Yes-associated Protein Protein Research Articles

Yes-associated Protein Mediates Acute Lung Injury in Adult but not Juvenile Mice

Objective: Adults with The Acute Respiratory Distress Syndrome (ARDS) have worse outcomes than children with Pediatric ARDS. This finding can be reproduced in animal models, suggesting a difference in Acute Lung Injury (ALI) mechanisms. Understanding these mechanistic differences could inform age-specific treatment. Hypothesis: Since Yes-associated protein (YAP) is important for lung development and reactive to mechanical stretch, we hypothesized that YAP is important for ALI mechanisms. Methods: To induce ALI, we intranasally instilled mice with Pseudomonas aeruginosa strain K (2.5 x 105 colony forming units (c.f.u.)) in juvenile (3-week-old) and adult (10-week-old) C57BL/6J mice. 24 h later, we collected bronchoalveolar lavage (BAL) fluid and measured protein, cell count, and c.f.u. To knockdown YAP expression, we injected siRNA 24 h before P. aeruginosa instillation. We removed the lungs and enriched for CD45+ and CD41+ fraction, and CD31+ fraction using Dynabeads sheep anti-rat IgG (Invitrogen). In separate experiments, lungs were separated into nuclear and cytosolic fractions for immunoblot analysis. For immunoprecipitation, the cytosolic fraction was added to beads coated with anti-YAP antibody (rabbit monoclonal). We washed the beads and eluted bound proteins, which were identified by label-free mass spectrometry. Data and results: While both juvenile and adult mice had significantly increased BAL cell counts and protein 24 h after P. aeruginosa instillation, adult mice had significantly greater cell counts and protein than juveniles. BAL c.f.u. were similar in both groups, suggesting equal instillation of P. aeruginosa. P. aeruginosa instillation increased whole lung YAP expression in both cytosolic and nuclear fractions in adult mice by >3-fold, but YAP expression was not increased in P. aeruginosa-treated juvenile mice. YAP protein increases occurred in CD31+ fraction (endothelial cells), but no YAP was quantified in CD45+/CD41+ fraction (leukocytes and platelets). Vascular knockdown of YAP expression by ~75% in adult mice blocked P. aeruginosa-induced BAL cell count increases. Proteomics analysis revealed 62 proteins that had significantly altered YAP binding upon P. aeruginosa treatment (47 with increased YAP binding). The complement factors H and I had a log2-fold-change for YAP binding of 4.56 and 2.26, respectively, upon P. aeruginosa treatment. Conclusions: Adult mice exhibited YAP-dependent Acute Lung Injury; juveniles did not. YAP-dependent mechanisms of injury may be secondary to YAP binding to the complement inhibitory proteins H and I. More work is needed to establish the cell-specificity of YAP-complement factor binding and to determine the location of complement signaling. HL148403, Parker B Francis, and Columbia University Department of Pediatrics Children’s Health Innovation Nucleation Fund to RFH; Ernest E Just Summer Scholarship to AMD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ACAT2 suppresses the ubiquitination of YAP1 to enhance the proliferation and metastasis ability of gastric cancer via the upregulation of SETD7

The contributions of aberrantly expressed metabolic enzymes to gastric cancer (GC) initiation and progression have been widely appreciated in recent years. Acetyl-CoA acetyltransferase 2 (ACAT2) is one member of the acetyl- CoA thiolase family. Previous studies demonstrated that ACAT2 either promotes or suppresses tumor progression in different conditions. However, the function and mechanisms of ACAT2 in GC remain unknown. We found that the expression of this enzyme was significantly increased in GC tissues compared with normal counterparts, which prompted us to further investigate the roles of this protein in GC biology. In vitro functional studies showed that ACAT2 knockdown markedly halted the proliferation and the motility of GC cells; these functions favoring malignant phenotypes of GC cells were further validated in animal experiments. Mechanistically, ACAT2 depletion significantly reduced the transcription of SETD7, which is a histone methyltransferase and plays critical roles in GC cells. We found that the pro-tumoral functions of ACAT2 were largely dependent on SETD7. Moreover, SETD7 decreased the ubiquitination level of Yes-associated protein 1 (YAP1), thereby protecting YAP1 from proteasome degradation. Increased YAP1 protein expression remarkably activated the YAP1/TAZ-TEAD1 signaling pathway, which further boosted the malignant phenotypes in GC cells. In conclusion, these findings highlight the pro-tumoral functions and molecular underpinnings of ACAT2 in GC cells, and suggest that ACAT2 could be a promising target in GC treatment.

Abstract 4386: Androgen hormone signaling regulates the association between the hippo pathway component YAP1 and RNA binding protein NPM1

Abstract Prostate cancer represents a major health concern for men in the United States and globally, disproportionately affecting men of African descent. Despite recent advances, the precise etiology and molecular mechanisms underpinning fatal prostate cancer in humans remain inadequately understood. RNA binding proteins (RBPs) regulate various aspects of RNA biology, and when their regulation is disrupted, they can lead to human diseases, including cancer. However, the comprehensive alterations in RBP-mRNA interactions in prostate cancer have not been explored. In this study, we investigated the dynamics of RBPs and their interactions with polyadenylated mRNA in the castration-sensitive LNCaP and castration-resistant C4-2 prostate cancer cell lines. Using an enhanced RNA interactome capture assay in combination with mass spectrometry, we discovered that LNCaP and C4-2 cells showed distinct RBP-mRNA interaction patterns in steady-state growth conditions and in response to androgen treatment. Additionally, we investigated the interplay between the oncogene Yes-associated protein 1 (YAP1) and the RNA-binding protein nucleophosmin (NPM1). We identified NPM1 as a component of the YAP1 protein complex through mass spectrometry-based proteomics. Our proximity ligation assay demonstrated that androgen signaling could regulate the interaction between YAP1 and NPM1. Also, our GST-pulldown revealed that NPM1 binds to the proline-rich domain of YAP1 in LNCaP cells, while the WW/SH3 domain of YAP1 mediates the interaction with NPM1 in C4-2 cells. Genetic and pharmacological inhibition of NPM1 activity reduced prostate cancer cell growth in culture by modulating cell cycle progression. Furthermore, our computational and immunological analysis of prostate tumor tissues indicated that the upregulation of NPM1 might be associated with the clinical advancement of human prostate cancer. These findings underscore the critical role of the molecular functional connection between YAP1 and RBPs and the global changes in RBP and RNA interaction patterns, in the progression and recurrence of prostate cancer. Citation Format: Abdulrahman M. Dwead, Marwah M. Al-Mathkour, Bekir Cinar. Androgen hormone signaling regulates the association between the hippo pathway component YAP1 and RNA binding protein NPM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4386.

Regulation mechanism of EBV-encoded EBER1 and LMP2A on YAP1 and the impact of YAP1 on the EBV infection status in EBV-associated gastric carcinoma

This study aims to explore the role and regulatory mechanism of Yes-associated protein 1 (YAP1) in the development of Epstein-Barr virus-associated gastric cancer (EBVaGC). Here we showed that EBV can upregulate the expression and activity of YAP1 protein through its encoded latent products EBV-encoded small RNA 1 (EBER1) and latent membrane protein 2A (LMP2A), enhancing the malignant characteristics of EBVaGC cells. In addition, we also showed that overexpression of YAP1 induced the expression of EBV encoding latent and lytic phase genes and proteins in the epithelial cell line AGS-EBV infected with EBV, and increased the copy number of the EBV genome, while loss of YAP1 expression reduced the aforementioned indicators. Moreover, we found that YAP1 enhanced EBV lytic reactivation induced by two known activators, 12-O-tetradecanoylhorbol-13-acetate (TPA) and sodium butyrate (NaB). These results indicated a bidirectional regulatory mechanism between EBV and YAP1 proteins, providing new experimental evidence for further understanding the regulation of EBV infection patterns and carcinogenic mechanisms in gastric cancer.

Myeloid SENP3 deficiency protects mice from diet and age-induced obesity via regulation of YAP1 SUMOylation

Obesity is characterized by chronic low-grade inflammation, which is driven by macrophage infiltration in adipose tissue and leads to elevated cytokines such as interleukin-1β (IL-1β) in the circulation and tissues. Previous studies demonstrate that SENP3, a redox-sensitive SUMO2/3-specific protease, is strongly implicated in the development and progression of cancer and cardiovascular diseases. However, the role of SENP3 in obesity-associated inflammation remains largely unknown. To better understand the effects of SENP3 on adipose tissue macrophage (ATM) activation and function within the context of obesity, we generated mice with myeloid-specific deletion of SENP3 (Senp3flox/flox;Lyz2-Cre mice). We found that the expression of SENP3 is dramatically increased in ATMs during high-fat diet (HFD)-induced obesity in mice. Senp3flox/flox;Lyz2-Cre mice show lower body weight gain and reduced adiposity and adipocyte size after challenged with HFD and during aging. Myeloid-specific SENP3 deletion attenuates macrophage infiltration in adipose tissue and reduces serum levels of inflammatory factors during diet and age-induced obesity. Furthermore, we found that SENP3 knockout markedly inhibits cytokine release from macrophage after lipopolysaccharide and palmitic acid treatment in vitro. Mechanistically, in cultured peritoneal macrophages, SENP3 protein level is enhanced by IL-1β, in parallel with the upregulation of Yes-associated protein 1 (YAP1). Moreover, we demonstrated that SENP3 modulates de-SUMO modification of YAP1 and SENP3 deletion abolishes the upregulation of YAP1 induced by IL-1β. Most importantly, SENP3 deficiency reduces YAP1 protein level in adipose tissue during obesity. Our results highlight the important role of SENP3 in ATM inflammation and diet and age-induced obesity.

Verteporfin regulates corneal neovascularization through inhibition of YAP protein activation

Corneal neovascularization (CNV) is a vision-threatening disease that is becoming a growing public health concern. While Yes-associated protein (YAP) plays a critical role in neovascular disease and allow for the sprouting angiogenesis. Verteporfin (VP) is a classical inhibitor of the YAP-TEAD complex, which is used for clinical treatment of neovascular macular degeneration through photodynamic therapy. The purpose of this study is to explore the effect of verteporfin (VP) on the inhibition of CNV and its potential mechanism. Rat CNV model were established by suturing in the central cornea and randomly divided into three groups (control, CNV and VP group). Neovascularization was observed by slit lamp to extend along the corneal limbus to the suture line. RNA-sequencing was used to reveal the related pathways on the CNV and the results revealed the vasculature development process and genes related with angiogenesis in CNV. In CNV group, we detected the nuclear translocation of YAP and the expression of CD31 in corneal neovascular endothelial cells through immunofluorescence. After the application of VP, the proliferation, migration and the tube formation of HUVECs were significantly inhibited. Furthermore, VP showed the CNV inhibition by tail vein injection without photoactivation. Then we found that the expression of phosphorylated YAP significantly decreased, and its downstream target protein connective tissue growth factor (CTGF) increased in the CNV group, while the expression was just opposite in other groups. Besides, both the expression of vascular endothelial growth factor receptor 2 (VEGFR2) and cofilin significantly increased in CNV group, and decreased after VP treatment. Therefore, we conclude that Verteporfin could significantly inhibited the CNV without photoactivation by regulating the activation of YAP.

MUC16 C terminal fragment activates YAP1 through Src signaling to promote gallbladder cancer growth

AbstractThe Hippo pathway is crucial to organ size control and its dysregulation contributes to tumorigenesis. The aberrant activation of YAP1 was identified in gallbladder cancer (GBC). However, the underlying mechanism and role in GBC remains unclear. The C terminal fragment of Mucin16, also known as carbohydrate antigen 125 (CA125) encoded product, MUC16c, plays extensive roles in tumor initiation and development. Our study showed that MUC16c binding with 14‐3‐3ε disrupted the interaction of 14‐3‐3ε and phosphorylated yes‐associated protein 1 (YAP1), which led to the activation of YAP1 in GBC. Furthermore, MUC16c decreased the phosphorylation of YAP1 at serine 397 (ser397) by inhibiting LATS1, which upregulated YAP1 protein stability. Interestingly, there was a potential Src kinase site in the MUC16c fragment. The MUC16c_del15Y polypeptides with the deletion of the Src kinase site promoted the interaction of YAP1 with 14‐3‐3ε and downregulated the YAP1 protein levels. Consistently, SU6656, a Src kinase inhibitor also blocked the activation of YAP1 by MUC16c. The MUC16c_del15Y polypeptides decreased GBC cell proliferation in vitro and the growth of xenograft tumors in vivo. Our study revealed the underlying mechanism of the activation of MUC16c on YAP1 mediated by Src signaling and the antitumor effect of MUC16c_del15Y, providing a potential target for GBC therapy.

The deubiquitinating enzyme USP19 facilitates hepatocellular carcinoma progression through stabilizing YAP

Hippo pathway plays a crucial role in the progression of hepatocellular carcinoma (HCC), and yes-associated protein (YAP) is one of the major factors of the Hippo pathway. However, the mechanism of abnormal YAP activation in HCC has not been well elucidated. Here, we screened a Deubiquitinating enzymes’ (DUB) siRNA library targeting DUBs, and identified Ubiquitin Specific Peptidase 19 (USP19) as a specific deubiquitinating enzyme of YAP in HCC, which could stabilize YAP at K76 and K90 sites via removing the K48- and K11-linked ubiquitin chains. USP19 knockdown decreased the expression of YAP protein and its target gene (CTGF, CYR61, ANKRD1) expression. Through substantial in vivo and in vitro experiments, we prove that USP19 facilities the proliferation and migration of HCC. More importantly, we found that USP19 was upregulated in HCC tissues and associated with poor prognosis. In general, our research revealed a novel post-translational mechanism between USP19 and YAP in HCC, suggesting that USP19 may be a pivotal therapeutic target for HCC treatment.

YAP suppresses human T-cell leukemia virus type 1 transcription.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that causes adult T-cell leukemia/lymphoma (ATL). HTLV-1 encodes Tax protein that activates transcription from viral long terminal repeats (LTR). Multiple cofactors are involved in the regulation of HTLV-1 transcription via association with Tax. Yes-associated protein (YAP), which is the key effector of Hippo pathway, is elevated and activated in ATL cells. In this study, we reported that YAP protein suppressed Tax activation of HTLV-1 5' LTR but not 3' LTR. The activation of the 5' LTR by Tax was potentiated when YAP was depleted. Moreover, overexpression of YAP repressed HTLV-1 plus-strand viral gene expression and virion production, whereas compromising YAP by RNA inference augmented the expression of HTLV-1 protein. As mechanisms of YAP-mediated viral transcription inhibition, we found that YAP interacted with Tax, and prevented the association between Tax and p300. It finally led to the inhibition of recruitment of Tax to the Tax-responsive element in the 5' LTR of HTLV-1. Taken together, our results demonstrate the negative regulatory function of YAP in Tax activation of HTLV-1 transcription. It may achieve sufficient transcriptional repression to maintain persistent infection and long-term latency of HTLV-1 in the host cells.

Abstract P1086: Induced Pluripotent Stem Cell-based Cardiac Tissue Modeling Of Mitogenic Cardiomyopathy In Alström Syndrome

Introduction: Cardiomyopathies are a prominent cause of heart failure and affect millions worldwide. Mutation in the Alström syndrome 1 (ALMS1) gene causing Alström syndrome (ALMS) , reported an extremely rare type of dilated cardiomyopathy - called mitogenic cardiomyopathy - leading to neonatal heart failure and death in early infancy due to delayed postnatal cardiomyocyte (CM) cell cycle arrest. Methods: Induced pluripotent stem cells (iPSCs) from ALMS patients with or without mitogenic cardiomyopathy were used to generate ALMS patient-specific cardiomyocytes (iPSC-CMs) . The iPSC-CMs were divided into two main groups based on the presence or absence of the mitogenic phenotype. After transcriptome sequencing, differentially expressed genes (FDR < 0.05, logFC > 0.5 | < -0.5) were found by testing (QL F-test) a robust fitted linear model (edgeR). Harnessing recent advances in stem cell differentiation and tissue engineering, we aimed at creating a human engineered heart tissue model as an unprecedented in vitro disease system to study mechanisms responsible for persistent CM proliferation. Results: ALMS1-deficient mitogenic iPSC-CMs exhibited an impaired ability to undergo cell cycle arrest, evidenced by a higher cell percentage in the G2/M phase (19.3% in mitogenic vs . 7.5% in non-mitogenic iPSC-CMs). Furthermore, increased extracellular matrix levels of the fibroblast-derived protein periostin (POSTN) were observed in co-cultures of ALMS1-deficient iPSC-CMs and ALMS1 fibroblasts. Finally, we found preliminary evidence of dysregulation of the Hippo signaling pathway, observing altered cellular localization of its key downstream effector Yes-associated protein (YAP) and dysregulated ratio between phosphor and total YAP protein levels that in combination with transcript alteration of PPIC, SH3BP4, NTN4, LRIG3 suggest perturbations in YAP signaling in ALMS patients. Conclusion: ALMS1-deficient mitogenic iPSC-CMs recapitulate postnatal proliferation, observed in ALMS patients with mitogenic cardiomyopathy. Preliminary molecular analyses in these in vitro (multicellular) models point to an important role for altered YAP signaling and provide novel cues to enhance CM proliferation, a much-sought objective in cardiac repair.

ECSIT Is a Critical Factor for Controlling Intestinal Homeostasis and Tumorigenesis through Regulating the Translation of YAP Protein.

The intestinal epithelium is the fastest renewing tissue in mammals and its regenerative process must be tightly controlled to minimize the risk of dysfunction and tumorigenesis. The orderly expression and activation of Yes-associated protein (YAP) are the key steps in driving intestinal regeneration and crucial for intestinal homeostasis. However, the regulatory mechanisms controlling this process remain largely unknown. Here, it is discovered that evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), a multi-functional protein, is enriched along the crypt-villus axis. Intestinal cell-specific ablation of ECSIT results in the dysregulation of intestinal differentiation unexpectedly accompanied with enhanced YAP protein dependent on translation, thus transforming intestinal cells to early proliferative stem "-like" cells and augmenting intestinal tumorigenesis. Loss of ECSIT leads to metabolic reprogramming in favor of amino acid-based metabolism, which results in demethylation of genes encoding the eukaryotic initiation factor 4F pathway and their increased expression that further promotes YAP translation initiation culminating in intestinal homeostasis imbalance and tumorigenesis. It is also shown that the expression of ECSIT is positively correlated with the survival of patients with colorectal cancer. Together, these results demonstrate the important role of ECSIT in regulating YAP protein translation to control intestinal homeostasis and tumorigenesis.

Yes-associated protein-1 overexpression in ocular surface squamous neoplasia; a potential diagnostic marker and therapeutic target.

Yes-associated protein-1 (YAP-1) is a Hippo system transcription factor, which serves as an oncogene in squamous cell carcinoma, and several solid tumors when the Hippo pathway is dysregulated. Yet, the activity of YAP-1 in ocular surface squamous neoplasia (OSSN) has not been determined. Here, we investigate the relationship between YAP-1 overexpression and OSSN. Using a cross-sectional study design, we recruited 227 OSSN patients from the University Teaching Hospitals in Lusaka, Zambia. Immunohistochemistry was used to assess YAP-1 protein overexpression in tumor tissue relative to surrounding benign squamous epithelium. OSSN patient samples (preinvasive, n = 62, 27% and invasive, n = 165, 73%) were studied. One hundred forty-nine invasive tumors contained adjacent preinvasive tissue, bringing the total number of preinvasive lesions examined to 211 (62 + 149). There was adjacent benign squamous epithelium in 50.2% (114/227) of OSSN samples. Nuclear YAP- 1 was significantly overexpressed in preinvasive (Fisher's (F): p <.0001, Monte Carlo (MC): p <.0001) and invasive (F: p <.0001, MC: p <.0001) OSSN in comparison to adjacent benign squamous epithelium when analyzed for basal keratinocyte positive count, staining intensity, expression pattern, and Immunostaining intensity-distribution index. YAP-1 expression did not differ between preinvasive and invasive OSSN (p >.05), keratinizing and non- keratinizing cancer (p >.05), or between T1/T2 and T3/T4 stages in invasive tumors (p >.05). However, grade 2 and 3 tumors had significantly stronger nucleus YAP-1 overexpression intensity than grade 1 tumors (F: p = .0078, MC: p = .0489). By immunohistochemistry, we identified significant overexpression (upregulation of YAP-1 protein expression) in preinvasive and invasive OSSN lesions compared to neighboring benign squamous epithelium. YAP-1 expression was significantly higher in poorly and moderately differentiated invasive squamous cancer than in well-differentiated carcinomas. Overexpression of YAP-1 within the margin of preinvasive and invasive OSSN, but not in the neighboring normal epithelium, indicates that it plays a role in the development and progression of OSSN.

Lipophilic statins inhibit YAP co-activator transcriptional activity in HCC cells through Rho-mediated modulation of the actin cytoskeleton.

Introduction: Hepatocellular carcinoma (HCC) is the third leading cause of liver-related death. Lipophilic statins have been associated with a decrease in HCC incidence, raising the possibility of their use as chemoprevention agents. The Yes associated protein (YAP) and Transcriptional co-Activator (TAZ) have emerged as an important pro-oncogenic mechanism in HCC. Statins modulate YAP/TAZ in other solid tumors but few studies have assessed their mechanisms in HCC. We aimed to delineate how lipophilic statins regulate YAP protein localization by interrogating the mevalonate pathway in a step-wise fashion using pharmacological and genetic approaches in HCC cells. Methods: Huh7 and Hep3B HCC cells were treated with the lipophilic statins cerivastatin and atorvastatin. YAP protein localization was determined using quantitative immunofluorescence (IF) imaging. The gene expression of CTGF and CYR61, known YAP/TEAD-regulated genes, were measured using quantitative real-time PCR. Rescue experiments were conducted using metabolites of the mevalonate pathway including mevalonic acid and geranylgeranyl pyrophosphate (GG-PP). The cellular cytoskeleton was assessed using F-actin IF staining. Results: YAP protein was extruded from the nucleus to the cytoplasm with statin treatment. Consistently, CTGF and CYR61 mRNA expression significantly decreased with statins. Cytoskeletal structure was also compromised with statins. Gene expression, YAP protein localization and cytoskeletal structure were all restored to baseline with exogenous GG-PP but not with other metabolites of the mevalonate pathway. Direct Rho GTPase inhibitor treatment mirrored the statin effects on YAP. Conclusions: YAP protein localization is regulated by lipophilic statins via Rho GTPases causing cytoskeletal structural changes and is independent of cholesterol metabolites.

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

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

Hepatitis C virus nonstructural protein 4B induces lipogenesis via the Hippo pathway

Hepatitis C virus (HCV) infection causes abnormal lipid metabolism in hepatocytes, which leads to hepatic steatosis and even hepatocellular carcinoma. HCV nonstructural protein 4B (NS4B) has been reported to induce lipogenesis, but the underlying mechanism is unclear. In this study, western blots were performed to investigate the effect of NS4B protein levels on key effectors of the Hippo and AKT signaling pathways. Yes-associated protein (YAP) and moesin-ezrin-radixin-like protein (Merlin) are effectors of the Hippo pathway. NS4B downregulated Merlin and phosphorylated YAP (p-YAP) protein expression while increasing the expression of the key AKT pathway proteins p-AKT and NF-κB. By observing the levels of AKT pathway proteins when Merlin was overexpressed or silenced, it was determined that Merlin mediates the AKT pathway. We suggest that HCV NS4B may mediate the AKT signaling pathway by inhibiting the Hippo pathway. Lipid droplets were observed in Huh7.5 cells overexpressing NS4B, and they increased significantly in number when Merlin was silenced. Overexpression of NS4B and Merlin silencing enhanced the expression of sterol regulatory element binding proteins (SREBPs), which have been demonstrated to be key regulatory factors controlling fatty acid synthesis. NS4B and Merlin silencing also enhanced the in vitro proliferative capacity of hepatocellular carcinoma cells. In conclusion, NS4B induces lipogenesis via the effect of the Hippo-YAP pathway on the AKT signaling pathway and thereby plays a significant role in the pathogenesis of HCV-associated diseases.

The deubiquitinating enzyme UCHL3 promotes anaplastic thyroid cancer progression and metastasis through Hippo signaling pathway.

Yes-associated protein (YAP) is one of major key effectors of the Hippo pathway and the mechanism supporting abnormal YAP expression in Anaplastic thyroid carcinoma (ATC) remains to be characterized. Here, we identified ubiquitin carboxyl terminal hydrolase L3 (UCHL3) as a bona fide deubiquitylase of YAP in ATC. UCHL3 stabilized YAP in a deubiquitylation activity-dependent manner. UCHL3 depletion significantly decreased ATC progression, stem-like and metastasis, and increased cell sensitivity to chemotherapy. Depletion of UCHL3 decreased the YAP protein level and the expression of YAP/TEAD target genes in ATC. UCHL3 promoter analysis revealed that TEAD4, through which YAP bind to DNA, activated UCHL3 transcription by binding to the promoter of UCHL3. In general, our results demonstrated that UCHL3 plays a pivotal role in stabilizing YAP, which in turn facilitates tumorigenesis in ATC, suggesting that UCHL3 may prove to be a potential target for the treatment of ATC.

Yes-Associated Protein 1 Inhibition Induces Immunogenic Cell Death and Synergizes With Radiation and PD-1 Blockade

Danger signals released by ionizing radiation (IR) can theoretically stimulate immune activation in the tumor environment (TME), but IR alone is not sufficient to induce an effective immune response in clinical practice. In this study, we investigated whether inhibition of yes-associated protein 1 (YAP1) could induce immunogenic cell death (ICD) and whether the combination of YAP1 inhibition with IR could increase in vivo immune infiltration and thereby boost a tumor response to immunotherapy. First, the expression of ICD markers, markers of T-cell activation, and key proteins involved in innate immune signaling were measured after YAP1 inhibition. Next, the expression level of YAP1 protein was measured after different doses of IR. Then, the antitumor effect of YAP1 inhibition combined with IR was investigated in vivo, and the immune status of the TME was evaluated. Finally, the efficacy of a triple therapy including YAP1 inhibition combined with IR and programmed cell death protein 1 blockade in the treatment of resistant tumors was determined. We found that YAP1 inhibition induced ICD and increased the levels of antigen presentation machinery, effectively causing the activation of T cells. Mechanistically, YAP1 inhibition induced cell DNA damage and activated the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. Surprisingly, IR upregulated YAP1 expression. IR combined with YAP1 inhibition significantly inhibited cancer growth and prolonged survival, which was related to the augmented infiltration, activation, and function of CD8+ T cells in the TME. Moreover, the addition of YAP1 inhibition significantly improved the efficacy of pancreatic cancer treatment when neither radiation nor programmed cell death protein 1 inhibitors were ideal. YAP1 inhibition could trigger ICD and is a potential approach to potentiating the therapeutic efficacy of radiation therapy and anti-PD1 immunotherapy.

UHRF1 plays an oncogenic role in small cell lung cancer.

Small cell lung cancer (SCLC) is a malignant tumor characterized by aggressiveness and dismal prognosis. The specific role of ubiquitin-like PHD and RING finger domain (UHRF1), a frequently overexpressed cancer-promoting gene in various tumors, is poorly understood in SCLC. Herein, we explored the potential carcinogenic role of UHRF1 in SCLC. First, public databases were used to analyze the expression of UHRF1 in SCLC, and tissue specimens in our center were examined to confirm the results while clinical outcomes were collected to analyze its relationship with UHRF1. Then, UHRF1 knockdown and overexpression cell lines were established to evaluate the carcinogenic function of UHRF1 in vitro and in vivo. The mechanism of the biological consequences was determined by co-inmunoprecipitation. Moreover, we also analyzed the influence of UHRF1 on cisplatin (DDP) sensitivity of SCLC. The expression of UHRF1 was significantly higher in SCLC tissues than in normal tissues, and high levels of UHRF1 suggested a poor prognosis for SCLC. Mechanistically, UHRF1 promoted SCLC growth through yes-associated protein 1 (YAP1). Specifically, UHRF1 bound to YAP1 and inhibited YAP1 ubiquitin degradation, thus stabilizing the YAP1 protein in SCLC cells. UHRF1 downregulation enhanced DDP sensitivity in SCLC cells and was correlated with a favorable prognosis in patients with SCLC treated with platinum-based chemotherapy. UHRF1 plays an oncogenic role in SCLC by modulating YAP1. Therefore, UHRF1 could be used as a biomarker to predict the prognosis of SCLC patients and serve as a potential therapeutic target for SCLC patients.

HBx and YAP expression could promote tumor development and progression in HBV-related hepatocellular carcinoma

BackgroundHepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) accounts for 10%–20% of the total HCC numbers. Its clinical features include the occurrence in the younger generation, large tumors, and poor prognosis. The contribution of hepatitis B virus X (HBx) protein in hepatocytes during activation of various oncogenic pathways has been reported. We aimed to assess the possible association between HBx and Yes-associated protein (YAP) expression in the liver tissue and the clinical features of HBV-related HCC. MethodsThe relationship between HBx and YAP expression was examined in vivo using HCC tumor and peritumor tissues (n = 55). The clinical information including tumor size, marker, and the prognosis was assessed with protein expressions. The in vitro gene expression analyses were conducted using HBx- and YAP-overexpressing HCC cell lines. ResultsAmong 19 cases of HBV-related, 17 cases of hepatitis C virus (HCV)-related, and 19 cases of nonviral-related HCC, the HBV-related tumor showed the largest size. The HBx-stained area in the tumor and peritumor tissue showed a significant correlation with tumor size and serum α-fetoprotein level. YAP expression was higher in HBV-related tumor tissue than in the peritumor tissue and HCV-related tumor. Additionally, HBx and YAP protein expressions are correlated and both expressions in the tumor contributed to the poor prognosis. An in vitro study demonstrated that HBx and YAP overexpression in the hepatocytes activate the various oncogenic signaling pathways. ConclusionsOur study demonstrated that YAP expression in the liver of HBV-infected patients might be the key factor in HBV-related HCC development and control of tumor-related features.

APE1 redox function is required for activation of Yes-associated protein 1 under reflux conditions in Barrett’s-associated esophageal adenocarcinomas

BackgroundEsophageal adenocarcinoma (EAC) is characterized by poor prognosis and low survival rate. Chronic gastroesophageal reflux disease (GERD) is the main risk factor for the development of Barrett’s esophagus (BE), a preneoplastic metaplastic condition, and its progression to EAC. Yes-associated protein 1 (YAP1) activation mediates stem-like properties under cellular stress. The role of acidic bile salts (ABS) in promoting YAP1 activation under reflux conditions remains unexplored.MethodsA combination of EAC cell lines, transgenic mice, and patient-derived xenografts were utilized in this study. mRNA expression and protein levels of APE1 and YAP1 were evaluated by qRT-PCR, western blot, and immunohistochemistry. YAP1 activation was confirmed by immunofluorescence staining and luciferase transcriptional activity reporter assay. The functional role and mechanism of regulation of YAP1 by APE1 was determined by sphere formation assay, siRNA mediated knockdown, redox-specific inhibition, and co-immunoprecipitation assays.ResultsWe showed that YAP1 signaling is activated in BE and EAC cells following exposure to ABS, the mimicry of reflux conditions in patients with GERD. This induction was consistent with APE1 upregulation in response to ABS. YAP1 activation was confirmed by its nuclear accumulation with corresponding up-regulation of YAP1 target genes. APE1 silencing inhibited YAP1 protein induction and reduced its nuclear expression and transcriptional activity, following ABS treatment. Further investigation revealed that APE1-redox-specific inhibition (E3330) or APE1 redox-deficient mutant (C65A) abrogated ABS-mediated YAP1 activation, indicating an APE1 redox-dependent mechanism. APE1 silencing or E3330 treatment reduced YAP1 protein levels and diminished the number and size of EAC spheroids. Mechanistically, we demonstrated that APE1 regulated YAP1 stability through interaction with β-TrCP ubiquitinase, whereas APE1-redox-specific inhibition induced YAP1 poly-ubiquitination promoting its degradation.ConclusionOur findings established a novel function of APE1 in EAC progression elucidating druggable molecular vulnerabilities via targeting APE1 or YAP1 for the treatment of EAC.