Yes-associated Protein Activation Research Articles

Decreased YAP activity reduces proliferative ability in human induced pluripotent stem cell of duchenne muscular dystrophy derived cardiomyocytes

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration accompanied by dilated cardiomyopathy. Recently, abnormality of yes-associated protein (YAP) has been reported as the pathogenesis of muscle degeneration of DMD; however YAP activity remains unclear in dystrophic heart of DMD. Herein, we investigated YAP activity using disease-specific induced pluripotent stem cell (iPSC) derived cardiomyocytes (CMs) in DMD. DMD-iPSCs were generated from DMD patient with exon 48–54 deletion in DMD, and genome-edited (Ed)-DMD-iPSCs with in-frame (Ed-DMD-iPSCs) were created using CRISPR/Cas9. Nuclear translocation of YAP [nuclear (N)/cytoplasmic (C) ratio] was significantly lower in DMD-iPSC-CMs than in Ed-DMD-iPSC-CMs. In addition, Ki67 expression, indicating proliferative ability, was significantly lower in DMD-iPSC-CMs than Ed-DMD-iPSC-CMs. Therefore, immunofluorescent staining showed that actin stress fibers associated with YAP activity by mechanotransduction were disorganized in DMD-iPSC-CMs. Lysophosphatidic acid (LPA), a known lipid mediator on induction of actin polymerization, significantly increased YAP activity and actin dynamics in DMD-iPSC-CMs using live cell imaging. These results suggested that altered YAP activity due to impaired actin dynamics reduced proliferative ability in DMD-iPSC-CMs. Hence, decreased YAP activity in dystrophic heart may contribute to DMD-cardiomyopathy pathogenesis.

Protein Phosphatase 2A Mediates YAP Activation in Endothelial Cells Upon VEGF Stimulation and Matrix Stiffness.

Angiogenesis is an essential process during development. Abnormal angiogenesis also contributes to many disease conditions such as tumor and retinal diseases. Previous studies have established the Hippo signaling pathway effector Yes-associated protein (YAP) as a crucial regulator of angiogenesis. In ECs, activated YAP promotes endothelial cell proliferation, migration and sprouting. YAP activity is regulated by vascular endothelial growth factor (VEGF) and mechanical cues such as extracellular matrix (ECM) stiffness. However, it is unclear how VEGF or ECM stiffness signal to YAP, especially how dephosphorylation of YAP occurs in response to VEGF stimulus or ECM stiffening. Here, we show that protein phosphatase 2A (PP2A) is required for this process. Blocking PP2A activity abolishes VEGF or ECM stiffening mediated YAP activation. Systemic administration of a PP2A inhibitor suppresses YAP activity in blood vessels in developmental and pathological angiogenesis mouse models. Consistently, PP2A inhibitor also inhibits sprouting angiogenesis. Mechanistically, PP2A directly interacts with YAP, and this interaction requires proper cytoskeleton dynamics. These findings identify PP2A as a crucial mediator of YAP activation in ECs and hence as an important regulator of angiogenesis.

Serine Metabolism Regulates YAP Activity Through USP7 in Colon Cancer.

Metabolic reprogramming is a vital factor in the development of many types of cancer, including colon cancer. Serine metabolic reprogramming is a major feature of tumor metabolism. Yes-associated protein (YAP) participates in organ size control and tumorigenesis. However, the relationship between YAP and serine metabolism in colon cancer is unclear. In this study, RNA sequencing and metabolomics analyses indicated significant enrichment of the glycine, serine, and threonine metabolism pathways in serine starvation–resistant cells. Short-term serine deficiency inhibited YAP activation, whereas a prolonged response dephosphorylated YAP and promoted its activity. Mechanistically, USP7 increases YAP stability under increased serine conditions by regulating deubiquitination. Verteporfin (VP) effectively inhibited the proliferation of colon cancer cells and organoids and could even modulate serine metabolism by inhibiting USP7 expression. Clinically, YAP was significantly activated in colon tumor tissues and positively correlated with the expression of phosphoglycerate dehydrogenase (PHGDH) and USP7. Generally, our study uncovered the mechanism by which serine metabolism regulates YAP via USP7 and identified the crucial role of YAP in the regulation of cell proliferation and tumor growth; thus, VP may be a new treatment for colon cancer.

YAP‐TEAD mediates peroxisome proliferator‐activated receptor α‐ induced hepatomegaly and liver regeneration in mice

Peroxisome proliferator-activated receptor a (PPARα, NR1C1) is a ligand-activated nuclear receptor involved in the regulation of lipid catabolism and energy homeostasis. PPARα activation induces hepatomegaly and plays an important role in liver regeneration, but the underlying mechanisms remain unclear. In this study, the effect of PPARα activation on liver enlargement and regeneration was investigated in several strains of genetically-modified mice. PPARα activation by the specific agonist WY-14643 significantly induced hepatomegaly in wild-type mice and Pparafl/fl mice, while this effect was abolished in hepatocyte-specific PPARα-deficient (PparaΔHep) mice. Furthermore, PPARα agonist accelerated liver regeneration after 70% partial hepatectomy (PHx). PPARα activation promoted hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area in wild-type, Pparafl/fl and PHx mice. Mechanistically, PPARα activation regulated expressions of yes-associated protein (YAP) and its downstream targets (CTGF, CYR61 and ANKRD1) as well as proliferation-related proteins (CCNA1, CCND1 and CCNE1). PPARα potentially interacted with YAP and PPARα activation induced nuclear translocation of YAP. In addition, PPARα failed to induce hepatomegaly in liver-specific YAP-deficient (Yap-/-) mice and AAV-Yap shRNA-treated mice. Blockade of YAP abolished PPARα-induced hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area. Additionally, disruption of YAP-transcriptional enhancer factor domain family member (TEAD) association significantly suppressed PPARα-induced hepatomegaly, as well as hepatocyte enlargement and proliferation. This study revealed a novel function of PPARα in regulating liver size and liver regeneration via activation of the YAP-TEAD signaling pathway. These findings have implications for understanding the physiological functions of PPARα and suggest its potential for manipulation of liver size and liver regeneration.

The Hippo effector YAP1 biochemically and functionally interacts with the nuclear factor‐kappa B/RELA transcription factor

The transcriptional co-activator YAP1 (yes-associated protein 1) is a critical nuclear effector of the Hippo pathway. The Hippo pathway regulates cell growth, cell motility, cell migration, and carcinogenesis, but poorly defined mechanism. 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 SDF-1a (stromal cell-derived factor-1 alpha) or RANKL (receptor activator of NF-kappa B ligand) enhanced the protein-protein interaction between YAP1 and RELA, as showed by proximity ligation assay. Our confocal microscopy experiment further showed that combined SDF-1a and androgen treatment promoted the YAP1 and RELA colocalization instead of single-agent treatment. Moreover, our promoter-reporter and RNAi experiments showed that knockdown of YAP1 or TEAD, a key mediator of the YAP transcription, significantly reduced the NF-Kappa B promoter-reporter gene activity. Also, disruption of YAP1 activity attenuated the TEAD-RELA interaction. Furthermore, controlled expression of MST1/STK4, a potent inhibitor of YAP1, attenuated the NF-Kappa B-promoter reporter activity. Additionally, our unbiased bioinformatics analysis of the exiting ChIP-seq (chromatin immunoprecipitation-sequencing) data sets identified several genes that are likely co-regulated by the YAP1/TEAD and NF-Kappa B transcription factor. These findings suggest that cooperative androgen and cytokine signaling regulates Hippo/YAP and NF-Kappa B interaction. The YAP1/TEAD and NF-Kappa B interaction may have critical roles in cellular biology and human diseases.

Vinculin Tension and YAP Activation are Altered in Dystrophic Muscle Cells

Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of dystrophin, a protein that links the actin cytoskeleton to the extracellular matrix. Cells generate, sense, and respond to forces via mechanosensitive protein complexes, and altered force transmission via these complexes such as focal adhesions (FAs) are known to contribute to the development and progression of muscular dystrophy. However, the interplay between loss of dystrophin and altered force transmission via FAs remains unknown. Here we show that FAs of dystrophin deficient muscle cells are under less mechanical load and have decreased activation of yes-associated protein 1 (YAP). Using a vinculin bioluminescent tension sensor, we measured FAs tension in transgenic muscle cells expressing wild type (WT) dystrophin, two patient derived dystrophin missense mutants (L172H and L54R), and a non-disease-causing mutation (I232M). We found that cells harboring WT dystrophin showed higher average tension across vinculin compared to the tension in dystrophic cells. I232M vinculin tension was the same as WT tension. YAP is a known FA mechano-target protein that functions in the nucleus as a transcriptional co-activator and regulates several cellular processes including cellular migration. Dystrophic cells had higher levels of cytoplasmic YAP when compared to nuclear YAP, increased YAP S127 inactivating phosphorylation and decreased migration rates, compared to WT and I23M. Our results suggest that the Hippo pathway may regulate YAP activation via LATS1/2 activity. LATS1/2 phosphorylates YAP in S172 sequestering it in the cytoplasm and preventing its translocation to the nucleus, decreasing the expression of YAP target genes in migration. Altogether, our results show that expression of disease-causing mutant dystrophins leads to decreased tension transmission and aberrant mechanotransduction via FAs. We propose that decreased vinculin tension leads to increased Hippo pathway signaling which causes decreased YAP activation that results in impaired migration. Therefore, our cell lines could be used as a platform to characterize the effectiveness of DMD therapies for patients with missense mutations by means of monitoring YAP and migration. Furthermore, they could also be used to explore novel therapeutic targets in the Hippo pathway.

Elaborating the Physiological Role of YAP as a Glucose Metabolism Regulator:A Systematic Review.

Yes-associated protein (YAP) is one of the Hippo pathway's two effectors, a pathway associated with organ size control. Research on YAP has focused on its oncogenic potential. However, in cancer cells, aside from inducing growth, YAP was also found to regulate glucose metabolism. The present review explores YAP's control of glucose metabolism and whether these findings are translatable to physiological conditions. According to current literature, YAP induces the transcriptional activity of most genes associated with glucose metabolism from enzymes to transport proteins. In glycolysis and gluconeogenesis, YAP upregulates all enzymes except for enolase and pyruvate kinase. Multiple research has also shown YAP's ability to regulate the expression of glucose transporter of the GLUT family. Additionally, glucose concentration, hypoxia, and hormones such as insulin and glucagon regulate YAP activity and depend on YAP to exert their biological activity. YAP is thus a central regulator of glucose metabolism, controlling both enzymes and proteins involved in glucose transport. YAP is also situated strategically in several pathways controlling glucose and was found to mediate their effects. If these results were consistent in physiological conditions and across glucose-associated metabolic disturbances, then YAP may become a prospective therapeutic target.

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.

Matrix stiffness changes affect astrocyte phenotype in an in vitro injury model

Injury to the central nervous system (CNS) usually leads to the activation of astrocytes, followed by glial scar formation. The formation of glial scars from active astrocytes in vivo has been found to be dependent on the cell microenvironment. However, how astrocytes respond to different microenvironmental cues during scar formation, such as changes in matrix stiffness, remains elusive. In this work, we established an in vitro model to assess the responses of astrocytes to matrix stiffness changes that may be related to pathophysiology. The investigated hydrogel backbones are composed of collagen type I and alginate. The stiffness of these hybrid hydrogels can be dynamically changed by association or dissociation of alginate chains through adding crosslinkers of calcium chloride or a decrosslinker of sodium citrate, respectively. We found that astrocytes obtain different phenotypes when cultured in hydrogels of different stiffnesses. The obtained phenotypes can be switched in situ when changing matrix stiffness in the presence of cells. Specifically, matrix stiffening reverts astrogliosis, whereas matrix softening initiates astrocytic activation in 3D. Moreover, the effect of matrix stiffness on astrocytic activation is mediated by Yes-associated protein (YAP), where YAP inhibition enhances the upregulation of GFAP and contributes to astrogliosis. To investigate the underlying mechanism of matrix stiffness-dependent GFAP expression, we also developed a mathematical model to describe the time-dependent dynamics of biomolecules involved in the matrix stiffness mechanotransduction process of astrocytes. The modeling results further indicate that the effect of matrix stiffness on cell fate and behavior may be related to changes in the cytoskeleton and subsequent activity of YAP. The results from this study will guide researchers to re-examine the role of matrix stiffness in reactive astrogliosis in vivo and inspire the development of a novel therapeutic approach for controlling glial scar formation following injury, enabling axonal regrowth and improving functional recovery by exploiting the benefits of mechanobiology studies.

Selective Targeting of Vascular Endothelial YAP Activity Blocks EndMT and Ameliorates Unilateral Ureteral Obstruction-Induced Kidney Fibrosis.

Kidney fibrosis is accompanied by vascular dysfunction. Discovering new ways to ameliorate dysfunctional angiogenesis may bypass kidney fibrosis. YAP (Yes-associated protein) plays a multifaceted role during angiogenesis. Here, we found that selectively targeting YAP signaling in the endothelium ameliorates unilateral ureteral obstruction (UUO)-induced kidney fibrosis. Genetic deletion of Yap1, encoding YAP protein, in VE-cadherin+ endothelial cells inhibited endothelial-to-mesenchymal transition (EndMT) and dysfunctional angiogenesis and improved obstructive nephropathy and kidney fibrosis. Treatment with the systemic YAP inhibitor verteporfin worsened kidney fibrosis symptoms because of its lack of cell specificity. In an attempt to identify endothelial-specific YAP modulators, we found that G-protein-coupled receptor coagulation factor II receptor-like 1 (F2RL1) was highly expressed in vessels after UUO-induced kidney fibrosis. The F2RL1 peptide antagonist FSLLRY-NH2 selectively blocked YAP activity in endothelial cells and ameliorated kidney fibrosis. Thus, selective antagonization of endothelial YAP activity might bypass kidney fibrosis and provide new avenues for the design of antifibrotic therapies.

Activation of AMPK inhibits Galectin-3-induced pulmonary artery smooth muscle cells proliferation by upregulating hippo signaling effector YAP.

Galectin-3(Gal-3) is an effective regulator in the pathological process of pulmonary arterial hypertension (PAH). However, the detailed mechanisms underlying Gal-3 contribution to PAH are not yet entirely clear. The aim of the present study was to explore these issues. Proliferation of rat pulmonary arterial smooth muscle cells (PASMCs) was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Small interfering RNA (siRNA) was applied to silence the expression of yes-associated protein (YAP) and Forkhead box M1 (FOXM1). The protein expression and phosphorylation were measured by immunoblotting. The subcellular location of YAP was determined using immunoblotting and immunofluorescence. Gal-3-stimulated PASMCs proliferation in a time- and dose-dependent manner, this was accompanied with, YAP upregulation, dephosphorylation, and nucleus translocation. Gal-3 further increased FOXM1 and cyclinD1 expression via YAP activation. Interfering YAP/FOXM1 axis suppressed Gal-3-induced PASMCs proliferation. Activation of AMPK also inhibited Gal-3-triggered cells proliferation by targeting YAP/FOXM1/cyclinD1 pathway. Gal-3 induced PASMCs proliferation by regulating YAP/FOXM1/cyclinD1 signaling cascade, and activation of AMPK targeted on this axis and suppressed Gal-3-stimulated PASMCs proliferation. Our study provides novel therapeutic targets for prevention and treatment of PAH.

SEPT6 drives hepatocellular carcinoma cell proliferation, migration and invasion via the Hippo/YAP signaling pathway.

Septin 6 (SEPT6) is a member of the GTP-binding protein family that is highly conserved in eukaryotes and regulates various biological functions, including filament dynamics, cytokinesis and cell migration. However, the functional importance of SEPT6 in hepatocellular carcinoma (HCC) is not completely understood. The present study aimed to investigate the expression levels and roles of SEPT6 in HCC, as well as the underlying mechanisms. The reverse transcription quantitative PCR, western blotting and immunohistochemistry staining results demonstrated that SEPT6 expression was significantly elevated in HCC tissues compared with corresponding adjacent non-tumor tissues, which indicated that SEPT6 expression may serve as a marker of poor prognosis for HCC. By performing plasmid transfection and G418 treatment, stable SEPT6-knockdown and SEPT6-overexpression cell lines were established. The Cell Counting Kit-8, flow cytometry and Transwell assay results demonstrated that SEPT6 overexpression significantly increased HCC cell proliferation, cell cycle transition, migration and invasion compared with the Vector group, whereas SEPT6 knockdown displayed significant suppressive effects on HCC cell lines in vitro compared with the control group. Mechanistically, SEPT6 might facilitate F-actin formation, which induced large tumor suppressor kinase 1 dephosphorylation, inhibited Hippo signaling, upregulated yes-associated protein (YAP) expression and nuclear translocation, and upregulated cyclin D1 and matrix metallopeptidase 2 (MMP2) expression. Furthermore, YAP overexpression significantly reversed SEPT6 knockdown-induced inhibitory effects on HCC, whereas YAP knockdown significantly inhibited the oncogenic effect of SEPT6 overexpression on HCC. Collectively, the present study demonstrated that SEPT6 may promote HCC progression by enhancing YAP activation, suggesting that targeting SEPT6 may serve as a novel therapeutic strategy for HCC.

MicroRNAs Regulating Hippo-YAP Signaling in Liver Cancer.

Liver cancer is one of the most common cancers worldwide, and its prevalence and mortality rate are increasing due to the lack of biomarkers and effective treatments. The Hippo signaling pathway has long been known to control liver size, and genetic depletion of Hippo kinases leads to liver cancer in mice through activation of the downstream effectors yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Both YAP and TAZ not only reprogram tumor cells but also alter the tumor microenvironment to exert carcinogenic effects. Therefore, understanding the mechanisms of YAP/TAZ-mediated liver tumorigenesis will help overcome liver cancer. For decades, small noncoding RNAs, microRNAs (miRNAs), have been reported to play critical roles in the pathogenesis of many cancers, including liver cancer. However, the interactions between miRNAs and Hippo-YAP/TAZ signaling in the liver are still largely unknown. Here, we review miRNAs that influence the proliferation, migration and apoptosis of tumor cells by modulating Hippo-YAP/TAZ signaling during hepatic tumorigenesis. Previous findings suggest that these miRNAs are potential biomarkers and therapeutic targets for the diagnosis, prognosis, and treatment of liver cancer.

NUPR1 participates in YAP-mediate gastric cancer malignancy and drug resistance via AKT and p21 activation.

To assess nuclear protein 1 (NUPR1) level in human gastric cancer (GC) cells, explore the effects of NUPR1 on GC progression, and investigate the possible regulatory mechanism. Immunohistochemistry (IHC), Immunoblot and quantitative PCR assays were conducted to detect the NUPR1 level in human GC tissues and corresponding normal tissues. Also, NUPR1 expression level correlates with clinical features of GC patients. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT), transwell assays, Immunoblot assays, and flow cytometry (FCM) assays were used to evaluate the effects of NUPR1 on the proliferation, invasion, epithelial-mesenchymal transformation (EMT) and apoptosis of GC cells in vitro. Immunoblot assays were performed to detect the potential mechanism in NUPR1-mediated drug resistance. We found the expression of NUPR1 was upregulated in human gastric cancer tissues and correlated with the clinical features including tumour size, tumour stage and, lymph node metastasis. We further noticed that the depletion of NUPR1 inhibited the invasion and EMT of gastric cancer cells and stimulated the apoptosis. In doxorubicin-resistant gastric cancer cells, yes-associated protein (YAP) activation was up-regulated, and YAP could regulate the expression of NUPR1 to affect drug-resistance. We further provided the evidence that overexpression of NUPR1 reversed the effect of YAP knockdown on cell malignancy and drug resistance via regulating AKT and p21 pathway. Our findings indicated the involvement of NUPR1 in the progression of gastric cancer and elucidated its molecular mechanism in regulating drug resistance.

LACTB suppresses melanoma progression by attenuating PP1A and YAP interaction.

Very limited progress has been made in the management of advanced melanoma, especially melanoma of uveal origin. Lactamase β (LACTB) is a novel tumor suppressor; however, its biological function in melanoma remains unknown. Herein we demonstrated markedly lower LACTB expression levels in melanoma tissues and cell lines. Overexpression of LACTB suppressed the proliferation, migration and invasion of melanoma cells in vitro. Mechanistically, LACTB inhibited the activity of yes-associated protein (YAP). We showed that the level of phospho-YAP (Serine 127) was increased upon LACTB overexpression, which prevented the translocation of YAP to the nucleus. Further, LACTB could directly bind to PP1A and attenuate the interaction between PP1A and YAP, resulting in decreased YAP dephosphorylation and inactivation in a LATS1-independent manner. Additionally, transfection of phosphorylation-defective YAP mutants reversed LACTB-induced tumor suppression. Upstream, we demonstrated that SOX10 binds to the LACTB promoter and negatively regulates its transcription. Overexpression of LACTB also suppressed the tumorigenicity and lung metastasis of MUM2B uveal melanoma cells in vivo. Taken together, our findings indicate a novel SOX10/LACTB/PP1A signaling cascade that renders YAP inactive and modulates melanoma progression, offering a new therapeutic target for melanoma treatment.

YAP promotes sorafenib resistance in hepatocellular carcinoma by upregulating survivin.

Sorafenib is the standard first-line treatment for advanced hepatocellular carcinoma (HCC), but its use is hampered by secondary drug resistance. Yes-associated protein (YAP) is a downstream effector of the Hippo signaling pathway, which is crucial for liver tumorigenesis. As yet, however, the mechanism underlying sorafenib resistance and the role of YAP therein is not fully understood and needs to be explored further. Western blotting, flow cytometry and CCK-8 assays were used to assess the role of YAP in HCC sorafenib resistance. Next, qRT-PCR and Western blotting were performed to identify survivin asa YAP downstream effector, and rescue experiments were performed to confirm that YAP induces sorafenib resistance via survivin. Additionally, Western blotting, flow cytometry and in vivo xenograft models were used to evaluate the effect of verteporfin in combination with sorafenib on HCC. We found that sorafenib enhances YAP nuclear accumulation and activation, thereby promoting sorafenib resistance through inhibiting apoptosis in HCC cells. In addition, we found that survivin acts as a downstream mediator of YAP to resist sorafenib-induced apoptosis. Pharmacological inhibition of YAP by verteporfin increased the sensitivity of HCC cells to sorafenib and reversed sorafenib resistance. Moreover, verteporfin in combination with sorafenib significantly suppressed in vivo HCC tumor growth. Our data indicate that YAP promotes sorafenib resistance through upregulation of survivin expression in HCC cells. Targeting YAP may be a therapeutic strategy to improve the antitumor effects of sorafenib in HCC.

Small extracellular vesicles with LncRNA H19 “overload”: YAP Regulation as a Tendon Repair Therapeutic Tactic

SummaryFunctional healing of tendon injuries remains a great challenge. Small extracellular vesicles (sEVs) have received attention as pro-regenerative agents. H19 overexpression could bring tendon regenerative ability, but the mechanism is still not fully elucidated, and reliable method for delivery of long non-coding RNAs (LncRNAs) was demanded. We identified the downstream mechanism of H19, the activation of yes-associated protein (YAP) via the H19-PP1-YAP axis. We established tendon stem/progenitor cells (TSPCs) stably overexpressing H19 with CRISPR-dCas9-based hnRNP A2/B1 activation (H19-CP-TSPCs). H19-OL-sEVs (H19 “overloading” sEVs) could be produced effectively from H19-CP-TSPCs. Only H19-OL-sEVs were able to significantly load large amounts of H19 rather than other competitors, and the potential of H19-OL-sEVs to promote tendon healing was far better than that of other competitors. Our study established a relatively reliable method for enrichment of LncRNAs into sEVs, providing new hints for modularized sEV-based therapies, and modularized sEVs represented a potential strategy for tendon regeneration.

Extracts of Perilla frutescens var. Acuta (Odash.) Kudo Leaves Have Antitumor Effects on Breast Cancer Cells by Suppressing YAP Activity.

Yes-associated protein (YAP)/WW domain-containing transcription factor (TAZ) is critical for cell proliferation, survival, and self-renewal. It has been shown to play a crucial oncogenic role in many different types of tumors. In this study, we investigated the antitumor effect of the extracts of Perilla frutescens var. acuta (Odash.) Kudo leaves (PLE) on Hippo-YAP/TAZ signaling. PLE induced the phosphorylation of YAP/TAZ, thereby inhibiting their activity. In addition, the treatment suppresses YAP/TAZ transcriptional activity via the dissociation of the YAP/TAZ-TEAD complex. To elucidate the molecular mechanism of PLE in the regulation of YAP activity, we treated WT and cell lines with gene knockout (KO) for Hippo pathway components with PLE. The inhibitory effects of PLE on YAP-TEAD target genes were significantly attenuated in LATS1/2 KO cells. Moreover, we found the antitumor effect of PLE on MDA-MB-231 and BT549, both of which are triple-negative breast cancer (TNBC) cell lines. PLE reduced the viability of TNBC cells in a dose-dependent manner and induced cell apoptosis. Further, PLE inhibited the migration ability in MDA-MB-231 cells. This ability was weakened in YAP and TEAD-activated clones suggesting that the inhibition of migration by PLE is mainly achieved by regulating YAP activity. Taken together, the results of this study indicate that PLE suppressed cell growth and increased the apoptosis of breast cancer (BC) cells via inactivation of YAP activity in a LATS1/2-dependent manner.

Spatio-selective activation of nuclear translocation of YAP with light directs invasion of cancer cell spheroids

SummaryThe mechanical properties of the extracellular matrix strongly influence tumor progression and invasion. Yes-associated protein (YAP) has been shown to be a key regulator of this process translating mechanical cues from the extracellular matrix into intracellular signals. Despite its apparent role in tumor progression and metastasis, it is not clear yet, whether YAP activation can actively trigger the onset of invasion. To address this question, we designed a photo-activatable YAP (optoYAP), which allows for spatiotemporal control of its activation. The activation mechanism of optoYAP is based on optically triggered nuclear translocation of the protein. Activation of optoYAP induces downstream signaling for several hours and leads to increased proliferation in two- and three-dimensional cultures. Applied to cancer spheroids, optoYAP activation induces invasion. Site-selective activation of optoYAP in cancer spheroids strikingly directs invasion into the activated direction. Thus, nuclear translocation of YAP may be enough to trigger the onset of invasion.

YAP1 Withdrawal in Hepatoblastoma Drives Therapeutic Differentiation of Tumor Cells to Functional Hepatocyte-Like Cells.

Despite surgical and chemotherapeutic advances, the 5-year survival rate for stage IV hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. Yes-associated protein 1 (YAP1) and β-catenin co-activation occurs in 80% of children's HB; however, a lack of conditional genetic models precludes tumor maintenance exploration. Thus, the need for a targeted therapy remains unmet. Given the predominance of YAP1 and β-catenin activation in HB, we sought to evaluate YAP1 as a therapeutic target in HB. We engineered the conditional HB murine model using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1S127A , constitutive β-cateninDelN90 , and a luciferase reporter to murine liver. Tumor regression was evaluated using bioluminescent imaging, tumor landscape characterized using RNA and ATAC sequencing, and DNA footprinting. Here we show that YAP1S127A withdrawal mediates more than 90% tumor regression with survival for 230+ days in mice. YAP1S127A withdrawal promotes apoptosis in a subset of tumor cells, and in remaining cells induces a cell fate switch that drives therapeutic differentiation of HB tumors into Ki-67-negative hepatocyte-like HB cells ("HbHeps") with hepatocyte-like morphology and mature hepatocyte gene expression. YAP1S127A withdrawal drives the formation of hbHeps by modulating liver differentiation transcription factor occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and rescue liver damage in mice. YAP1S127A withdrawal, without silencing oncogenic β-catenin, significantly regresses hepatoblastoma, providing in vivo data to support YAP1 as a therapeutic target for HB. YAP1S127A withdrawal alone sufficiently drives long-term regression in HB, as it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes.