Yes-associated Protein Inhibitor Research Articles

Retinoic Acid Improves Vascular Endothelial Dysfunction by Inhibiting PI3K/AKT/YAP-Mediated Ferroptosis in Diabetes Mellitus.

Vascular endothelial dysfunction is the initial factor involved in cardiovascular injury in patients with diabetes. Retinoic acid is involved in improving vascular complications in patients with diabetes, but its protective mechanism is still unclear. This study aimed to evaluate the effect and mechanism of All-Trans Retinoic Acid (ATRA) on endothelial dysfunction induced by diabetes. In the present study, streptozotocin (STZ)-induced diabetic rats and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs) were observed, and the effects of ATRA on HG-induced endothelial dysfunction and ferroptosis were evaluated. ATRA treatment improved impaired vasorelaxation in diabetic aortas in an endothelium-dependent manner, and this effect was accompanied by an increase in the NO concentration and eNOS expression. Ferroptosis, characterized by lipid peroxidation and iron overload induced by HG, was improved by ATRA administration, and a ferroptosis inhibitor (ferrostatin-1, Fer-1) improved endothelial function to a similar extent as ATRA. In addition, the inactivation of phosphoinositol-3-kinase (PI3K)/protein kinases B (AKT) and Yes-Associated Protein (YAP) nuclear localization induced by HG were reversed by ATRA administration. Vascular ring relaxation experiments showed that PI3K/AKT activation and YAP inhibition had similar effects on ferroptosis and endothelial function. However, the vasodilative effect of retinoic acid was affected by PI3K/AKT inhibition, and the inhibitory effects of ATRA on ferroptosis and the improvement of endothelial function were dependent on the retinoic acid receptor. ATRA could improve vascular endothelial dysfunction by inhibiting PI3K/AKT/YAP-mediated ferroptosis induced by HG, which provides a new idea for the treatment of vascular lesions in diabetes.

Circ_0002722-induced regulation of YAP promotes platinum resistance in oral squamous cell carcinoma: Implications for verteporfin therapy

Oral squamous cell carcinoma (OSCC) poses a significant public health burden due to its high prevalence and poor prognosis. Platinum resistance is one of the major challenges in OSCC treatment. Yes-associated protein (YAP) has been identified as a pivotal player in OSCC tumorigenesis and progression. Circular RNA (circRNA) has been implicated in chemoresistance in various cancers by regulation the function of microRNA. Nevertheless, the specific mechanisms linking circRNA to YAP expression in OSCC remain poorly understood. In this study, we detected the YAP and circRNA hsa_circ_0002722 (circ_0002722) expression by western blot (WB) and quantitative polymerase chain reaction (qPCR). We found that YAP and circ_0002722 were up-regulated in platinum resistance in OSCC tissues. Furthermore, transfection of circ_0002722 siRNA into platinum-resistant cells revealed that circ_0002722 acted as a regulator of miR-1305, which influenced YAP expression and thereby affected platinum sensitivity. In vivo experiments corroborated the synergistic effects of cisplatin and verteporfin (a YAP inhibitor) in combating platinum resistance. Targeting YAP emerges as a promising therapeutic strategy for addressing platinum resistance in OSCC, with circ_0002722 serving as a potential therapy target and valuable diagnostic marker. These findings shed light on the underlying mechanisms of platinum resistance, paving the way for the development of effective treatment approaches.

Yes-associated protein inhibition ameliorates carbon tetrachloride-induced acute liver injury in mice by reducing VDR

Carbon tetrachloride (CCl4) has a wide range of toxic effects, especially causing acute liver injury (ALI), in which rapid compensation for hepatocyte loss ensures liver survival, but proliferation of surviving hepatocytes (known as endoreplication) may imply impaired residual function. Yes-associated protein (YAP) drives hepatocytes to undergo endoreplication and ploidy, the underlying mechanisms of which remain a mystery. In the present study, we uncover during CCl4-mediated ALI accompanied by increased hepatocytes proliferation and YAP activation. Notably, bioinformatics analyses elucidate that hepatic-specific deletion of YAP substantially ameliorated CCl4-induced hepatic proliferation, effectively decreased the vitamin D receptor (VDR) expression. Additionally, a mouse model of acute liver injury substantiated that inhibition of YAP could suppress hepatocytes proliferation via VDR. Furthermore, we also disclosed that the VDR agonist nullifies CCl4-induced ALI alleviated by the YAP inhibitor in vivo. Importantly, hepatocytes were isolated from mice, and it was spotlighted that the anti-proliferative impact of the YAP inhibitor was abolished by the activation of VDR within these hepatocytes. Similarly, primary hepatic stellate cells (HSCs) were isolated and it was manifested that YAP inhibitor suppressed HSC activation via VDR during acute liver injury. Our findings further elucidate the YAP's role in ALI and may provide new avenues for protection against CCl4-drived acute liver injury.

Suppression of YAP Ameliorates Cartilage Degeneration in Ankle Osteoarthritis via Modulation of the Wnt/β-Catenin Signaling Pathway.

Ankle osteoarthritis is a relatively understudied condition and the molecular mechanisms involved in its development are not well understood. This investigation aimed to explore the role and underlying molecular mechanisms of Yes-associated protein (YAP) in rat ankle osteoarthritis. The results demonstrated that YAP expression levels were abnormally increased in the ankle osteoarthritis cartilage model. In addition, knockdown of YAP expression was shown to hinder the imbalance in ECM metabolism induced by IL-1β in chondrocytes, as demonstrated by the regulation of matrix metalloproteinase (MMP)-3, MMP-9, and MMP-13, a disintegrin, metalloprotease with thrombospondin motifs, aggrecan, and collagen II expression. Additional studies revealed that downregulation of YAP expression markedly inhibited the overexpression of β-catenin stimulated by IL-1β. Furthermore, inhibition of the Wnt/β-catenin signaling pathway reversed the ECM metabolism imbalance caused by YAP overexpression in chondrocytes. It is important to note that the YAP-specific inhibitor verteporfin (VP) significantly delayed the progression of ankle osteoarthritis. In conclusion, the findings highlighted the crucial role of YAP as a regulator in modulating the progression of ankle osteoarthritis via the Wnt/β-catenin signaling pathway. These findings suggest that pharmacological inhibition of YAP can be an effective and critical therapeutic target for alleviating ankle osteoarthritis.

LncRNA HULC augments high glucose‐associated pancreatic cancer progression and drug resistance by enhancing YAP activity and autophagy

AbstractBackground Information: One of the confounding factors in pancreatic cancer (PC) pathogenesis is hyperglycemia. The molecular mechanism by which high glucose (HG) influences PC severity is poorly understood. Our investigation delved into the impact of lncRNA highly upregulated in liver cancer (HULC) and its interaction with yes‐associated protein (YAP) in regulating the fate of pancreatic ductal adenocarcinoma cells (PDAC) under HG‐induced conditions. PDAC cells were cultured under normal or HG conditions. We thereafter measured the effect of HG on the viability of PDAC cells, their migration potential and drug resistance properties. The lncRNAs putatively dysregulated in PC and diabetes were shortlisted by bioinformatics analysis followed by wet lab validation of function. Results: HG led to enhanced proliferation and drug refractoriness in PDAC cells. HULC was identified as one of the major deregulated lncRNAs following bioinformatics analysis. HULC was found to regulate the expression of the potent transcriptional regulator – YAP through selective histone modifications at the YAP promoter. siRNA‐mediated ablation of HULC resulted in a concurrent decrease in YAP transcriptional activity. Importantly, HULC and YAP were found to co‐operatively regulate the cellular homeostatic process autophagy, thus inculcating drug resistance and proliferative potential in PDAC cells. Moreover, inhibition of autophagy or YAP led to a decrease in HULC levels, suggesting the existence of an inter‐regulatory feedback loop. Conclusions: We observed that HG triggers aggressive properties in PDAC cells. Mechanistically, up‐regulation of lncRNA HULC resulted in activation of YAP and differential regulation of autophagy coupled to increased proliferation of PDAC cells. Significance: Inhibition of HULC and YAP may represent a novel therapeutic strategy for PDAC. Furthermore, this study portrays the intricate molecular interplay between HULC, YAP and autophagy in PDAC pathogenesis.

IQGAP1 overexpression attenuates chemosensitivity through YAP-mediated ferroptosis inhibition in esophageal squamous cell cancer cells

Chemoresistance is one of the major hindrances to many cancer therapies, including esophageal squamous cell carcinoma (ESCC). Ferroptosis, a new programmed cell death, plays an essential role in chemoresistance. IQ-domain GTPase activating protein 1 (IQGAP1) is a scaffold protein and functions as an oncogene in various human malignancies. However, the underlying effect and molecular mechanisms of IQGAP1 on paclitaxel (PTX) resistance and ferroptosis in ESCC remain to be elucidated. In this study, we found that IQGAP1 was highly expressed in ESCC tissues and could as a potential biomarker for diagnosis and predicting the prognosis of ESCC. Functional studies revealed that IQGAP1 overexpression reduced the sensitivity of ESCC cells to PTX by enhancing ESCC cell viability and proliferation and inhibiting cell death, and protected ESCC cells from ferroptosis, whereas IQGAP1 knockdown exhibited contrary effects. Importantly, reductions of chemosensitivity and ferroptosis caused by IQGAP1 overexpression were reversed with ferroptosis inducer RSL3, while the increases of chemosensitivity and ferroptosis caused by IQGAP1 knockdown were reversed with ferroptosis inhibitor ferrostatin-1 (Fer-1) in ESCC cells, indicating that IQGAP1 played a key role in resistance to PTX through regulating ferroptosis. Mechanistically, we demonstrated that IQGAP1 overexpression upregulated the expression of Yes-associated protein (YAP), the central mediator of the Hippo pathway. YAP inhibitor Verteporfin (VP) could reverse the effects of IQGAP1 overexpression on ESCC chemoresistance and ferroptosis. Taken together, our findings suggest that IQGAP1 promotes chemoresistance by blocking ferroptosis through targeting YAP. IQGAP1 may be a novel therapeutic target for overcoming chemoresistance in ESCC.

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.

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

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

Combination of Molecule-Targeted Therapy and Photodynamic Therapy Using Nanoformulated Verteporfin for Effective Uveal Melanoma Treatment.

Uveal melanoma (UM) is the most common primary ocular malignancy in adults and has high mortality. Recurrence, metastasis, and therapeutic resistance are frequently observed in UM, but no beneficial systemic therapy is available, presenting an urgent need for developing effective therapeutic drugs. Verteporfin (VP) is a photosensitizer and a Yes-Associated Protein (YAP) inhibitor that has been used in clinical practice. However, VP's lack of tumor targetability, poor biocompatibility, and relatively low treatment efficacy hamper its application in UM management. Herein, we developed a biocompatible CD44-targeting hyaluronic acid nanoparticle (HANP) carrying VP (HANP/VP) to improve UM treatment efficacy. We found that HANP/VP showed a stronger inhibitory effect on cell proliferation than that of free VP in UM cells. Systemic delivery of HANP/VP led to targeted accumulation in the UM-tumor-bearing mouse model. Notably, HANP/VP mediated photodynamic therapy (PDT) significantly inhibited UM tumor growth after laser irradiation compared with no treatment or free VP treatment. Consistently, in HANP/VP treated tumors after laser irradiation, the tumor proliferation and YAP expression level were decreased, while the apoptotic tumor cell and CD8+ immune cell levels were elevated, contributing to effective tumor growth inhibition. Overall, the results of this preclinical study showed that HANP/VP is an effective nanomedicine for tumor treatment through PDT and inhibition of YAP in the UM tumor mouse model. Combining phototherapy and molecular-targeted therapy offers a promising approach for aggressive UM management.

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.

Nuclear Localization of Yes-Associated Protein Is Associated With Tumor Progression in Cutaneous Melanoma

Yes-associated protein (YAP), an effector molecule of the Hippo signaling pathway, is expressed at high levels in cutaneous melanoma. However, the role of YAP in melanoma progression according to cellular localization is poorly understood. Tissues from 140 patients with invasive melanoma were evaluated by immunohistochemistry. Flow cytometry, western blotting, viability assays, wound healing assays, verteporfin treatment, and xenograft assays were conducted using melanoma cell lines B16F1 and B16F10 subjected to YapS127A transfection and siYap knockdown. Nuclear YAP localization was identified in 63 tumors (45.0%) and was more frequent than cytoplasmic YAP in acral lentiginous and nodular subtypes (P = .007). Compared with cytoplasmic YAP melanomas, melanomas with nuclear YAP had higher mitotic activity (P = .016), deeper invasion (P < .001), and more frequently metastasized to lymph nodes (P < .001) and distant organs (P < .001). Patients with nuclear YAP melanomas had poorer disease-free survival (P < .001) and overall survival (P < .001). Nuclear YAP was an independent risk factor for distant metastasis (hazard ratio: 3.206; 95% CI, 1.032-9.961; P = .044). Proliferative ability was decreased in siYapB16F1 (P < .001) and siYapB16F10 (P = .001) cells and increased in YapS127AB16F1 (P = .003) and YapS127AB16F10 (P = .002) cells. Cell cycle analysis demonstrated relative G1 retention in siYapB16F1 (P < .001) and siYapB16F10 (P < .001) cells and S retention in YapS127AB16F1 cells (P = .008). Wound healing assays showed that Yap knockdown inhibited cell invasion (siYapB16F1, P = .001; siYapB16F10, P < .001), whereas nuclear YAP promoted it (YapS127AB16F, P < .001; YapS127AB16F1, P = .017). Verteporfin, a direct YAP inhibitor, reduced cellular proliferation in B16F1 (P = .003) and B16F10 (P < .001) cells. Proliferative effects of nuclear YAP were confirmed in xenograft mice (P < .001). In conclusion, nuclear YAP in human melanomas showed subtype specificity and correlated with proliferative activity and proinvasiveness. It is expected that YAP becomes a useful prognostic marker, and its inhibition may be a potential therapy for melanoma patients.

YAP/TEAD involvement in resistance to paclitaxel chemotherapy in lung cancer.

The Yes-associated protein (YAP) oncoprotein has been linked to both metastases and resistance to targeted therapy of lung cancer cells. We aimed to investigate the effect of YAP pharmacological inhibition, using YAP/TEA domain (TEAD) transcription factor interaction inhibitors in chemo-resistant lung cancer cells. YAP subcellular localization, as a readout for YAP activation, cell migration, and TEAD transcription factor functional transcriptional activity were investigated in cancer cell lines with up-regulated YAP, with and without YAP/TEAD interaction inhibitors. Parental (A549) and paclitaxel-resistant (A549R) cell transcriptomes were analyzed. The half-maximal inhibitory concentration (IC50) of paclitaxel or trametinib, which are Mitogen-Activated protein kinase and Erk Kinase (MEK) inhibitors, combined with a YAP/TEAD inhibitor (IV#6), was determined. A three-dimensional (3D) microfluidic culture device enabled us to study the effect of IV#6/paclitaxel combination on cancer cells isolated from fresh resected lung cancer samples. YAP activity was significantly higher in paclitaxel-resistant cell lines. The YAP/TEAD inhibitor induced a decreased YAP activity in A549, PC9, and H2052 cells, with reduced YAP nuclear staining. Wound healing assays upon YAP inhibition revealed impaired cell motility of lung cancer A549 and mesothelioma H2052 cells. Combining YAP pharmacological inhibition with trametinib in K-Ras mutated A549 cells recapitulated synthetic lethality, thereby sensitizing these cells to MEK inhibition. The YAP/TEAD inhibitor lowered the IC50 of paclitaxel in A549R cells. Differential transcriptomic analysis of parental and A549R cells revealed an increased YAP/TEAD transcriptomic signature in resistant cells, downregulated upon YAP inhibition. The YAP/TEAD inhibitor restored paclitaxel sensitivity of A549R cells cultured in a 3D microfluidic system, with lung cancer cells from a fresh tumor efficiently killed by YAP/TEAD inhibitor/paclitaxel doublet. Evidence of the YAP/TEAD transcriptional program's role in chemotherapy resistance paves the way for YAP therapeutic targeting.

Assessment of human embryonic stem cells differentiation into definitive endoderm lineage on the soft substrates.

Pluripotent stem cells (PSCs) hold enormous potential for treating multiple diseases owing to their ability to self-renew and differentiate into any cell type. Albeit possessing such promising potential, controlling their differentiation into a desired cell type continues to be a challenge. Recent studies suggest that PSCs respond to different substrate stiffness and, therefore, can differentiate towards some lineages via Hippo pathway. Human PSCs can also differentiate and self-organize into functional cells, such as organoids. Traditionally, human PSCs are differentiated on stiff plastic or glass plates towards definitive endoderm and then into functional pancreatic progenitor cells in the presence of soluble growth factors. Thus, whether stiffness plays any role in differentiation towards definitive endoderm from human pluripotent stem cells(hPSCs) remains unclear. Our study found that the directed differentiation of human embryonic stem cells towards endodermal lineage on the varying stiffness did not differ from the differentiation on stiff plastic dishes. We also observed no statistical difference between the expression of yes-associated protein(YAP) and phosphorylatedYAP. Furthermore, we demonstrate that lysophosphatidic acid, a YAP activator, enhanced definitive endoderm formation, whereas verteporfin, a YAP inhibitor, did not have the significant effect on the differentiation. In summary, our results suggest that human embryonic stem cells may not differentiate in response to changes in stiffness, and that such cues may not have as significant impact on the level of YAP. Our findings indicate that more research is needed to understand the direct relationship between biophysical forces and hPSCs differentiation.

Inhibition of transcriptional coactivator YAP Impairs the expression and function of transcription factor WT1 in diabetic podocyte injury

Podocyte injury and loss are hallmarks of diabetic nephropathy (DN). However, the molecular mechanisms underlying these phenomena remain poorly understood. YAP (Yes-associated protein) is an important transcriptional coactivator that binds with various other transcription factors, including the TEAD family members (nuclear effectors of the Hippo pathway), that regulate cell proliferation, differentiation, and apoptosis. The present study found an increase in YAP phosphorylation at S127 of YAP and a reduction of nuclear YAP localization in podocytes of diabetic mouse and human kidneys, suggesting dysregulation of YAP may play a role in diabetic podocyte injury. Tamoxifen-inducible podocyte-specific Yap gene knockout mice (YappodKO) exhibited accelerated and worsened diabetic kidney injury. YAP inactivation decreased transcription factor WT1 expression with subsequent reduction of Tead1 and other well-known targets of WT1 in diabetic podocytes. Thus, our study not only sheds light on the pathophysiological roles of the Hippo pathway in diabetic podocyte injury but may also lead to the development of new therapeutic strategies to prevent and/or treat DN by targeting the Hippo signaling pathway.

NRP1 Induces Enhanced Stemness and Chemoresistance in Glioma Cells via YAP.

Neuropilin-1 (NRP1), a transmembrane glycoprotein, plays an important role in the malignant progression of gliomas; however, its role in chemoresistance is not fully understood. In this study, we observed the effects of NRP1 on the stemness and chemoresistance of glioma cells and the mediating role of Yes-associated protein (YAP). We constructed NRP1 overexpressing LN-229 glioma cells. Cells were treated with recombinant NRP1 protein (rNRP1) and the YAP inhibitor Super-TDU when necessary. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the sensitivity of cells to temozolomide (TMZ). Sphere and clone formation assays were performed to detect the sphere- and clone-forming abilities of cells. Western blotting was performed to detect cellular CD133, CD44, p-LATS1, and p-YAP protein expression. Immunofluorescence and flow cytometry were used to detect the subcellular localization of YAP and apoptosis, respectively. We found that both NRP1 overexpression and rNRP1 treatment enhanced self-renewal, TMZ resistance, and CD133 and CD44 protein expression in LN-229 cells. NRP1 overexpression and rNRP1 treatment also induced LATS1 and YAP dephosphorylation and YAP nuclear translocation. Super-TDU inhibits NRP1 overexpression-induced enhanced self-renewal and TMZ resistance in LN-229 cells. Our study suggests that NRP1 induces increased stemness in glioma cells, resulting in chemoresistance, and that this effect is associated with YAP activation.

Verteporfin induces lipid peroxidation and ferroptosis in pancreatic cancer cells

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

The inhibition of YAP Signaling Prevents Chronic Biliary Fibrosis in the Abcb4-/- Model by Modulation of Hepatic Stellate Cell and Bile Duct Epithelium Cell Pathophysiology.

Primary sclerosing cholangitis (PSC) represents a chronic liver disease characterized by poor prognosis and lacking causal treatment options. Yes-associated protein (YAP) functions as a critical mediator of fibrogenesis; however, its therapeutic potential in chronic biliary diseases such as PSC remains unestablished. The objective of this study is to elucidate the possible significance of YAP inhibition in biliary fibrosis by examining the pathophysiology of hepatic stellate cells (HSC) and biliary epithelial cells (BEC). Human liver tissue samples from PSC patients were analyzed to assess the expression of YAP/connective tissue growth factor (CTGF) relative to non-fibrotic control samples. The pathophysiological relevance of YAP/CTGF in HSC and BEC was investigated in primary human HSC (phHSC), LX-2, H69, and TFK-1 cell lines through siRNA or pharmacological inhibition utilizing verteporfin (VP) and metformin (MF). The Abcb4-/- mouse model was employed to evaluate the protective effects of pharmacological YAP inhibition. Hanging droplet and 3D matrigel culture techniques were utilized to investigate YAP expression and activation status of phHSC under various physical conditions. YAP/CTGF upregulation was observed in PSC patients. Silencing YAP/CTGF led to inhibition of phHSC activation and reduced contractility of LX-2 cells, as well as suppression of epithelial-mesenchymal transition (EMT) in H69 cells and proliferation of TFK-1 cells. Pharmacological inhibition of YAP mitigated chronic liver fibrosis in vivo and diminished ductular reaction and EMT. YAP expression in phHSC was effectively modulated by altering extracellular stiffness, highlighting YAP's role as a mechanotransducer. In conclusion, YAP regulates the activation of HSC and EMT in BEC, thereby functioning as a checkpoint of fibrogenesis in chronic cholestasis. Both VP and MF demonstrate effectiveness as YAP inhibitors, capable of inhibiting biliary fibrosis. These findings suggest that VP and MF warrant further investigation as potential therapeutic options for the treatment of PSC.

Dysregulated Hippo Signaling Pathway and YAP Activation in Atopic Dermatitis: Insights from Clinical and Animal Studies.

The yes-associated protein (YAP) of the Hippo pathway regulates a variety of target genes involved in cell proliferation, survival, and inflammation. YAP and transcription activator with a PDZ-binding motif (TAZ) proteins act as mediators of the inflammatory response. Still, their role in atopic dermatitis (AD)-particularly, the association with the nuclear factor kappa-B and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways-is not fully understood. In this study, we found that YAP, is upregulated in AD patients and NC/Nga mouse model of AD. In addition, inhibition of YAP significantly reduced epidermal cell proliferation by 58% and mast cell numbers by 51% and attenuated the upregulation of both Th1- and Th2-associated cytokines. Among the JAK-STAT family proteins, the expressions of JAK1 and JAK2 and those of STAT1, STAT2, and STAT3 were also downregulated. These findings may explain the role of YAP in AD and suggest YAP inhibitors as promising therapeutic agents for AD.

Understanding the Role of Yes-Associated Protein (YAP) Signaling in the Transformation of Lens Epithelial Cells (EMT) and Fibrosis.

Fibrotic cataracts, posterior capsular opacification (PCO), and anterior subcapsular cataracts (ASC) are mainly attributed to the transforming growth factor-β (TGFβ)-induced epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs). Previous investigations from our laboratory have shown the novel role of non-canonical TGFβ signaling in the progression of EMT in LECs. In this study, we have identified YAP as a critical signaling molecule involved in lens fibrosis. The observed increase in nuclear YAP in capsules of human ASC patients points toward the involvement of YAP in lens fibrosis. In addition, the immunohistochemical (IHC) analyses on ocular sections from mice that overexpress TGFβ in the lens (TGFβtg) showed a co-expression of YAP and α-SMA in the fibrotic plaques when compared to wild-type littermate lenses, which do not. The incubation of rat lens explants with verteporfin, a YAP inhibitor, prevented a TGFβ-induced fiber-like phenotype, α-SMA, and fibronectin expression, as well as delocalization of E-cadherin and β-catenin. Finally, LECs co-incubated with TGFβ and YAP inhibitor did not exhibit an induction in matrix metalloproteinase 2 compared to those LECs treated with TGFβ alone. In conclusion, these data demonstrate that YAP is required for TGFβ-mediated lens EMT and fibrosis.

Yes-Associated Protein Mediates the Transition from Inflammation to Fibrosis in Graves' Orbitopathy.

Background: In Graves' orbitopathy (GO), localized orbital inflammation within the fixed orbit often leads to a fibrotic phenotype resulting in restrictive myopathy or refractory proptosis. However, the molecular pathways related to the transition from inflammation to fibrosis in GO are less understood. Yes-associated protein (YAP) and its homolog, transcriptional coactivator with PDZ-binding motif (TAZ; a Hippo pathway effector), are critical mechanosensors of mechanical stimuli and activate signaling cascades for cell proliferation, differentiation, and transformation. In this study, we aimed to examine the role of YAP in both inflammatory and fibrotic GO pathogenesis. Methods: Based on RNA sequencing performed on freshly obtained orbital adipose tissue from patients with GO and healthy individuals, Gene Ontology analysis and gene set-enrichment analysis were performed to analyze gene-expression differences between GO and normal orbital tissues. The role of YAP in GO-related inflammation and fibrosis was studied in primary cultured orbital fibroblasts. The effects of interleukin-1β (IL-1β)-induced inflammation and transforming growth factor-beta (TGF-β)-induced fibrosis on YAP expression were evaluated using real-time polymerase chain reaction and Western blotting analyses. The effects of YAP on inflammatory and fibrotic responses were also examined by YAP silencing or treatment with pharmacological YAP inhibitors. Results: RNA sequencing revealed enhanced YAP expression in GO orbital tissues. Gene Ontology analysis indicated that "response to mechanical stimulus"-related genes were overexpressed in GO orbital tissues, along with those enriched for the "adipose proliferation," "inflammatory responses," and "hormone stimulus responses" terms. IL-1β did not enhance YAP expression, and YAP silencing decreased IL-1β-induced IL-6 expression while increasing prostaglandin-endoperoxide synthase 2 expression, leading to paradoxical pro-inflammatory effects. Conversely, TGF-β enhanced YAP expression, and YAP silencing and pharmacological YAP inhibitor (cerivastatin, verteporfin, TED-347, and CA3) treatment significantly reduced TGF-β-induced myofibroblast differentiation and collagen formation. Conclusion: YAP, a mechanotransducer responding to mechanical stimuli, was strongly expressed in GO orbital tissues, and YAP was induced by TGF-β in orbital fibroblasts. Our study establishes YAP as a novel mediator of GO pathobiology, potentially mediating the transition from early inflammation to chronic fibrosis in GO. The finding that YAP inhibition suppressed TGF-β-induced fibrotic response suggests YAP as a therapeutic target against the fibrotic mechanism of GO.