Yes-associated Protein Pathway Research Articles

Fenofibrate induces liver enlargement in aging mice via activating the PPARα-YAP signaling pathway

Fenofibrate is a clinically prescribed drug for treating hypertriglyceridemia, which is also a classic peroxisome proliferator-activated receptor α (PPARα) agonist. We previously reported that fenofibrate induced liver enlargement in adult mice partially through activation of the yes-associated protein (YAP) signaling pathway. However, the effects of fenofibrate on liver enlargement and the YAP signaling pathway in aging mice remain unclear. In this study, D-galactose-induced aging mice, naturally aging mice, and senescence-accelerated mice P8 (SAMP8) were used to investigate the effects of aging on fenofibrate-induced liver enlargement and YAP signaling activation. The results showed that fenofibrate-induced liver enlargement in aging mice was consistent with that of adult mice. The effects of fenofibrate on hepatocyte enlargement around the central vein (CV) area and hepatocyte proliferation around the portal vein (PV) area were comparable between adult and aging mice. There was no significant difference in the upregulation of PPARα downstream proteins between the two groups following fenofibrate treatment. Fenofibrate treatment also increased the expression of proliferation-related proteins and activated the YAP signaling pathway to a similar degree in both groups. In summary, these results demonstrate that the fenofibrate-induced liver enlargement and activation of the YAP pathway are consistent between adult and aging mice, indicating that the effects of fenofibrate on promoting liver enlargement and its activation of the PPARα and YAP pathway were independent of aging. These findings offer a new perspective for the clinical use of fenofibrate in elderly patients and provide a new insight for the role of PPARα in liver enlargement.

Signalling of the neuropeptide calcitonin gene-related peptide (CGRP) through RAMP1 promotes liver fibrosis via TGFβ1/Smad2 and YAP pathways

The liver is innervated by primary sensory nerve fibres releasing the neuropeptide calcitonin gene-related peptide (CGRP). Elevated plasma levels of CGRP have been found in patients with liver fibrosis or cirrhosis. We hypothesised that signalling of CGRP and its receptors might regulate liver fibrosis and propose a novel potential target for the treatment. In this study, hepatic expression of CGRP and its receptor component, the receptor activity-modifying protein 1 (RAMP1), was dramatically increased in diseased livers of patients. In a murine liver fibrosis model, deficiency of RAMP1 resulted in attenuated fibrogenesis characterized by less collagen deposition and decreased activity of hepatic stellate cells (HSC). Mechanistically, activity of the TGFβ1 signalling core component Smad2 was severely impaired in the absence of RAMP1, and Yes-associated protein (YAP) activity was found to be diminished in RAMP1-deficient liver parenchyma. In vitro, stimulation of the HSC line LX-2 cells with CGRP induces TGFβ1 production and downstream signalling as well as HSC activation documented by increased α-SMA expression and collagen synthesis. We further demonstrate in LX-2 cells that CGRP promotes YAP activation and its nuclear translocation subsequent to TGFβ1/Smad2 signals. These data support a promotive effect of CGRP signalling in liver fibrosis via stimulation of TGFβ1/Smad2 and YAP activity.

YAP inhibition overcomes adaptive resistance in HER2-positive gastric cancer treated with trastuzumab via the AKT/mTOR and ERK/mTOR axis

BackgroundHuman epidermal growth factor receptor 2 (HER2)-positive gastric cancer (GC) is a heterogeneous GC subtype characterized by the overexpression of HER2. To date, few specific targeted therapies have demonstrated durable efficacy in HER2-positive GC patients, with resistance to trastuzumab typically emerging within 1 year. However, the mechanisms of resistance to trastuzumab remain incompletely understood, presenting a significant challenge to clinical practice.MethodsIn this study, we integrated genetic screening and bulk transcriptome and epigenomic profiling to define the mechanisms mediating adaptive resistance to HER2 inhibitors and identify potential effective therapeutic strategies for treating HER2-positive GCs.ResultsWe revealed a potential association between adaptive resistance to trastuzumab in HER2-positive GC and the expression of YES-associated protein (YAP). Notably, our investigation revealed that long-term administration of trastuzumab triggers extensive chromatin remodeling and initiates YAP gene transcription in HER2-positive cells characterized by the initial inhibition and subsequent reactivation. Furthermore, treatment of HER2-positive GC cells and cell line-derived xenografts (CDX) models with YAP inhibitors in combination with trastuzumab was found to induce synergistic effects through the AKT/mTOR and ERK/mTOR pathways.ConclusionThese findings underscore the pivotal role of reactivated YAP and mTOR signaling pathways in the development of adaptive resistance to trastuzumab and may serve as a promising joint target to overcome resistance to trastuzumab.

Fenofibrate-promoted hepatomegaly and liver regeneration are PPARα-dependent and partially related to the YAP pathway

Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, is widely prescribed for hyperlipidemia management. Recent studies also showed that it has therapeutic potential in various liver diseases. However, its effects on hepatomegaly and liver regeneration and the involved mechanisms remain unclear. Here, the study showed that fenofibrate significantly promoted liver enlargement and regeneration post-partial hepatectomy in mice, which was dependent on hepatocyte-expressed PPARα. Yes-associated protein (YAP) is pivotal in manipulating liver growth and regeneration. We further identified that fenofibrate activated YAP signaling by suppressing its K48-linked ubiquitination, promoting its K63-linked ubiquitination, and enhancing the interaction and transcriptional activity of the YAP–TEAD complex. Pharmacological inhibition of YAP–TEAD interaction using verteporfin or suppression of YAP using AAV Yap shRNA in mice significantly attenuated fenofibrate-induced hepatomegaly. Other factors, such as MYC, KRT23, RAS, and RHOA, might also participate in fenofibrate-promoted hepatomegaly and liver regeneration. These studies demonstrate that fenofibrate-promoted liver enlargement and regeneration are PPARα-dependent and partially through activating the YAP signaling, with clinical implications of fenofibrate as a novel therapeutic agent for promoting liver regeneration.

GPX4-mediated bone ferroptosis under mechanical stress decreased bone formation via the YAP-TEAD signalling pathway.

Fracture of the alveolar bone resorption is a common complication in orthodontic treatment, which mainly caused by extreme mechanical loading. However, the ferroptosis with orthodontic tooth movement(OTM) relationship has not been thoroughly described. We here analysed whether ferroptosis is involved in OTM-associated alveolar bone loss. Mouse osteoblasts (MC-3T3) and knockdown glutathione peroxidase 4 (GPX4) MC-3T3 were stimulated with compressive force loading and ferrostatin-1 (Fer-1, a ferroptosis inhibitor), and the changes in lipid peroxidation morphology, expression of ferroptosis-related factors and osteogenesis levels were detected. After establishing the rat experimental OTM model, the changes in ferroptosis-related factors and osteogenesis levels were reevaluated in the same manner. Ferroptosis was involved in mechanical stress regulating osteoblast remodelling, and Fer-1 and erastin affected osteoblasts under compression force loading. Fer-1 regulated ferroptosis and autophagy in MC-3T3 and promoted bone proliferation. GPX4-dependent ferroptosis stimulated the YAP (homologous oncoproteins Yes-associated protein) pathway, and GPX4 promoted ferroptosis via the YAP-TEAD (transcriptional enhanced associate domain) signal pathway under mechanical compression force. The invivo experiment results were consistent with the invitro experiment results. Ferroptosis transpires during the motion of orthodontic teeth, with compression force side occurring earlier than stretch side within 4 h. GPX4 plays an important role in alveolar bone loss, while Fer-1 can inhibit the compression force-side alveolar bone loss. GPX4's Hippo-YAP pathway is activated by the lack of compression force in the lateral alveolar bone.

The LKB1–TSSK1B axis controls YAP phosphorylation to regulate the Hippo–YAP pathway

The Hippo pathway’s main effector, Yes-associated protein (YAP), plays a crucial role in tumorigenesis as a transcriptional coactivator. YAP’s phosphorylation by core upstream components of the Hippo pathway, such as mammalian Ste20 kinase 1/2 (MST1/2), mitogen-activated protein kinase kinase kinase kinases (MAP4Ks), and their substrate, large tumor suppressor 1/2 (LATS1/2), influences YAP’s subcellular localization, stability, and transcriptional activity. However, recent research suggests the existence of alternative pathways that phosphorylate YAP, independent of these core upstream Hippo pathway components, raising questions about additional means to inactivate YAP. In this study, we present evidence demonstrating that TSSK1B, a calcium/calmodulin-dependent protein kinase (CAMK) superfamily member, is a negative regulator of YAP, suppressing cellular proliferation and oncogenic transformation. Mechanistically, TSSK1B inhibits YAP through two distinct pathways. Firstly, the LKB1–TSSK1B axis directly phosphorylates YAP at Ser94, inhibiting the YAP–TEAD complex’s formation and suppressing its target genes’ expression. Secondly, the TSSK1B–LATS1/2 axis inhibits YAP via phosphorylation at Ser127. Our findings reveal the involvement of TSSK1B-mediated molecular mechanisms in the Hippo–YAP pathway, emphasizing the importance of multilevel regulation in critical cellular decision-making processes.

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.

Deletion of Endothelial TRPV4 Protects Heart From Pressure Overload-Induced Hypertrophy.

Left ventricular hypertrophy is a bipolar response, starting as an adaptive response to the hemodynamic challenge, but over time develops maladaptive pathology partly due to microvascular rarefaction and impaired coronary angiogenesis. Despite the profound influence on cardiac function, the mechanotransduction mechanisms that regulate coronary angiogenesis, leading to heart failure, are not well known. We subjected endothelial-specific knockout mice of mechanically activated ion channel, TRPV4 (transient receptor potential cation channel subfamily V member 4; TRPV4ECKO) to pressure overload via transverse aortic constriction and examined cardiac function, cardiomyocyte hypertrophy, cardiac fibrosis, and apoptosis. Further, we measured microvascular density and underlying TRPV4 mechanotransduction mechanisms using human microvascular endothelial cells, extracellular matrix gels of varying stiffness, unbiased RNA sequencing, small interfering RNA, Western blot, quantitative-PCR, and confocal immunofluorescence techniques. We demonstrate that endothelial-specific deletion of TRPV4 preserved cardiac function, cardiomyocyte structure, and reduced cardiac fibrosis compared with TRPV4lox/lox mice, 28 days post-transverse aortic constriction. Interestingly, comprehensive RNA sequencing analysis revealed an upregulation of proangiogenic factors (VEGFα [vascular endothelial growth factor α], NOS3 [nitric oxide synthase 3], and FGF2 [fibroblast growth factor 2]) with concomitant increase in microvascular density in TRPV4ECKO hearts after transverse aortic constriction compared with TRPV4lox/lox. Further, an increased expression of VEGFR2 (vascular endothelial growth factor receptor 2) and activation of the YAP (yes-associated protein) pathway were observed in TRPV4ECKO hearts. Mechanistically, we found that downregulation of TRPV4 in endothelial cells induced matrix stiffness-dependent activation of YAP and VEGFR2 via the Rho/Rho kinase/large tumor suppressor kinase pathway. Our results suggest that endothelial TRPV4 acts as a mechanical break for coronary angiogenesis, and uncoupling endothelial TRPV4 mechanotransduction attenuates pathological cardiac hypertrophy by enhancing coronary angiogenesis.

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).

Cyclic tensile strain-induced yes-associated protein activity modulates the response of human periodontal ligament mesenchymal stromal cells to tumor necrosis factor-α

ObjectivesThis study aimed to evaluate the role of yes-associated protein (YAP) in the inflammatory processes induced in human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) by cyclic tensile strain (CTS). DesignhPDL-MSCs from five periodontally healthy individuals were stimulated with 12% CTS and/or TNF-α for 24 h. YAP activity was determined by analyzing the YAP nuclear localization and the target genes expression, using immunofluorescence and qPCR, respectively. Verteporfin was used to inhibit the activation of YAP. The gene expression of interleukin (IL)-6, IL-8, vascular cell adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1 was analyzed by qPCR. ResultsIn the absence of TNF-α, application of CTS resulted in the nuclear YAP translocation and upregulation of YAP target genes. Verteporfin inhibited the activation of YAP pathway and upregulated the basal expression of IL-6 and IL-8. TNF-α induced the activation of YAP pathway, which was inhibited by verteporfin. However, application of CTS under these conditions diminished TNF-α-induced YAP activation. TNF-α-induced expression of IL-6, VCAM-1, and ICAM-1 was inhibited after the application of CTS. Inhibition of YAP activation by verteporfin diminished TNF-α-induced gene expression of IL-6, VCAM-1, and ICAM-1, and under these conditions no inhibitory effect of CTS on these parameters was observed. ConclusionsYAP is at least partially involved in the CTS-activated mechanotransduction pathway. The effects of CTS and YAP on the inflammatory responses depend on the inflammatory environment. A better understanding of the inflammatory modulation by mechanical stress may help improve the orthodontic strategies, especially in the patient with periodontitis.

EGF Receptor Signaling Modulates YAP Activation and Promotes Experimental Proliferative Vitreoretinopathy.

PurposeBoth epidermal growth factor receptor (EGFR) and the Yes-associated protein (YAP) signaling pathway are implicated in cell proliferation and differentiation. In this study, we explored whether the formation of proliferative vitreoretinopathy (PVR) depends on the interaction of the EGFR receptor and YAP pathway.MethodsWe studied the effects of EGFR and YAP activation on retinal fibrosis in a PVR mouse model as well as in knockout mice (conditional deletion of EGFR or YAP specifically in RPE cells). Reversal and knockdown experiments were performed to induce a model of ARPE-19 cells treated with TGF-β2 in vitro. The effect of EGFR/YAP signaling blockade on the PVR-induced cell cycle and TGF-β2–induced ARPE-19 cell activation was determined.ResultsThe EGFR inhibitor erlotinib or conditional deletion of EGFR attenuated YAP activation and decreased the expression of YAP and its downstream target Cyr61 and of connective tissue growth factor in vivo and in vitro. EGFR-PI3K-PDK1 signaling induced by PVR promoted YAP activation and cell cycle progression. Furthermore, activated EGFR signaling bypassed RhoA to increase the protein levels of YAP, C-Myc, CyclinD1, and Bcl-xl.ConclusionsOur work highlights that EGFR-PI3K-PDK1–dependent YAP activation plays a crucial role in the formation of PVR. Targeting EGFR and the YAP pathway provides promising therapeutic treatments for PVR.

CTNNAL1 participates in the regulation of mucus overproduction in HDM-induced asthma mouse model throughthe YAP-ROCK2 pathway.

Our previous study indicated that adhesion molecule catenin alpha‐like 1(CTNNAL1) is downregulated in airway epithelial cells of asthma patients and asthma animal model but little is known about how the CTNNAL1 affects asthma pathogenesis. To reveal the direct relationship between asthma and CTNNAL1, CTNNAL1‐deficient mouse model in bronchopulmonary tissue was constructed by introducing CTNNAL1‐siRNA sequence using adeno‐associated virus (AAV) as vector. The mouse model of asthma was established by stimulation of house dust mite (HDM). After HDM‐challenged, there was marked airway inflammation, especially mucus hypersecretion in the CTNNAL1‐deficient mice. In addition, the CTNNAL1‐deficient mice exhibited an increase of lung IL‐4 and IL‐13 levels, as well as a significant increase of goblet cell hyperplasia and MUC5AC after HDM exposure. The expression of Yes‐associated protein (YAP), protein that interacted with α‐catenin, was downregulated after CTNNAL1 silencing and was upregulated due to its overexpression. In addition, the interaction between CTNNAL1 and YAP was confirmed by CO‐IP. Besides, inhibition of YAP could decrease the secretion of MUC5AC, IL‐4 and IL‐13 in CTNNAL1‐deficient 16HBE14o‐cells. Above results indicated us that CTNNAL1 regulated mucus hypersecretion through YAP pathway. In addition, the expression of ROCK2 increased when CTNNAL1 was silenced and decreased after YAP silencing, and inhibition of YAP decreased the expression of ROCK2 in CTNNAL1‐deficient HBE cells. Inhibition of ROCK2 decreased MUC5AC expression and IL‐13 secretion. In all, our study demonstrates that CTNNAL1 plays an important role in HDM‐induced asthma, mediating mucus secretion through the YAP‐ROCK2 pathway.

Dopamine receptor D2 antagonism normalizes profibrotic macrophage-endothelial crosstalk in non-alcoholic steatohepatitis

Currently there is no effective treatment for liver fibrosis, which is one of the main histological determinants of non-alcoholic steatohepatitis (NASH). While Hippo/YAP (Yes-associated protein) signaling is essential for liver regeneration, its aberrant activation frequently leads to fibrosis and tumorigenesis. Unravelling "context-specific" contributions of YAP in liver repair might help selectively bypass fibrosis and preserve the pro-regenerative YAP function in hepatic diseases. We used murine liver fibrosis and minipig NASH models, and liver biopsies from patients with cirrhosis. Single-cell RNA-sequencing (scRNA-Seq) was performed, and a G-protein-coupled receptor (GPCR) ligand screening system was used to identify cell-selective YAP inhibitors. YAP levels in macrophages are increased in the livers of humans and mice with liver fibrosis. The increase in type I interferon and attenuation of hepatic fibrosis observed in mice specifically lacking Yap1 in myeloid cells provided further evidence for the fibrogenic role of macrophage YAP. ScRNA-Seq further showed that defective YAP pathway signaling in macrophages diminished a fibrogenic vascular endothelial cell subset that exhibited profibrotic molecular signatures such as angiocrine CTGF and VCAM1 expression. To specifically target fibrogenic YAP in macrophages, we utilized a GPCR ligand screening system and identified a dopamine receptor D2 (DRD2) antagonist that selectively blocked YAP in macrophages but not hepatocytes. Genetic and pharmacological targeting of macrophage DRD2 attenuated liver fibrosis. In a large animal (minipig) NASH model recapitulating human pathology, the DRD2 antagonist blocked fibrosis and restored hepatic architecture. DRD2 antagonism selectively targets YAP-dependent fibrogenic crosstalk between macrophages and CTGF+VCAM1+ vascular niche, promoting liver regeneration over fibrosis in both rodent and large animal models. Fibrosis in the liver is one of the main histological determinants of non-alcoholic steatohepatitis (NASH), a disease paralleling a worldwide surge in metabolic syndromes. Our study demonstrates that a macrophage-specific deficiency in Yes-associated protein (YAP) attenuates liver fibrosis. Dopamine receptor D2 (DRD2) antagonism selectively blocks YAP in macrophages and thwarts liver fibrosis in both rodent and large animal models, and thus holds potential for the treatment of NASH.

Mechanical Strain Regulates Myofibroblast Differentiation of Human Scleral Fibroblasts by YAP.

Scleral extracellular matrix (ECM) remodeling is thought to play a critical role in the pathogenesis of glaucoma. Mechanical strain induced by elevated intraocular pressure can promote myofibroblast differentiation of fibroblasts and result in scleral ECM remodeling; however, the underlying mechanism remains poorly understood. Yes-associated protein (YAP) is a mechanosensory protein and the key downstream transcriptional effector of the Hippo signaling pathway. Here, we investigated the role of YAP in mechanical strain-induced myofibroblast transformation during glaucoma scleral ECM remodeling. Integrative bioinformatics analyses were performed to identify the key pathways for the ECM remodeling of the sclera in glaucoma. Sprague–Dawley rats were used to establish a chronic ocular hypertension model, and the expression of collagen type I (COL1) and YAP in the sclera was analyzed by immunohistochemical analysis and Western blotting. Furthermore, human scleral fibroblasts (HSFs) were cultured and subjected to mechanical strain. In groups with or without the YAP siRNA or YAP inhibitor, cell proliferation, migration capacity, and the expression levels of YAP, COL1, and α-smooth muscle actin (α-SMA) were evaluated by Cell Counting Kit-8 assay, scratch assay, and Western blotting. The interactions between YAP and Smad3 were demonstrated by coimmunoprecipitation, and the expression levels of COL1 and α-SMA were evaluated in groups treated with or without the Smad3 inhibitor. We first revealed that the Hippo signaling pathway may be involved in mechanical strain-induced scleral ECM remodeling through bioinformatics analysis. Furthermore, the in vivo study showed upregulated YAP, COL1, and α-SMA expression in the hypertensive sclera of rats. In vitro, mechanical strain increased YAP and COL1 expression in HSFs and promoted myofibroblast differentiation. After YAP knockdown or inhibition with verteporfin, mechanical strain-induced fibrotic changes in HSFs were markedly suppressed. Additionally, YAP showed a protein interaction with Smad3, and the upregulation of a-SMA and COL1 in response to mechanical strain was also significantly downregulated following the inhibition of Smad3. In conclusion, mechanical strain activated scleral myofibroblast differentiation via YAP. The YAP pathway may play an important role in regulating scleral myofibroblast differentiation and ECM remodeling of the sclera in glaucoma.

Efficient fabrication of stretching hydrogels with programmable strain gradients as cell sheet delivery vehicles

Fabricating functional cell sheets with excellent mechanical strength for tissue regeneration remains challenging. Therefore, we devised a novel 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide/N-hydroxy-succinimide crosslinked hydrogel carrier composed of gelatin (Ge) and beta-cyclodextrin (β-CD) that promoted the adhesion and proliferation of keratinocytes (Kcs) compared with those cultured on a Ge hydrogel due to significantly higher pore size, porosity, and stiffness, as confirmed using field emission scanning electron microscopy (FE-SEM) and shear wave elastography (SWE). Upon exposure to a programmable gradient microenvironment, cells displayed a stress/strain-dependent spatial-temporal distribution of extended cellular phenotypes and cytoskeletons. The promoted proliferation of Kcs and the increased retention of the undifferentiated cell phenotype on Ge-β-CD composite hydrogels under a 15% strain led to the accelerated detachment of cell sheets with retained cell-cell junctions. Moreover, the stretch-triggered upregulated expression of phosphorylated yes-associated protein (YAP) 1 suggested that this effect might be associated with the mechanical stimulation-induced activation of the YAP pathway.

Abstract LB194: Analysis of Hippo/YAP abnormalities in head and neck cancer

Abstract Introduction: Squamous cell carcinomas (HNSCC) comprise more than 95% of all head and neck cancers. Over the past decades the incidence and mortality rates of HNSCC have been steadily increasing worldwide. HNSCCs account for 6% of all human cancers and for 3% of all cancers in the United States. Leukoplakia, also known as smoker's keratosis are described as non-cancerous, pre-malignant inflammatory patches is a major risk factor in the development of HNSCC. In fact, up to 31% of all leukoplakias are believed to transform into HNSCC. Our interest into the molecular contributions to head and neck carcinogenesis has led us to follow up intriguing findings on the contributions of Yes Associated Protein (YAP) pathways that were found to upregulate proliferation, invasion/migration, and survival in head and neck cancer. More recent studies have found that Hippo/Yap activation occurs in many human cancers as well. Materials and Methods: We performed several in silico analyses of available human cancer genetic data. We interrogated data from various Head and Neck Cancer studies. Specifically, we included data from the Recurrent and Metastatic Head & Neck Cancer (MSKCC, JAMA Oncol 2016), Head and Neck Squamous Cell Carcinoma (Broad, Science 2011), Head and Neck Squamous Cell Carcinoma (Johns Hopkins, Science 2011), Head and Neck Squamous Cell Carcinoma (TCGA, PanCancer Atlas), and the Oral Squamous Cell Carcinoma (MD Anderson, Cancer Discov 2013) using CBioPortal web tool. We explored factors responsible for Hippo pathway activation via loss of negative regulators or amplification of Hippo drivers. Results: We found many negative regulators, such as TAOK1, LATS1/2, among others to be mutated, exclusive of FAT1 loss, a known event in HNSCC. We also found amplification of YAP1, a driver of the Hippo pathway. We saw a loss of FAT1 in 22% of tumors, mutations in TAOK1 and LATS1/2 were seen in over 5% of tumors, exclusive of other mutations or gene expression derangements. YAP1 amplification was observed in up to 5% of tumors. Conclusions: These findings substantiate earlier findings where the YAP pathway was found to be upregulated in head and neck cancer subsets. Intriguingly, the loss of negative regulators or amplification of oncogenes, such as YAP1, likely contribute to Hippo/YAP upregulation. Since they are important pathways that may be activated at the crux of oral carcinoma in situ/invasion, findings from our in silico interrogation shed light on genetic abnormalities that probably occur earlier in the carcinogenesis spectrum. Further interrogation of leukoplakia preclinical models or data sets could shed additional light on contributions of Hippo/YAP to oral carcinogenesis. Citation Format: German Velez Reyes, Beverly Wuertz, Frank G. Ondrey. Analysis of Hippo/YAP abnormalities in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB194.

Yes-activated protein promotes primary resistance of BRAF V600E mutant metastatic colorectal cancer cells to mitogen-activated protein kinase pathway inhibitors.

Most colorectal cancer (CRC) patients with the BRAF V600E mutation display resistance to chemotherapy and targeted medicinal treatments. Thus, exploring new drugs and drug resistance mechanisms for the BRAF V600E mutation has become an urgent clinical priority. MTS experiment, cell cloning experiment, cell scratching experiment, Transwell experiment, chromatin immunoprecipitation (ChIP), quantitative polymerase chain reaction (qPCR) and flow cytometry are used. Detect the transcription and protein expression of YAP in colorectal cancer cell lines, establish a transient cell line with YAP gene overexpression and knockdown, and detect the effect of YAP gene expression on the biological functions of colorectal cancer cells RKO and HT-29. And further study the mechanism of YAP regulating the response of RAF and MEK targeted therapy. In this study, for the first time, we verified that the expression of transcription factor yes-associated protein (YAP) was upregulated in BRAF V600E mutant CRC cells. After knocking down YAP, we observed a reduction in the growth rate, proliferation, and invasion ability of colon cancer cells. We further verified that YAP knockdown increased sensitivity of BRAF V600E mutant CRC cells to mitogen-activated protein kinase (MAPK) pathway inhibitors. In addition, we clarified the mechanism underlying YAP regulation of RAF and MAPK/extracellular signal-regulated kinase (MEK)-targeted therapy response: YAP cooperates with RAF→MEK pathway inhibitors to regulate the cell cycle, increase cell G1/S phase arrest, and increase apoptosis. These results suggest that YAP expression may be related to the primary resistance of MAPK inhibitors in metastatic CRC with the BRAF V600E mutation. Therefore, the combination of YAP and MAPK pathway inhibitors in BRAF V600E mutant metastatic CRC may present a promising treatment method.

Microconvex Dot-Featured Silk Fibroin Films for Promoting Human Umbilical Vein Endothelial Cell Angiogenesis via Enhancing the Expression of bFGF and VEGF.

Insufficient vascularization of grafts often leads to delayed tissue ingrowth and impaired tissue function in tissue engineering. The surface topography of grafts plays critical roles in angiogenesis. In the present study, we prepared silk fibroin (SF)-based microtopography films with the number of convex dots ranging from 37 to 4835/mm2. The convex dot-featured topography surfaces were characterized by scanning electron microscopy, a Profilm3D optical profilometer, atomic force microscopy, and a contact angle goniometer. The effect of microtopographic films on the proliferation, adhesion, and expression of angiogenic factors of human umbilical vein endothelial cells (HUVECs) was investigated. Our results demonstrated that the SF film surface with 2899 convex dots/mm2 significantly enhanced adhesion, viability, and levels of vascular endothelial growth factors and basic fibroblast growth factors of HUVECs and significantly downregulated the level of α-SMA in human aortic smooth muscle cells, indicating that the microtopographic films could promote angiogenesis. Furthermore, in vitro results showed that HUVEC proliferation was positively correlated with yes-associated protein (YAP) activation, suggesting that the enhanced angiogenesis was mediated via the YAP pathway. Finally, mice subcutaneous embedding model results indicated that the SF film surface with 2899 convex dots/mm2 could significantly enhance angiogenesis in vivo. Altogether, our results showed that the SF film surface with 2899 convex dots/mm2 promoted the angiogenesis of HUVECs and offered a novel angiogenesis-promoting strategy of implant surface design for tissue engineering.

PXR mediates mifepristone-induced hepatomegaly in mice.

Mifepristone (Mif), an effective synthetic steroidal antiprogesterone drug, is widely used for medical abortion and pregnancy prevention. Due to its anti-glucocorticoid effect, high-dose Mif is also used to treat Cushing's syndrome. Mif was reported to active pregnane X receptor (PXR) in vitro and PXR can induce hepatomegaly via activation and interaction with yes-associated protein (YAP) pathway. High-dose Mif was reported to induce hepatomegaly in rats and mice, but the underlying mechanism remains unclear. Here, the role of PXR was studied in Mif-induced hepatomegaly in C57BL/6 mice and Pxr-knockout mice. The results demonstrated that high-dose Mif (100 mg · kg-1 · d-1, i.p.) treatment for 5 days significantly induced hepatomegaly with enlarged hepatocytes and promoted proliferation, but low dose of Mif (5 mg · kg-1 · d-1, i.p.) cannot induce hepatomegaly. The dual-luciferase reporter gene assays showed that Mif can activate human PXR in a concentration-dependent manner. In addition, Mif could promote nuclear translocation of PXR and YAP, and significantly induced the expression of PXR, YAP, and their target proteins such as CYP3A11, CYP2B10, UGT1A1, ANKRD, and CTGF. However, Mif (100 mg · kg-1 · d-1, i.p.) failed to induce hepatomegaly in Pxr-knockout mice, as well as hepatocyte enlargement and proliferation, further indicating that Mif-induced hepatomegaly is PXR-dependent. In summary, this study demonstrated that PXR-mediated Mif-induced hepatomegaly in mice probably via activation of YAP pathway. This study provides new insights in Mif-induced hepatomegaly, and provides novel evidence on the crucial function of PXR in liver enlargement and regeneration.

SAA1 regulates pro-labour mediators in term labour by activating YAP pathway.

Term labour is associated with activation of inflammation which results in myometrial contractility, cervical ripening and decidual/membrane rupture. Serum amyloid A1 (SAA1) is an acute response protein, whose role and underlying regulatory mechanisms in human labour remain unknown. In this study, we found that the mRNA and protein expression of SAA1 in human myometrium at term was increased in labouring tissues compared to non-labouring tissues. In addition, the expression of SAA1 was significantly increased in human primary myometrial cells treated with the pro-inflammatory cytokines interleukin-1 beta (IL-1β) or tumour necrosis factor-alpha (TNF-α). Knockdown of SAA1 using siRNA (siSAA1) resulted in a significant reduction in the expression and secretion of pro-inflammatory cytokines (IL8, IL6), chemokines (CXCL5, CCL2), adhesion molecules (ICAM1, ICAM5) and contraction-associated factors (COX2, PGE2). Mechanistically, the effects of SAA1 were mediated through activation of the Yes-associated protein (YAP) pathway. There was a decrease in the protein expression of phosphorylated YAP (pYAP) after treatment of siSAA1-transfected human primary myometrial cells with IL-1β or TNF-α. Moreover, enhanced expression of YAP reversed the effect of siSAA1 on pro-labour mediators. In conclusion, these experiments demonstrated that SAA1 accelerates the inflammatory response associated with parturition by activating YAP pathway, which may be a novel understanding of the molecular mechanism of labour onset.