed that genetic silencing YAP1 or TEAD protein, a key mediator of the YAP1 transcription, significantly reduced the NF-Kappa B promoter-reporter gene activity. Also, disruption of YAP1 activity by genetic and small molecule attenuated endogenous protein-protein interaction between TEAD and RELA. Furthermore, controlled expression of MST1/STK4, a potent inhibitor of YAP1, suppressed the NF-Kappa B-promoter reporter activity. Additionally, our unbiased bioinformatics analysis of the exiting ChIP-seq (chromatin immunoprecipitation-sequencing) data identified several genes likely co-regulated by the TEAD1-4 and NF-Kappa B transcription factors. Besides, our computational analysis of the TCGA and Stand Up To Cancer (SU2C) prostate cancer data sets indicated that expression of YAP/TEAD and NF-kappa B/RELA correlates in clinical cases. These findings suggest that cooperative androgen and cytokine signaling regulates Hippo/YAP and NF-Kappa B interaction and their functions. These may have critical roles in advanced prostate cancer downstream of the Hippo pathway. Citation Format: Bekir Cinar, Marwah M. Al-Mathkour, Abdulrahman M. Dwead, Ava M. Boston, Michael S. Lewis, Carlos S. Moreno. The YAP1/TEAD and NF-Kappa B transcription factors may cooperate to mediate prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1448.

Mir-302a/TWF1 Axis Impairs the Myogenic Differentiation of Progenitor Cells through F-Actin-Mediated YAP1 Activation

Actin cytoskeleton dynamics have been found to regulate myogenesis in various progenitor cells, and twinfilin-1 (TWF1), an actin-depolymerizing factor, plays a vital role in actin dynamics and myoblast differentiation. Nevertheless, the molecular mechanisms underlying the epigenetic regulation and biological significance of TWF1 in obesity and muscle wasting have not been explored. Here, we investigated the roles of miR-302a in TWF1 expression, actin filament modulation, proliferation, and myogenic differentiation in C2C12 progenitor cells. Palmitic acid, the most prevalent saturated fatty acid (SFA) in the diet, decreased the expression of TWF1 and impeded myogenic differentiation while increasing the miR-302a levels in C2C12 myoblasts. Interestingly, miR-302a inhibited TWF1 expression directly by targeting its 3'UTR. Furthermore, ectopic expression of miR-302a promoted cell cycle progression and proliferation by increasing the filamentous actin (F-actin) accumulation, which facilitated the nuclear translocation of Yes-associated protein 1 (YAP1). Consequently, by suppressing the expressions of myogenic factors, i.e., MyoD, MyoG, and MyHC, miR-302a impaired myoblast differentiation. Hence, this study demonstrated that SFA-inducible miR-302a suppresses TWF1 expression epigenetically and impairs myogenic differentiation by facilitating myoblast proliferation via F-actin-mediated YAP1 activation.

Tyrosine phosphorylation of YAP-1 in biliary epithelial cells mediates posthepatectomy liver regeneration and is affected by serotonin.

Experimental data suggested activation of yes-associated protein (YAP-1) as a critical regulator of liver regeneration (LR). Serotonin (5-HT) promotes LR in rodent models and has been proposed to act via YAP-1. How 5-HT affects LR is incompletely understood. A possible mechanism how 5-HT affects human LR was explored. Sixty-one patients were included. Tissue samples prior and 2 h after induction of LR were collected. Circulating levels of 5-HT and osteopontin (OPN) were assessed. YAP-1, its phosphorylation states, cytokeratin 19 (CK-19) and OPN were assessed using immunofluorescence. A mouse model of biliary epithelial cells (BECs) specific deletion of YAP/TAZ was developed. YAP-1 increased as early as 2 h after induction of LR (p = 0.025) predominantly in BECs. BEC specific deletion of YAP/TAZ reduced LR after 70% partial hepatectomy in mice (Ki67%, p < 0.001). SSRI treatment, depleting intra-platelet 5-HT, abolished YAP-1 and OPN induction upon LR. Portal vein 5-HT levels correlated with intrahepatic YAP-1 expression upon LR (R = 0.703, p = 0.035). OPN colocalized with YAP-1 in BECs and its circulating levels increased in the liver vein 2 h after induction of LR (p = 0.017). In the context of LR tyrosine-phosphorylated YAP-1 significantly increased (p = 0.042). Stimulating BECs with 5-HT resulted in increased YAP-1 activation via tyrosine-phosphorylation and subsequently increased OPN expression. BECs YAP-1 appears to be critical for LR in mice and humans. Our evidence suggests that 5-HT, at least in part, exerts its pro-regenerative effects via YAP-1 tyrosine-phosphorylation in BECs and subsequent OPN-dependent paracrine immunomodulation.

Topographic Cues of a PLGA Scaffold Promote Odontogenic Differentiation of Dental Pulp Stem Cells through the YAP/β-Catenin Signaling Axis.

The underlying mechanism of how topographic cues of artificial scaffolds regulate cell function remains poorly understood. Yes-associated protein (YAP) and β-catenin signaling have both been reported to play important roles in mechano-transduction and dental pulp stem cells (DPSCs) differentiation. We investigated the effects of YAP and β-catenin in spontaneous odontogenic differentiation of DPSCs induced by topographic cues of a poly(lactic-co-glycolic acid) (PLGA) membrane. The topographic cues and function of a fabricated PLGA scaffold were explored via scanning electron microscopy (SEM), alizarin red staining (ARS), reverse transcription-polymerase chain reaction (RT-PCR), and pulp capping. Immunohistochemistry (IF), RT-PCR, and western blotting (WB) were used to observe the activation of YAP and β-catenin when DPSCs were cultured on the scaffolds. Further, YAP was inhibited or overexpressed on either side of the PLGA membrane, and YAP, β-catenin, and odontogenic marker expression were analyzed using IF, ARS, and WB. The closed side of the PLGA scaffold promoted spontaneous odontogenic differentiation and nuclear translocation of YAP and β-catenin in vitro and in vivo compared to the open side. The YAP antagonist verteporfin inhibited β-catenin expression, nuclear translocation, and odontogenic differentiation on the closed side, but the effects were rescued by LiCl. YAP overexpressing DPSCs on the open side activated β-catenin signaling and promoted odontogenic differentiation. The topographic cue of our PLGA scaffold promotes odontogenic differentiation of DPSCs and pulp tissue through the YAP/β-catenin signaling axis.

Lipophilic Statins Inhibit YAP Nuclear Localization, Coactivator Activity, and Migration in Response to Ligation of HLA Class I Molecules in Endothelial Cells: Role of YAP Multisite Phosphorylation.

Solid-organ transplant recipients exhibiting HLA donor-specific Abs are at risk for graft loss due to chronic Ab-mediated rejection. HLA Abs bind HLA molecules expressed on the surface of endothelial cells (ECs) and induce intracellular signaling pathways, including the activation of the transcriptional coactivator yes-associated protein (YAP). In this study, we examined the impact of lipid-lowering drugs of the statin family on YAP localization, multisite phosphorylation, and transcriptional activity in human ECs. Exposure of sparse cultures of ECs to cerivastatin or simvastatin induced striking relocalization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEA domain DNA-binding transcription factor-regulated genes connective tissue growth factor and cysteine-rich angiogenic inducer 61. In dense cultures of ECs, statins prevented YAP nuclear import and expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61 stimulated by the mAb W6/32 that binds HLA class I. Exposure of ECs to either cerivastatin or simvastatin completely blocked the migration of ECs stimulated by ligation of HLA class I. Exogenously supplied mevalonic acid or geranylgeraniol reversed the inhibitory effects of statins on YAP localization either in low-density ECs or high-density ECs challenged with W6/32. Mechanistically, cerivastatin increased the phosphorylation of YAP at Ser127, blunted the assembly of actin stress fiber, and inhibited YAP phosphorylation at Tyr357 in ECs. Using mutant YAP, we substantiated that YAP phosphorylation at Tyr357 is critical for YAP activation. Collectively, our results indicate that statins restrain YAP activity in EC models, thus providing a plausible mechanism underlying their beneficial effects in solid-organ transplant recipients.

Targeting YAP-mediated HSC death susceptibility and senescence for treatment of liver fibrosis.

Liver fibrosis results from the accumulation of myofibroblasts (MFs) derived from quiescent HSCs, and yes-associated protein (YAP) controls this state transition. Although fibrosis is also influenced by HSC death and senescence, whether YAP regulates these processes and whether this could be leveraged to treat liver fibrosis are unknown. YAP activity was manipulated in MF-HSCs to determine how YAP impacts susceptibility to pro-apoptotic senolytic agents or ferroptosis. Effects of senescence on YAP activity and susceptibility to apoptosis versus ferroptosis were also examined. CCl 4 -treated mice were treated with a ferroptosis inducer or pro-apoptotic senolytic to determine the effects on liver fibrosis. YAP was conditionally disrupted in MFs to determine how YAP activity in MF-HSC affects liver fibrosis in mouse models. Silencing YAP in cultured MF-HSCs induced HSC senescence and vulnerability to senolytics, and promoted ferroptosis resistance. Conversely, inducing HSC senescence suppressed YAP activity, increased sensitivity to senolytics, and decreased sensitivity to ferroptosis. Single-cell analysis of HSCs from fibrotic livers revealed heterogeneous sensitivity to ferroptosis, apoptosis, and senescence. In mice with chronic liver injury, neither the ferroptosis inducer nor senolytic improved fibrosis. However, selectively depleting YAP in MF-HSCs induced senescence and decreased liver injury and fibrosis. YAP determines whether MF-HSCs remain activated or become senescent. By regulating this state transition, Yap controls both HSC fibrogenic activity and susceptibility to distinct mechanisms for cell death. MF-HSC-specific YAP depletion induces senescence and protects injured livers from fibrosis. Clarifying determinants of HSC YAP activity may facilitate the development of novel anti-fibrotic therapies.

Hypoxia-induced YAP activation and focal adhesion turnover to promote cell migration in mesenchymal TNBC cells.

Hypoxia is commonly characterized by malignant tumors that promote the aggressiveness and metastatic potential of cancer. Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with approximately 46% capacity related to distant metastasis. Transcriptional factor yes-associated protein (YAP), a core component of the Hippo pathway, is associated with poor prognosis and outcome in cancer metastasis. Here, we explored the effect of hypoxia-mediated YAP activation and focal adhesions (FAs) turnover in mesenchymal TNBC cell migration. We characterized the effect of hypoxia on YAP in different breast cancer cell lines using a hypoxia chamber and CoCl2 . Hypoxia-induced YAP nuclear translocation is significantly observed in normal breast epithelial cells, non-TNBC cells, mesenchymal TNBC cells, but not in basal-like TNBC cells. Functionally, we demonstrated that YAP activation was required for hypoxia to promote mesenchymal TNBC cell migration. Furthermore, hypoxia induced the localization of FAs at the leading edge of mesenchymal TNBC cells. In contrast, verteporfin (VP), a YAP inhibitor, significantly reduced the migration and the recruitment of nascent FAs at the cell periphery under hypoxia conditions, which only showed in mesenchymal TNBC cells. Our data support the hypothesis that YAP is novel factor and positively responsible for hypoxia-promoting mesenchymal TNBC cell migration. Our findings provide further evidence and outcomes to help prevent the progression of TNBC.

Stretch-activated nuclear YAP localization in cardiomyocytes.

Yes Associated Protein (YAP) is a mechanosensitive transcriptional activator regulating a variety of cell fates, including proliferation and cell growth. YAP is activated (localized to the nucleus) in both physiological development (e.g., organ growth mediated by cell proliferation) and pathological heart diseases (e.g., organ growth mediated by cell enlargement). Cardiomyocytes (CMs) generate the mechanical work that powers each heartbeat, and each contraction shortens the CM and strains the nuclei. Mechanical forces and nuclear deformation are considered putative drivers of YAP activation. However, the mechanoregulation of YAP's dual roles in organ growth - through proliferation during development and via hypertrophic enlargement in adulthood - remain unknown. Both contractile output and strain increase as the heart works harder under increased load (e.g., with exercise, disease, aging) and over time can lead to remodeling characteristic of hypertrophic cardiomyopathy. To mimic this additional loading, we first patterned extracellular matrix (Matrigel) single-cell rectangles of 7:1 aspect ratio on silicon substrate of a previously published, microfabricated, silicone pneumatic cell-stretching device (Hart, CAMB, 2021). Then, we then seeded human induced pluripotent stem cell-derived CMs (hiPSC-CM) on these patterns to elicit an elongated, anisotropic subcellular structure. We applied 15% stretch strain in static and cyclic, at 1 Hz frequency, for 10 mins to hiPSC-CM aligned in perpendicular or in parallel to the direction of stretch. Finally, we fixed and immunostained with YAP antibodies to quantify YAP localization. We quantified the ratio of nuclear/non-nuclear YAP in hiPSC-CM and found an upregulation of nuclear YAP in parallel, 97±10% in static and 42±5% in cyclic, and no significant change in regulation in perpendicular. Ultimately, these experiments will help us understand how mechanical cues and directionality of forces drive nuclear YAP, and the role of YAP signaling in driving hypertrophic hiPSC-CM remodeling.

Effect of phosphorylation on the assembly of Hippo signaling regulatory complexes.

The Hippo pathway is an evolutionarily conserved signaling network that plays a crucial role in maintaining tissue homeostasis by regulating cell growth/proliferation, differentiation, and apoptosis in response to environmental stimuli. The Hippo pathway integrates a variety of cellular cues including cell contact/density, growth factors, oxidative stress, hormones, and metabolic conditions, to regulate the function of the transcriptional co-activator protein YAP (yes-associated protein) in humans, and its orthologue Yki (yorkie) in Drosophila. Many upstream signals regulate YAP activity by influencing YAP phosphorylation at S127 (S168 in Yki). Although it is understood that phosphorylation of YAP at S127 induces YAP-14-3-3 binding and subsequent cytoplasmic retention, little is known about how phosphorylation at S127 impacts YAP local structure or the assembly of Hippo signaling regulatory complexes. Here we use traditional biophysical techniques such as solution state nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography multi-angle light scattering, to determine the structure, map the binding interface, and characterize the binding thermodynamics of pS127 YAP and Hippo pathway binding partner LATS1 (large tumor suppressor kinase 1).

Extracellular signal-Regulated Kinase 5 (ERK5) is required for the Yes-associated protein (YAP) co-transcriptional activity

YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of several physio-pathological cellular processes, by integrating multiple cell autonomous and microenvironmental cues. YAP is the main downstream effector of the Hippo pathway, a tumor-suppressive signaling able to transduce several extracellular signals. The Hippo pathway acts restraining YAP activity, since its activation induces YAP phosphorylation and cytoplasmic sequestration. However, recent observations indicate that YAP activity can be also modulated by Hippo independent/integrating pathways, still largely unexplored. In this study, we demonstrated the role of the extracellular signal-regulated kinase 5 (ERK5)/mitogen-activated protein kinase in the regulation of YAP activity. By means of ERK5 inhibition/silencing and overexpression experiments, and by using as model liver stem cells, hepatocytes, and hepatocellular carcinoma (HCC) cell lines, we provided evidence that ERK5 is required for YAP-dependent gene expression. Mechanistically, ERK5 controls the recruitment of YAP on promoters of target genes and its physical interaction with the transcriptional partner TEAD; moreover, it mediates the YAP activation occurring in cell adhesion, migration, and TGFβ-induced EMT of liver cells. Furthermore, we demonstrated that ERK5 signaling modulates YAP activity in a LATS1/2-independent manner. Therefore, our observations identify ERK5 as a novel upstream Hippo-independent regulator of YAP activity, thus unveiling a new target for therapeutic approaches aimed at interfering with its function.

Protein tyrosine phosphatase 1B modulates yes associated protein and cholangiocarcinoma cell growth

Background: Cholangiocarcinoma (CCA) is a cancer of the biliary tract that is increasing in incidence. The Hippo pathway effector Yes-associated protein (YAP) has been identified by us and others as a driver for oncogenesis. YAP canonical regulatory pathways consist of serine kinases. These kinases are rarely altered in CCA but increased YAP activity is consistently observed. We have previously demonstrated that YAP tyrosine phosphorylation serves as an important, activating, pathway in CCA. We have also identified the YAP-interacting tyrosine phosphatase, SHP2, as an inhibitory modulator of this pathway and CCA growth. Herein, we explore an additional tyrosine phosphatase, protein tyrosine phosphatase 1B (PTP1B), and its role in regulating YAP activity and CCA cell growth.

The roles and regulation of Yes-associated protein 1 in stem cells

Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo signaling pathway, and it is involved in tumorigenesis, tissue repair, growth, and development. In this review, the biological roles and the mechanisms of YAP1 in mediating stem cell fate decisions are discussed, including cell proliferation, differentiation, and apoptosis. In general, YAP1 promotes the proliferation and differentiation of stem cells, including embryonic stem cells and adult stem cells. It inhibits apoptosis by binding to the transcription factors, e.g., transcriptional enhanced associate domain (TEAD), Smad, runt-related transcription factor 1/2, p73, p63, and Erb84, to maintain tissue homeostasis. The translocalization of YAP1 in cellular nuclei and the phosphorylation in the cytoplasm work as important and unusual events for the activation of YAP1. Moreover, YAP1 serves as the crosstalk for the Hippo pathway and other signaling pathways, including the Wnt and Notch pathways. It is highlighted in this review that YAP1 is an essential regulator for stem cells that have significant applications in regenerative medicine and reproductive medicine.