Nuclear Yes-associated Protein Research Articles

Role of MiR-325-3p in the Regulation of CFL2 and Myogenic Differentiation of C2C12 Myoblasts.

Skeletal myogenesis is required to maintain muscle mass and integrity, and impaired myogenesis is causally linked to the etiology of muscle wasting. Recently, it was shown that excessive uptake of saturated fatty acids (SFA) plays a significant role in the pathogenesis of muscle wasting. Although microRNA (miRNA) is implicated in the regulation of myogenesis, the molecular mechanism whereby SFA-induced miRNAs impair myogenic differentiation remains largely unknown. Here, we investigated the regulatory roles of miR-325-3p on CFL2 expression and myogenic differentiation in C2C12 myoblasts. PA impeded myogenic differentiation, concomitantly suppressed CFL2 and induced miR-325-3p. Dual-luciferase analysis revealed that miR-325-3p directly targets the 3′UTR of CFL2, thereby suppressing the expression of CFL2, a crucial factor for actin dynamics. Transfection with miR-325-3p mimic resulted in the accumulation of actin filaments (F-actin) and nuclear Yes-associated protein (YAP) in myoblasts and promoted myoblast proliferation and cell cycle progression. Consequently, miR-325-3p mimic significantly attenuated the expressions of myogenic factors and thereby impaired the myogenic differentiation of myoblasts. The roles of miR-325-3p on CFL2 expression, F-actin modulation, and myogenic differentiation suggest a novel miRNA-mediated regulatory mechanism of myogenesis and PA-inducible miR-325-3p may be a critical mediator between obesity and muscle wasting.

Neuronal let-7b-5p acts through the Hippo-YAP pathway in neonatal encephalopathy

Despite increasing knowledge on microRNAs, their role in the pathogenesis of neonatal encephalopathy remains to be elucidated. Herein, we identify let-7b-5p as a significant microRNA in neonates with moderate to severe encephalopathy from dried blood spots using next generation sequencing. Validation studies using Reverse Transcription and quantitative Polymerase Chain Reaction on 45 neonates showed that let-7b-5p expression was increased on day 1 in neonates with moderate to severe encephalopathy with unfavourable outcome when compared to those with mild encephalopathy. Mechanistic studies performed on glucose deprived cell cultures and the cerebral cortex of two animal models of perinatal brain injury, namely hypoxic-ischaemic and intrauterine inflammation models confirm that let-7b-5p is associated with the apoptotic Hippo pathway. Significant reduction in neuronal let-7b-5p expression corresponded with activated Hippo pathway, with increased neuronal/nuclear ratio of Yes Associated Protein (YAP) and increased neuronal cleaved caspase-3 expression in both animal models. Similar results were noted for let-7b-5p and YAP expression in glucose-deprived cell cultures. Reduced nuclear YAP with decreased intracellular let-7b-5p correlated with neuronal apoptosis in conditions of metabolic stress. This finding of the Hippo-YAP association with let-7b needs validation in larger cohorts to further our knowledge on let-7b-5p as a biomarker for neonatal encephalopathy.

Yes-Associated Protein Is Required for ZO-1-Mediated Tight-Junction Integrity and Cell Migration in E-Cadherin-Restored AGS Gastric Cancer Cells.

Yes-associated protein (YAP) regulates numerous cellular homeostasis processes and malignant transformation. We found that YAP influences ZO-1-mediated cell migration using E-cadherin-restored EC96 cells derived from gastric malignant AGS cells. Ectopic expression of E-cadherin enhanced straightforward migration of cells, in comparison to the meandering movement of parental AGS cells. In EC96 cells, YAP and ZO-1 expression increased but nuclear YAP levels and activity were reduced. Nuclear factor-κB (NF-κB) mediated the increase in ZO-1 expression, possibly stabilizing cytoplasmic YAP post-translationally. Downregulation of YAP expression using siYAP RNA or stable knock-down inhibited straightforward cell migration by fragmenting ZO-1 containing tight junctions (TJs) but not adherens junctions, implying involvement of YAP in ZO-1-mediated cell migration. The association of YAP with ZO-1 was mediated by angiomotin (AMOT) because downregulation of AMOT dissociated YAP from ZO-1 and reduced cell migration. E-cadherin restoration in malignant cancer cells induced NF-κB signaling to enhance ZO-1 expression and subsequently stabilize YAP. At high expression levels, YAP associates with ZO-1 via AMOT at TJs, influencing ZO-1-mediated cell migration and maintaining TJ integrity.

P04.20 The role of YAP in Glioblastoma cell lines

Abstract BACKGROUND Glioblastoma (GBM) is a primary human malignant brain tumor, the most common in adults. Several studies have highlighted the Hippo-pathway as a cancer signalling network. The Hippo pathway is an evolutionarily conserved signal cascade, which is involved in the control of organ growth. Dysregulations among this pathway have been found in lung, ovarian, liver and colorectal cancer. The key downstream effector of the Hippo-pathway is the Yes-associated protein (YAP); in the nucleus, its function as transcription co-activator is to interact with transcription factors, resulting in the expression of target genes involved in pro-proliferating and anti-apoptotic programs. MATERIAL AND METHODS Using western blotting analysis, we determined the nuclear expression of YAP on three GBM cell lines (U87MG, T98G and A172). To investigate which inhibitors against the Hippo-pathway were the most efficient, we performed a cytotoxic assay: we treated all the three cell lines with different inhibitors such as Verteporfin (VP), Cytochalasin D (CIT), Latrunculin A (LAT), Dobutamine (DOB) and Y27632. Afterwards, we performed a treatment using Doxorubicin (DOX) combined with the inhibitors, evaluating its cytotoxic effect on our cell lines, through cell viability experiments. More western blotting experiments were performed to investigate the oncogenic role of YAP at nucleus level. Furthermore, preliminary experiments have been conducted in order to investigate the apoptosis, senescence and autophagy modulation due to the Hippo-pathway. RESULTS We showed our cell lines express nuclear YAP. We assessed the efficiency of the main inhibitors against Hippo-pathway, proving that VP, LAT A and CIT show a strong cytostatic effect, linked to time increase; plus we saw a cytotoxic effect on T98G. The association of DOX with selected inhibitors is able to reduce cell viability and nuclear YAP expression rate in all three GBM lines. Finally, preliminary experiments were set up to assess how and if the mechanisms of apoptosis, autophagy and senescence were affected by the Hippo-pathway. The combination of DOX with inhibitors promotes resistance to apoptosis. CONCLUSION Our results show that nuclear YAP is present in all tumor lines, thus confirming that this molecular pathway is functioning in GBM lines. Nuclear YAP is more highly expressed after DOX administration. Moreover, the combined treatment (DOX with Hippo-pathway inhibitors) reduces both cell proliferation and viability, and increases the rate of apoptosis. Preliminary experiments on senescence and autophagy were used to determine the best Hippo-pathway inhibitor. These data demonstrate that the Hippo-pathway plays a crucial role in GBM proliferation and resistance to apoptosis. Inhibiting this pathway and in particular the transcription factor YAP, in association with DOX, might be an excellent therapeutic target.

Keloid fibroblasts have elevated and dysfunctional mechanotransduction signaling that is independent of TGF-β

BackgroundFibroblasts found in keloid tissues are known to present an altered sensitivity to microenvironmental stimuli. However, the impact of changes in extracellular matrix stiffness on phenotypes of normal fibroblasts (NFs) and keloid fibroblasts (KFs) is poorly understood. ObjectivesInvestigation the impact of matrix stiffness on NFs and KFs mainly via detecting yes-associated protein (YAP) expression. MethodsWe used fibronectin-coated polyacrylamide hydrogel substrates with a range from physiological to pathological stiffness values with or without TGF-β (fibrogenic inducer). Atomic force microscopy was used to measure the stiffness of fibroblasts. Cellular mechanoresponses were screened by immunocytochemistry, Western blot and Luminex assay. ResultsKFs are stiffer than NFs with greater expression of α-SMA. In NFs, YAP nuclear translocation was induced by increasing matrix stiffness as well as by stimulation with TGF-β. In contrast, KFs showed higher baseline levels of nuclear YAP that was not responsive to matrix stiffness or TGF-β. TGF-β1 induced p-SMAD3 in both KFs and NFs, demonstrating the pathway was functional and not hyperactivated in KFs. Moreover, blebbistatin suppressed α-SMA expression and cellular stiffness in KFs, linking the elevated YAP signaling to keloid phenotype. ConclusionsThese data suggest that whilst normal skin fibroblasts respond to matrix stiffness in vitro, keloid fibroblasts have elevated activation of mechanotransduction signaling insensitive to the microenvironment. This elevated signaling appears linked to the expression of α-SMA, suggesting a direct link to disease pathogenesis. These findings suggest changes to keloid fibroblast phenotype related to mechanotransduction contribute to disease and may be a useful therapeutic target.

Delayed Senescence of Human Vascular Endothelial Cells by Molecular Mobility of Supramolecular Biointerfaces.

Yes-associated protein (YAP), a transcriptional coactivator of the Hippo signaling pathway, has been widely implicated in vascular aging and diseases. For preventing vascular endothelial cell senescence, the design and development of biomaterials to regulate YAP activity are required. This study prepares polyrotaxane-coated surfaces with molecular mobility and clarifies the role of the mobility on vascular endothelial cell senescence through Hippo-YAP signaling. The polyrotaxane surface with high mobility induces cytoplasmic YAP localization in endothelial cells, whereas the surface with low mobility induces nuclear YAP localization. After serial cultivation of endothelial cells using polyrotaxane surfaces with different mobilities for 35 d, the endothelial cells aged on the polyrotaxane surface with high mobility exhibit higher proliferative potential, smaller spreading size, and lower activity of senescence-associated β-galactosidase than those aged on the surface with low mobility. These findings suggest that cellular senescence can be delayed by modulating the molecular mobility on polyrotaxane surfaces.

Fluid shear stress activates YAP to promote epithelial-mesenchymal transition in hepatocellular carcinoma.

Epithelial–mesenchymal transition (EMT) mediated by fluid shear stress (FSS) in the tumor microenvironment plays an important role in driving metastasis of the malignant tumor. As a mechanotransducer, Yes‐associated protein (YAP) is known to translocate into the nucleus to initiate transcription of genes involved in cell proliferation upon extracellular biophysical stimuli. Here, we showed that FSS facilitated cytoskeleton rearrangement in hepatocellular carcinoma cells, which led to the release of YAP from its binding partner, integrin β subunit, in the cytomembrane. Moreover, we found that upregulation of guanine nucleotide exchange factor (GEF)‐H1, a microtubule‐associated Rho GEF, is a critical step in the FSS‐induced translocation of YAP. Nuclear YAP activated the expression of the EMT‐regulating transcription factor SNAI1, but suppressed the expression of N6‐methyladenosine (m6A) modulators; together, this promoted the expression of EMT‐related genes. We also observed that FSS‐treated HepG2 cells showed markedly increased tumorigenesis and metastasis in vivo. Collectively, our findings unravel the underlying molecular processes by which FSS induces translocation of YAP from the cytomembrane to the nucleus, contributes to EMT and enhances metastasis in hepatocellular carcinoma.

Visudyne™: a new therapeutic strategy for the treatment of ovarian cancer

Objectives: Yes-associated protein (YAP) is a transcriptional coactivator regulated via the Hippo pathway that has been shown to be tumorigenic in ovarian cancer (OC). YAP has also been identified to be uniquely expressed within induced regulatory T cells (Tregs). A conditional knockout of YAP in T cells decreased Tregs within the tumor microenvironment and increased antitumor immune response in a subcutaneous mouse melanoma model. Visudyne™ is a liposomal derivative of verteporfin (VP), a known YAP inhibitor that is FDA approved for the treatment of adult macular degeneration. The objective of our study is to: 1) evaluate the effect of Visudyne™ on YAP and its transcriptional targets, 2) determine cytotoxic effects of Visudyne™ in platinum sensitive and resistant OC cell lines, and 3) to evaluate its effect on immune regulatory cells. Methods: Western blot analysis and immunofluorescence were performed to determine YAP expression and localization in R182 and MR182 OC cell lines using specific antibodies for YAP, phosphorylated YAP (pYAP), and connective tissue growth factor (CTGF), a transcriptional target of YAP. Cell proliferation was measured using Incucyte real-time imaging following treatment with increasing doses of cisplatin, VP and Visudyne™ in A2780 wildtype and platinum resistant OC cells. IC50 was calculated using GraphPad PRISM. Three C57/B6 mice were injected intraperitoneally (IP) with triple knockout p53mut/PTENmut/mCherry OC cells. Tumor progression was monitored by mCherry signal with fluorescence spectroscopy. Visudyne™ was administered IP at 2 mg/kg and 3 days later at 4 mg/kg. Flow cytometry was performed using antibodies for CD4, CD8, FOXP3, and interferon gamma to identify Treg and cytolytic T cells in peritoneal lavage and spleen of the control and Visudyne™ treated mice. Download : Download high-res image (329KB) Download : Download full-size image Results: Treatment of OC cells with 1uM Visudyne™ is associated with a significant decrease expression of total YAP, pYAP, and CTGF (Figure 1A). Immunofluorescence demonstrated loss of nuclear YAP and increased pYAP in the cytoplasm of R182 and MR182 cells after VP treatment (Figure 1B). Increasing doses of Visudyne™ significantly decreased cell growth in platinum resistant A2780 OC cells when compared to cells treated with cisplatin alone. In vivo treatment of tumor bearing mice with Visudyne™ is associated with a significant decrease of Treg CD4+/FOXP3+ cells in peritoneal lavage of treated mice compared to the control (85.5% vs. 92.9%, Figure IC). No changes were observed in the spleen in treated mice vs. control. Conclusions: We propose that Visudyne™ should be further investigated as a novel therapeutic agent for the treatment of OC. Its effect on immune function, specifically Tregs, suggests a promising effect either as a single agent or in combination with other checkpoint inhibitors in platinum sensitive and resistant OC.

The diverse roles of YAP in the regulation of human nasal epithelial remodeling

Yes-associated protein (YAP) is essential in maintaining tissue size. Aberrant epithelial remodeling is a key pathological alteration in both inflammation and benign tumors in nasal mucosa. We sought to investigate the expression and localization patterns of YAP in remodeled nasal epithelium of basal cell hyperplasia, goblet cell metaplasia and squamous metaplasia. YAP expression patterns were evaluated in tissues obtained from patients with NP (n = 45) and IP (n = 27), and control subjects with septal deviation (n = 17) and tissue-derived primary cell cultures. Compared to the normal epithelium, expressions of YAP were significantly higher in basal cell hyperplasia (NP, 11.4-fold; IP, 19.6-fold), followed by squamous metaplasia (8.2-fold) and mild to moderate goblet cell metaplasia (2.9-fold); while their expression was lower in severe goblet cell metaplasia (3.3-fold). Our resultsshowed that: 1) ectopic nuclear YAP expression associated with p63+ basal cell hyperplasia and the high proliferative potential epithelial cells; 2) increase of cytoplasmic YAP correlated with mild to moderate goblet cell metaplasia; 3) increase of cytoplasmic YAP correlated with squamous cell metaplasia. The in vitro cell model also demonstrated almost concordant changes of YAP with the mucosa findings. Different YAP expression and localization patterns should play critical but differential roles in the nasal epithelial remodeling processes under mucosal inflammation and benign tumor formation.

Abstract 2005: ZDHHC7 contributes to ferroptosis resistance through YAP localization in ovarian clear cell carcinoma

Abstract [Objective] Ovarian clear cell carcinoma (CCC) shows unique clinical features including an association with benign endometriosis which contains iron-rich fluid, and poor prognosis because of resistance to chemotherapy. Therefore, elucidating the basic mechanism as well as new treatment strategy of ovarian CCC is urgently needed. Recently, ferroptosis, which is characterized by the iron-dependent accumulation of lipid ROS, was reported to affect the malignant behaviors. Also, some reports showed ferroptosis regulation by Hippo signaling. ZDHHC7 is reported as the molecule regulating yes-associated protein (YAP), a key molecule of Hippo signature and is one of the ovarian CCC gene signature which we previously identified. We explored the impact of ferroptosis in ovarian CCC by investigating YAP localization.[Methods] We performed immunohistochemistry (IHC) of malondialdehyde (MDA; a marker of lipid reactive oxygen species, ROS) and 8-Oxo-2'-deoxyguanosine (8-OHdG, a marker of oxidative stress DNA damage) to analyze the status of lipid ROS with ovarian cancer patients. We also performed IHC of YAP and ZDHHC7 to evaluate them in clinical status. We calculated IC50 values of erastin (ferroptosis inducer) with seventeen ovarian cancer cell lines. Functional annotation clustering of microarray expression data was performed using Metascape. ZDHHC7 knockdown and YAP-5SA mutant, or YAP-activated form of ovarian CCC cell lines were generated and applied for in vitro assays.[Results] IHC analysis indicated higher MDA expression (p=0.0354) and 8-OHdG (p<0.0001) in CCC compared to high-grade serous carcinoma (HGSC), suggesting higher lipid ROS as well as oxidative stress status in ovarian CCC. IC50 values of erastin were significantly higher in CCC than in other types of ovarian cancer (p<0.01). Categorical analysis revealed that genes related to Hippo signaling were enriched in erastin-resistant cell lines. IHC analyses indicated low nuclear expression of YAP and high expression of ZDHHC7 in CCC samples (p=0.0010). The cases with low expression of nuclear YAP showed significantly poorer prognosis compared to high expression cases (p=0.0075) in ovarian CCC. YAP-5SA mutant (YAP-active-mutant) CCC cells showed higher sensitivity to erastin and ML210 (ferroptosis inducer) than control in ovarian CCC cell lines. ZDHHC7 knockdown of CCC cells induced nuclear localization of YAP and showed decreased IC50 values of erastin and ML210 compared to control cells.[Conclusion] Although lipid ROS is accumulated in ovarian CCC, ovarian CCC showed ferroptosis resistance through YAP localization regulated by ZDHHC7. Citation Format: Yoko Furutake, Ken Yamaguchi, Masayo Ukita, Mana Taki, Koji Yamanoi, Junzo Hamanishi, Takashi Kobayashi, Masaki Mandai. ZDHHC7 contributes to ferroptosis resistance through YAP localization in ovarian clear cell carcinoma [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 2005.

An Updated Understanding of the Role of YAP in Driving Oncogenic Responses.

Simple SummaryIn 2020, the global cancer database GLOBOCAN estimated 19.3 million new cancer cases worldwide. The discovery of targeted therapies may help prognosis and outcome of the patients affected, but the understanding of the plethora of highly interconnected pathways that modulate cell transformation, proliferation, invasion, migration and survival remains an ambitious goal. Here we propose an updated state of the art of YAP as the key protein driving oncogenic response via promoting all those steps at multiple levels. Of interest, the role of YAP in immunosuppression is a field of evolving research and growing interest and this summary about the current pharmacological therapies impacting YAP serves as starting point for future studies.Yes-associated protein (YAP) has emerged as a key component in cancer signaling and is considered a potent oncogene. As such, nuclear YAP participates in complex and only partially understood molecular cascades that are responsible for the oncogenic response by regulating multiple processes, including cell transformation, tumor growth, migration, and metastasis, and by acting as an important mediator of immune and cancer cell interactions. YAP is finely regulated at multiple levels, and its localization in cells in terms of cytoplasm–nucleus shuttling (and vice versa) sheds light on interesting novel anticancer treatment opportunities and putative unconventional functions of the protein when retained in the cytosol. This review aims to summarize and present the state of the art knowledge about the role of YAP in cancer signaling, first focusing on how YAP differs from WW domain-containing transcription regulator 1 (WWTR1, also named as TAZ) and which upstream factors regulate it; then, this review focuses on the role of YAP in different cancer stages and in the crosstalk between immune and cancer cells as well as growing translational strategies derived from its inhibitory and synergistic effects with existing chemo-, immuno- and radiotherapies.

RAB11A-mediated YAP localization to adherens and tight junctions is essential for colonic epithelial integrity

Within the intestinal epithelium, regulation of intracellular protein and vesicular trafficking is of utmost importance for barrier maintenance, immune responses, and tissue polarity. RAB11A is a small GTPase that mediates the anterograde transport of protein cargos to the plasma membrane. Loss of RAB11A-dependent trafficking in mature intestinal epithelial cells results in increased epithelial proliferation and nuclear accumulation of Yes-associated protein (YAP), a key Hippo-signaling transducer that senses cell–cell contacts and regulates tissue growth. However, it is unclear how RAB11A regulates YAP intracellular localizations. In this report, we examined the relationship of RAB11A to epithelial junctional complexes, YAP, and the associated consequences on colonic epithelial tissue repair. We found that RAB11A controls the biochemical associations of YAP with multiple components of adherens and tight junctions, including α-catenin, β-catenin, and Merlin, a tumor suppressor. In the absence of RAB11A and Merlin, we observed enhanced YAP–β-catenin complex formation and nuclear translocation. Upon chemical injury to the intestine, mice deficient in RAB11A were found to have reduced epithelial integrity, decreased YAP localization to adherens and tight junctions, and increased nuclear YAP accumulation in the colon epithelium. Thus, RAB11A-regulated trafficking regulates the Hippo–YAP signaling pathway for rapid reparative response after tissue injury.

The association between the expression of nuclear Yes-associated protein 1 (YAP1) and p53 protein expression profile in breast cancer patients.

BackgroundYes-associated protein 1 (YAP1) is a key effector molecule regulated by the Hippo pathway and described as a poor prognostic factor in breast cancer. Tumor protein 53 (TP53) mutation is well known as a biomarker related to poor survival outcomes. So far clinical characteristics and survival outcome according to YAP1 and TP53 mutation have been poorly identified in breast cancer.Patients and methodsRetrospectively, 533 breast tumor tissues were collected at the Seoul St Mary’s hospital and Gangnam Severance Hospital from 1992 to 2017. Immunohistochemistry with YAP1 and p53 specific antibodies were performed, and the clinical data were analyzed.ResultsMutant p53 pattern was associated with aggressive tumor features and advanced anatomical stage. Inferior overall survival (OS) and recurrence free survival (RFS) were related with mutant p53 pattern cases with low nuclear YAP1 expression (P = 0.0009 and P = 0.0011, respectively). Multivariate analysis showed that mutant p53 pattern was an independent prognostic marker for OS [hazard ratios (HR): 2.938, 95% confidence intervals (CIs): 1.028–8.395, P = 0.044] and RFS (HR: 1.842, 95% CIs: 1.026–3.304). However, in cases with high nuclear YAP1 expression, there were no significantly difference in OS and RFS according to p53 staining pattern.ConclusionWe found that mutant p53 pattern is a poor prognostic biomarker in breast tumor with low nuclear YAP1 expression. Our findings suggest that interaction between nuclear YAP1 and p53 expression pattern impact survival outcomes.

Geometrically defined environments direct cell division rate and subcellular YAP localization in single mouse embryonic stem cells

Mechanotransduction via yes-associated protein (YAP) is a central mechanism for decision-making in mouse embryonic stem cells (mESCs). Nuclear localization of YAP is tightly connected to pluripotency and increases the cell division rate (CDR). How the geometry of the extracellular environment influences mechanotransduction, thereby YAP localization, and decision-making of single isolated mESCs is largely unknown. To investigate this relation, we produced well-defined 2D and 2.5D microenvironments and monitored CDR and subcellular YAP localization in single mESCs hence excluding cell–cell interactions. By systematically varying size and shape of the 2D and 2.5D substrates we observed that the geometry of the growth environment affects the CDR. Whereas CDR increases with increasing adhesive area in 2D, CDR is highest in small 2.5D micro-wells. Here, mESCs attach to all four walls and exhibit a cross-shaped cell and nuclear morphology. This observation indicates that changes in cell shape are linked to a high CDR. Inhibition of actomyosin activity abrogate these effects. Correspondingly, nuclear YAP localization decreases in inhibitor treated cells, suggesting a relation between cell shape, intracellular forces, and cell division rate. The simplicity of our system guarantees high standardization and reproducibility for monitoring stem cell reactions and allows addressing a variety of fundamental biological questions on a single cell level.

Oxidative Stress-Mediated YAP Dysregulation Contributes to the Pathogenesis of Pemphigus Vulgaris

Pemphigus Vulgaris (PV) is a life-threatening autoimmune disease manifested with blisters in the skin and mucosa and caused by autoantibodies against adhesion protein desmoglein-3 (Dsg3) expressed in epithelial membrane linings of these tissues. Despite many studies, the pathogenesis of PV remains incompletely understood. Recently we have shown Dsg3 plays a role in regulating the yes-associated protein (YAP), a co-transcription factor and mechanical sensor, and constraining reactive oxygen species (ROS). This study investigated the effect of PV sera as well as the anti-Dsg3 antibody AK23 on these molecules. We detected elevated YAP steady-state protein levels in PV cells surrounding blisters and perilesional regions and in keratinocytes treated with PV sera and AK23 with concomitant transient ROS overproduction. Cells treated with hydrogen peroxide also exhibited augmented nuclear YAP accompanied by reduction of Dsg3 and α-catenin, a negative regulator of YAP. As expected, transfection of α-catenin-GFP plasmid rendered YAP export from the nucleus evoked by hydrogen peroxide. In addition, suppression of total YAP was observed in hydrogen peroxide treated cells exposed to antioxidants with enhanced cell-cell adhesion being confirmed by decreased fragmentation in the dispase assay compared to hydrogen peroxide treatment alone. On the other hand, the expression of exogenous YAP disrupted intercellular junction assembly. In contrast, YAP depletion resulted in an inverse effect with augmented expression of junction assembly proteins, including Dsg3 and α-catenin capable of abolishing the effect of AK23 on Dsg3 expression. Finally, inhibition of other kinase pathways, including p38MAPK, also demonstrated suppression of YAP induced by hydrogen peroxide. Furthermore, antioxidant treatment of keratinocytes suppressed PV sera-induced total YAP accumulation. In conclusion, this study suggests that oxidative stress coupled with YAP dysregulation attributes to PV blistering, implying antioxidants may be beneficial in the treatment of PV.

Increased nuclear translation of YAP might act as a potential therapeutic target for NF1-related plexiform neurofibroma.

Plexiform neurofibroma (pNF) in the head and neck is a characteristic feature in patients with neurofibromatosis type 1 (NF1) and is associated with significant disfigurement and psychological distress. Yes-associated protein (YAP), the key molecule involved in the Hippo pathway, is a vital transductor that regulates the proliferation and remyelinating of Schwann cells. The functional status of YAP and its feasibility as a potential target are still unknown in pNF. A total of 17 pNF tumor tissue specimens from the head and neck were collected at the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. Histologically, diagnosis of the Schwann cell region in pNF was achieved with hematoxylin-eosin staining, positive reactions for S100, SOX10, ERK and p-ERK, and low identification of Ki67 and SMA. Compared with normal nerve tissue, obviously increased nuclear YAP was detected in the Schwann cell region of pNF, with a mean nuclear staining rate of 67.11%. Based on the shNF1 Schwann cell model (the RSC96 cell line), with upregulated expression of RAS, ERK and p-ERK, p-YAP (Ser127) and p-YAP (Ser397) were significantly decreased and total YAP and nuclear YAP were increased. According to a confocal assay, the interference of shNF1 substantially promoted YAP nuclear translocation. Compared with control Schwann cells, the YAP inhibitor CA3 might have a more sensitive effect (IC50: NC=0.96±0.04, shNF1=0.71±0.02, P<0.05) on the shNF1 Schwann cell model than the classic MEK1/2 inhibitor selumetinib (IC50: NC=14.36±0.95, shNF1=24.83±0.98, P>0.05). For in vivo inhibition, the CA3 group and the selumetinib group displayed a similar inhibition effect with no significant difference. Increased nuclear translation and the functional state of YAP implies that the YAP-Hippo pathway might play an important role in the formation and remyelination of pNF. Compared with selumetinib, the YAP inhibitor can exhibit a similar but more sensitive effect on NF1-/- Schwann cells. These observations imply that YAP as a novel or adjuvant therapy target in the treatment of pNF.

Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development.

Autophagy and glycolysis are two catabolic processes that manipulate pancreatic ductal adenocarcinoma (PDAC) development in response to hypoxia sensing, yet the underlying mechanism of how they are interlinked remain elusive.Methods: The functional roles of Unc-51 like kinase 1 and 2 (ULK1/2) in pyruvate kinase M2 (PKM2) transcription and glycolysis under hypoxia were assessed by chromatin immunoprecipitation, luciferase reporter, glucose consumption and lactate production assay. Co-immunoprecipitation, cellular ubiquitination, His-pulldown, in vitro protein kinase assay, immunofluorescence, immunohistochemistry, CRISPR technology, in silico studies were adopted to determine the molecular mechanism. Correlation analyses were performed in KPC (Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+) mice and clinical samples from PDAC patients. Therapeutic potential of ULK1/2 inhibitor and 2-deoxyglucose (2-DG) or 3-bromopyruvate (3-BP) was evaluated in cell-derived xenograft (CDX) and the patient-derived xenograft (PDX) models of nude mice.Results: ULK1/2, but not ULK3, augments hypoxic glycolysis in PDAC cells mediated by PKM2 independent of BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). Mechanistically, hypoxia stimulates ULK1 to translocate into nucleus, where it interacts with and phosphorylates yes-associated protein (YAP) at Ser227, resulting in YAP stabilization through blockade of ubiquitin-proteasome system (UPS), which in turn facilitates PKM2 transcription, glycolysis, cell proliferation in vitro as well as PDAC growth in mice. ULK1/2 is positively correlated with YAP and PKM2 in tumor tissues from KPC mice and clinical samples from PDAC patients. Pharmacological deactivation of ULK1/2 potentiates the antineoplastic efficacy of 2-DG and 3-BP in CDX and PDX models.Conclusion: Our findings underscore the Ser227 autophosphorylation-dependent nuclear YAP stabilization as a central node that couples ULK1/2-initiated autophagy to hypoxic glycolysis during PDAC development and propose that targeting ULK1/2 combined with 2-DG or 3-BP might be a feasible therapeutic strategy against PDAC.

Improved epithelial cell-cell adhesion using molecular mobility of supramolecular surfaces.

Cells can sense the surrounding microenvironmental properties including contact with biomaterials. Although in vitro cell fates in response to the physical properties of cell-adhesive materials have been widely reported, their influence on cell-cell adhesion is unclear. Here, we investigated the role of molecular mobility on polyrotaxane surfaces in epithelial cell-cell adhesion. Polyrotaxane surfaces with high mobility induced cytoplasmic yes-associated protein (YAP) localization in epithelial cells, whereas those with low mobility induced nuclear YAP localization, suggesting that YAP localization is switched by the mobility of the polyrotaxane surface. The cytoplasmic YAP localization increased the expression of tight junction-associated genes. A scratch assay revealed that although the epithelial cells on the low mobile surface rapidly initiated their migration, the cells on the highly mobile surface delayed their migration. Thus, this finding suggests that polyrotaxane surfaces with higher mobility induce cytoplasmic YAP localization, leading to stronger cell-cell adhesion. The polyrotaxane biointerface is promising as a powerful tool to improve the physical immune system and repair biological tissues.

Low-intensity vibration restores nuclear YAP levels and acute YAP nuclear shuttling in mesenchymal stem cells subjected to simulated microgravity

Reducing the musculoskeletal deterioration that astronauts experience in microgravity requires countermeasures that can improve the effectiveness of otherwise rigorous and time-expensive exercise regimens in space. The ability of low-intensity vibrations (LIV) to activate force-responsive signaling pathways in cells suggests LIV as a potential countermeasure to improve cell responsiveness to subsequent mechanical challenge. Mechanoresponse of mesenchymal stem cells (MSC), which maintain bone-making osteoblasts, is in part controlled by the “mechanotransducer” protein YAP (Yes-associated protein), which is shuttled into the nucleus in response to cyto-mechanical forces. Here, using YAP nuclear shuttling as a measurement outcome, we tested the effect of 72 h of clinostat-induced simulated microgravity (SMG) and daily LIV application (LIVDT) on the YAP nuclear entry driven by either acute LIV (LIVAT) or Lysophosphohaditic acid (LPA), applied after the 72 h period. We hypothesized that SMG-induced impairment of acute YAP nuclear entry would be alleviated by the daily application of LIVDT. Results showed that while both acute LIVAT and LPA treatments increased nuclear YAP entry by 50 and 87% over the basal levels in SMG-treated MSCs, nuclear YAP levels of all SMG groups were significantly lower than non-SMG controls. LIVDT, applied in parallel to SMG, restored the SMG-driven decrease in basal nuclear YAP to control levels as well as increased the LPA-induced but not LIVAT-induced YAP nuclear entry over SMG only, counterparts. These cell-level observations suggest that daily LIV treatments are a feasible countermeasure for restoring basal nuclear YAP levels and increasing the YAP nuclear shuttling in MSCs under SMG.

Increased Cytoplasmic Yes-associated Protein (YAP) Expression in Mismatch Repair Protein-Proficient Colorectal Cancer With High-grade Tumor Budding and Reduced Autophagy Activity.

Yes-associated protein (YAP) is a transcriptional coactivator regulated by autophagy that stimulates colorectal cancer (CRC) progression through activation of epithelial-mesenchymal transition (EMT), represented by tumor budding. The associations between these components in CRC are unknown. Archived surgically resected CRCs with known mismatch repair protein (MMR) status were retrieved (n=81; 2010 to 2016). Electronic medical records were reviewed for clinicopathologic variables including pathologic TNM stage and clinical stage. Tumor budding was graded according to consensus guidelines. Cytoplasmic and nuclear YAP and p62 (autophagy substrate) immunoreactivity were semiquantitatively scored within tumor samples. The Student t test, Fisher exact test, χ2 test, and Spearman correlation coefficient were performed with P<0.05 as a significance level. MMR proficiency (MMR-P) status correlated with high-grade tumor budding. The extent of cytoplasmic YAP staining and pathologic N stage was associated with tumor budding in multivariate analysis. Cytoplasmic YAP expression correlated with higher cytoplasmic p62 expression, suggesting an inverse correlation between autophagy activation and cytoplasmic YAP expression. Nuclear YAP expression correlated with pathologic N stage and clinical stage. A correlation between MMR-P status and tumor budding, combined with correlations between cytoplasmic YAP, tumor budding and p62 raise the possibility of 2 distinct neoplastic pathways concerning autophagy and YAP; one displaying relative activation of YAP and EMT, being commonly observed in MMR-P, and another with less active YAP and EMT, but active autophagy, being commonly seen in MMR-deficient CRC. Nuclear YAP staining could be useful in prognostication.