YAP Knockdown Research Articles

Naringenin alleviates intestinal ischemia/reperfusion injury by inhibiting ferroptosis via targeting YAP/STAT3 signaling axis

BackgroundIntestinal ischemia/reperfusion injury (IRI) is a significant clinical emergency, and investigating novel therapeutic approaches and understanding their underlying mechanisms is essential for improving patient outcomes. Naringenin (Nar), a flavanone present in tomatoes and citrus fruits, is frequently consumed in the human diet and recognized for having immunomodulatory, anti-inflammatory, and antioxidant properties. Despite Nar being able to alleviate intestinal IRI, the exact molecular mechanisms remain elusive. PurposeTo investigate Nar's protective properties on intestinal IRI and elucidate the mechanisms, a comprehensive approach that combines network pharmacology analysis with experimental verification in vitro and in vivo was adopted. MethodsThe oxygen-glucose deprivation/reoxygenation (OGD/R) model in IEC-6 cells and a murine model of intestinal IRI were used. Nar's effects on intestinal IRI were assessed through histological analysis using H&E staining and tight junction (TJ) protein expression. Ferroptosis-related parameters, including iron levels, superoxide dismutase (SOD), glutathione (GSH), reactive oxygen species (ROS), malondialdehyde (MDA), and mitochondrial morphology, were analyzed. Network pharmacology was utilized to predict the pathways through which Nar exerts its anti-ferroptosis effects. Further mechanistic insights were obtained through si-RNA transfection, YAP inhibitor (verteporfin, VP) treatment, ferroptosis inhibitor (Ferrostatin-1) and ferroptosis inducer (Erastin) application, co-immunoprecipitation (Co-IP) and Western blotting. ResultsOur results revealed that pretreatment with Nar significantly mitigated intestinal tissue damage and improved gut barrier function, as evidenced by increased TJ proteins (ZO-1 and Occludin). Nar reduced iron, MDA, and ROS, while it increased GSH and SOD levels. Additionally, Nar alleviated mitochondrial damage in mice. Nar treatment increased GPX4 and SLC7A11, while decreasing ACSL4 levels both in vivo and in vitro. Network pharmacology analysis suggested that Nar may target the Hippo signaling pathway. Notably, YAP, a key transcriptional co-activator within the Hippo pathway, was downregulated in intestinal IRI mice and OGD/R-induced IEC-6 cells. Nar pretreatment activated YAP, thereby augmenting anti-ferroptosis effects. The inhibition of YAP activation by VP or YAP knockdown increased p-STAT3 expression, thereby diminishing Nar's efficacy. Co-IP and immunofluorescence studies confirmed the interaction between YAP and STAT3. ConclusionThis study shows that Nar can inhibit ferroptosis in intestinal IRI via activating YAP, which in turn suppresses STAT3 phosphorylation, thereby unveiling a novel mechanism and supporting Nar's potential to be a promising therapeutic agent for intestinal IRI.

SOX9 promotes hypoxic pulmonary hypertension through stabilization of DPP4 in pulmonary artery smooth muscle cells

Pulmonary hypertension (PH) is a progressive cardiopulmonary disorder characterized by pulmonary vascular remodeling (PVR), primarily due to the excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). This study aimed to investigate the role and molecular mechanism of SOX9 in hypoxic PH in rats. The findings revealed that SOX9 was upregulated in the pulmonary arteries and PASMCs of hypoxia-exposed rats. SOX9 knockdown inhibited hypoxia-induced proliferation and migration of PASMCs, reduced PVR, and subsequently alleviated hypoxia-induced PH in rats, suggesting that SOX9 plays a critical role in PH. Further investigation demonstrated that SOX9 interacted with DPP4, preventing its ubiquitin degradation in hypoxia-exposed PASMCs. DPP4 knockdown inhibited hypoxia-induced PASMC proliferation and migration, and administration of the DPP4 inhibitor sitagliptin (5 mg/kg) significantly reduced PVR and alleviated hypoxia-induced PH in rats, indicating that SOX9 contributes to PH by stabilizing DPP4. The results also showed that hypoxia induced YAP1 expression and dephosphorylation, leading to YAP1 nuclear localization. YAP1 knockdown promoted the degradation of HIF-1α in hypoxia-exposed PASMCs and inhibited hypoxia-induced proliferation and migration of PASMCs. Additionally, HIF-1α, as a transcription factor, promoted SOX9 expression by binding to the SOX9 promoter in hypoxia-exposed PASMCs. In conclusion, hypoxia promotes the proliferation and migration of PASMCs through the regulation of the YAP1/HIF-1α/SOX9/DPP4 signaling pathway, leading to PH in rats. These findings suggest that SOX9 may serve as a potential prognostic marker and therapeutic target for PH.

Aspirin attenuates the detrimental effects of TNF-α on BMMSC stemness by modulating the YAP-SMAD7 axis

BackgroundBone marrow mesenchymal stem cells (BMMSCs) are commonly used for cell transplantation to treat refractory diseases. However, the presence of inflammatory factors, such as tumour necrosis factor-alpha (TNF-α), at the transplantation site severely compromises the stemness of BMMSCs, thereby reducing the therapeutic effect of cell transplantation. Aspirin (AS) is a drug that has been in use for over a century and has a wide range of effects, including the regulation of cell proliferation, multidirectional differentiation, and immunomodulatory properties of stem cells. However, it is still unclear whether AS can delay the damaging effects of TNF-α on BMMSC stemness.MethodsThis study investigated the effects of AS and TNF-α on BMMSC stemness and the molecular mechanisms using colony formation assay, western blot, qRT-PCR, and overexpression or knockdown of YAP and SMAD7.ResultsThe results demonstrated that TNF-α inhibited cell proliferation, the expression of stemness, osteogenic and chondrogenic differentiation markers of BMMSCs. Treatment with AS was shown to mitigate the TNF-α-induced damage to BMMSC stemness. Mechanistic studies revealed that AS may reverse the damage caused by TNF-α on BMMSC stemness by upregulating YAP and inhibiting the expression of SMAD7.ConclusionAS can attenuate the damaging effects of TNF-α on BMMSC stemness by regulating the YAP-SMAD7 axis. These findings are expected to promote the application of AS to improve the efficacy of stem cell therapy.

Induced expression of AMOT reverses adriamycin resistance in breast cancer cells.

Adriamycin (ADR) is widely used against breast cancer, but subsequent resistance always occurs. YAP, a downstream protein of angiomotin (AMOT), importantly contributes to ADR resistance, whereas the mechanism is largely unknown. MCF-7 cells and MDA-MB-231 cells were used to establish ADR-resistant cell. Then, mRNA and protein expressions of AMOT and YAP expressions were determined. After AMOT transfection alone or in combination with YAP, the sensitivity of the cells to ADR were evaluated in vitro by examining cell proliferation, apoptosis, and cell cycle, as well as in vivo by examining tumor growth. Additionally, the expressions of proteins in YAP pathway were determined in AMOT-overexpressing cells. In the ADR-resistant cells, the expression of AMOT was decreased while YAP was increased, respectively, and the nucleus localization of YAP was increased at the same time. After AMOT overexpression, these were inhibited, whereas the cell sensitivity to ADR was enhanced. However, the AMOT-induced changes were significantly suppressed by YAP knockdown. The consistent results in vivo showed that AMOT enhanced the inhibition of ADR on tumor growth, and inhibited YAP signaling, evidenced by decreased levels of YAP, CycD1, and p-ERK. Our data revealed that decreased AMOT contributed to ADR resistance in breast cancer cells, which was importantly negatively mediated YAP. These observations provide a potential therapy against breast cancer with ADR resistance.

Human trophoblast invasion and migration are mediated by the YAP1-CCN1 pathway: defective signaling in trophoblasts during early-onset severe preeclampsia†.

The transcription coactivator YAP1 mediates the major effects of the Hippo signaling pathway. The CCN family is a small group of glycoproteins known to be downstream effectors of YAP1 in diverse tissues. However, whether CCN family members mediate the effects of YAP1 in human trophoblasts is unknown. In this study, placental expression of both YAP1 and CCN1 was found to be impaired in pregnancies complicated by early-onset severe preeclampsia. CCN1 was expressed not only in cytotrophoblasts, trophoblast columns, and mesenchymal cells, similar to active YAP1, but also in syncytiotrophoblasts of normal first-trimester placental villi; moreover, decidual staining of active YAP1 and CCN1 was found in both interstitial and endovascular extravillous trophoblasts. In cultured immortalized human trophoblastic HTR-8/SVneo cells, knockdown of YAP1 decreased CCN1 mRNA and protein expression and led to impaired cell invasion and migration. Also, CCN1 knockdown negatively affected HTR-8/SVneo cell invasion and migration but not viability. YAP1 knockdown was further found to impair HTR-8/SVneo cell viability via G0/G1 cell cycle arrest and apoptosis, while CCN1 knockdown had minimal effect on cell cycle arrest and no effect on apoptosis. Accordingly, treatment with recombinant CCN1 partially reversed the YAP1 knockdown-induced impairment in trophoblast invasion and migration but not in viability. Thus, CCN1 mediates the effects of YAP1 on human trophoblast invasion and migration but not apoptosis, and decreased placental expression of YAP1 and CCN1 in pregnancies complicated by early-onset severe preeclampsia might contribute to the pathogenesis of this disease.

YAP promotes the early development of temporomandibular joint bony ankylosis by regulating mesenchymal stem cell function

To explore the role of YAP, a key effector of the Hippo pathway, in temporomandibular joint (TMJ) ankylosis. The temporal and spatial expression of YAP was detected via immunohistochemistry and multiplex immunohistochemistry on postoperative Days 1, 4, 7, 9, 11, 14 and 28 in a sheep model. Isolated mesenchymal stem cells (MSCs) from samples of the Day 14. The relative mRNA expression of YAP was examined before and after the osteogenic induction of MSCs. A YAP-silenced MSC model was constructed, and the effect of YAP knockdown on MSC function was examined. YAP is expressed in the nucleus of the key sites that determine the ankylosis formation, indicating that YAP is activated in a physiological state. The expression of YAP increased gradually over time. Moreover, the number of cells coexpressing of RUNX2 and YAP—with the osteogenic active zone labelled by RUNX2—tended to increase after Day 9. After the osteogenic induction of MSCs, the expression of YAP increased. After silencing YAP, the osteogenic, proliferative and migratory abilities of the MSCs were inhibited. YAP is involved in the early development of TMJ bony ankylosis. Inhibition of YAP using shRNA might be a promising way to prevent or treat TMJ ankylosis.

Disruption of the Hippo pathway promotes the proliferation of childhood acute lymphoblastic leukemia cells, inhibits apoptosis and chemosensitivity

ABSTRACT Background Despite advancements in chemotherapy and stem cell transplantation, the recurrence and chemoresistance of childhood acute lymphoblastic leukemia (cALL) remain a significant challenge, thus indicating the need for novel therapeutic targets. Research design and methods The protein levels of YAP1, p-YAP1, TAZ, and Cyr61 of cALL patients and healthy volunteers were measured by western blot analysis. Then the leukemic cell line SUP-B15 was transfected with sh-YAP1 and pcDNA3.1-YAP1 to knockdown or overexpress YAP1. The viability, chemosensitivity, apoptosis, migration, and invasion of SUP-B15 cells were determined by MTT, flow cytometry, and Transwell assay. Results The cALL patients had higher YAP1, TAZ, and Cyr61 protein expression and lower p-YAP1 protein expression in bone marrow tissues compared with healthy volunteers (p < 0.01). In SUP-B15 cells, YAP1 knockdown upregulated p-YAP1 protein expression (p < 0.01) and downregulated TAZ and Cyr61 protein expression (p < 0.01). In addition, knocking down YAP1 significantly inhibited cell viability, migration, and invasion, and induced apoptosis (p < 0.01). YAP1 knockdown also reduced the IC50 value following treatment with vincristine, daunorubicin, cyclophosphamide, and dexamethasone (p < 0.05). Conclusions Disruption of the Hippo pathway attenuates the development of cALL by promoting cell proliferation while suppressing apoptosis and drug sensitivity.

Dihydroartemisinin breaks the positive feedback loop of YAP1 and GLUT1-mediated aerobic glycolysis to boost the CD8+ effector T cells in hepatocellular carcinoma

Aerobic glycolysis is a hallmark of hepatocellular carcinoma (HCC). Dihydroartemisinin (DHA) exhibits antitumor activity towards liver cancer. Our previous studies have shown that DHA inhibits the Warburg effect in HCC cells. However, the mechanism still needs to be clarified. Our study aimed to elucidate the interaction between YAP1 and GLUT1-mediated aerobic glycolysis in HCC cells and focused on the underlying mechanisms of DHA inhibiting aerobic glycolysis in HCC cells. In this study, we confirmed that inhibition of YAP1 expression lowers GLUT1-mediated aerobic glycolysis in HCC cells and enhances the activity of CD8+T cells in the tumor niche. Then, we found that DHA was bound to cellular YAP1 in HCC cells. YAP1 knockdown inhibited GLUT1-mediated aerobic glycolysis, whereas YAP1 overexpression promoted GLUT1-mediated aerobic glycolysis in HCC cells. Notably, liver-specific Yap1 knockout by AAV8-TBG-Cre suppressed HIF-1α and GLUT1 expression in tumors but not para-tumors in DEN/TCPOBOP-induced HCC mice. Even more crucial is that YAP1 forms a positive feedback loop with GLUT1-mediated aerobic glycolysis, which is associated with HIF-1α in HCC cells. Finally, DHA reduced GLUT1-aerobic glycolysis in HCC cells through YAP1 and prevented the binding of YAP1 and HIF-1α. Collectively, our study revealed the mechanism of DHA inhibiting glycolysis in HCC cells from a perspective of a positive feedback loop involving YAP1 and GLUT1 mediated-aerobic glycolysis and provided a feasible therapeutic strategy for targeting enhanced aerobic glycolysis in HCC.

Babaodan overcomes cisplatin resistance in cholangiocarcinoma via inhibiting YAP1

Context Cholangiocarcinoma with highly heterogeneous, aggressive, and multidrug resistance has a poor prognosis. Although babaodan (BBD) combined with cisplatin improved non-small cell lung cancer efficacy, its impact on overcoming resistance in cholangiocarcinoma remains unexplored. Objective This study explored the role and mechanism of BBD on cisplatin resistance in cholangiocarcinoma cells (CCAs). Materials and methods Cisplatin-resistant CCAs were exposed to varying concentrations of cisplatin (25–400 μg/mL) or BBD (0.25–1.00 mg/mL) for 48 h. IC50 values, inhibition ratios, apoptosis levels, DNA damage, glutathione (GSH) levels, oxidized forms of GSH, total GSH content, and glutaminase relative activity were evaluated using the cell counting kit 8, flow cytometry, comet assay, and relevant assay kits. Results BBD-reduced the cisplatin IC50 in CCAs from 118.8 to 61.83 μg/mL, leading to increased inhibition rate, apoptosis, and DNA damage, and decreased expression of B-cell lymphoma-2, p-Yes-associated protein 1/Yes-associated protein 1, solute carrier family 1 member 5, activating transcription factor 4, and ERCC excision repair 1 in a dose-dependent manner with maximum reductions of 78.97%, 51.98%, 54.03%, 56.59%, and 63.22%, respectively; bcl2-associated X and gamma histone levels were increased by 0.43–115.77% and 22.15–53.39%. The impact of YAP1 knockdown on cisplatin-resistant CCAs resembled BBD. GSH, oxidized GSH species, total GSH content, and glutaminase activity in cisplatin-resistant CCAs with BBD treatment also decreased, while YAP1 overexpression countered BBD’s effects. Discussion and conclusion This study provides a scientific basis for BBD clinical application and provides a new direction for BBD biological mechanism research.

Mechanisms of YAP1-mediated trophoblast ferroptosis in recurrent pregnancy loss.

The purpose of our study is to investigate the function of YAP1 in the trophoblast ferroptosis and maternal-fetal interface communication of RPL. We collected 25 villous tissues and detected the expression of YAP1. Cell counting kit-8 assay, scratch wound-healing assay, and Matrigel invasion assay were performed to observe the proliferation, migration, and invasion of HTR-8/SVneo and JAR cells. Subsequently, measured the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH), SLC7A11, SOD2, and GPX4. Ultimately, the use of ferroptosis activator (erastin) and inhibitor (Ferrostatin-1, fer-1) further confirmed the regulation by YAP1. In addition, established an in vitro-induced cell model to study the effect of YAP1 on the decidualization process. Finally, animal models were implemented for further confirmation. We found that YAP1 was downregulated in RPL patients. Overexpression of YAP1 could significantly enhance the proliferation, migration, and invasion of trophoblasts, and inhibit ferroptosis. Knocking down YAP1 exhibited the opposite effect. Rescue experiments have shown that YAP1 could upregulate the expression of SLC7A11 and GPX4, which are key molecules in the classic pathway of ferroptosis. In addition, the decidualization was impaired when hESCs were treated with conditioned medium of YAP1 knockdown trophoblasts. Moreover, we found that Yap1, Slc7a11, and Gpx4 were downregulated in the RPL mice, along with increased MDA and decreased GSH. Downregulation of YAP1 induces ferroptosis, thereby damaging the trophoblast invasion processes, which also disturbs the communication at the maternal-fetal interface. Our study identified YAP1 as a potential key molecule in the pathogenesis of RPL.

Abstract 6959: Acetylcholine potentiates invasion and migration of human pancreatic cancer cells under hypoxia through α7 nAChR-mediated increases in HIF-1α expression

Abstract The most significant nerve-related problem in clinical pancreatic cancer management is perineural invasion (PNI), characterized as the neoplastic invasion of cancer cells into or surrounding the nerves. It is a characteristic feature of pancreatic ductal adenocarcinoma (PDAC) associated with a poor prognosis, tumor recurrence, and emergent pain. However, why PDAC is associated with a higher incidence of PNI remains unclear. HIF-1 (hypoxia-inducible factor-1) regulates the expression of ~1000 genes involved in angiogenesis, metastasis, tumor growth, chemoresistance and radioresistance, underscoring the growing interest in targeting HIF-1 for cancer control. In the present study, we investigated the molecular mechanisms underlying neurotransmitter acetylcholine (Ach)-potentiated HIF-1α expression and subsequent increase in invasion and migration of human pancreatic cancer cells in hypoxia (2% O2). The expression of various acetylcholine receptors in six types of pancreatic cancer cell lines (MIA-PaCa-2, PANC-1, HPAF-II, Capan-1, AsPC-1, and BxPC-3 cells) was observed using qPCR, and α7 nAChRs was observed to be expressed in all pancreatic cancer cells. Therefore, we chose MIA-PaCa-2 cells, expressing high levels of α7 nAChRs, and HPAF-II cells, expressing low levels of α7 nAChRs, for loss- and gain-of-function studies, respectively. In vitro assays showed that, under hypoxic conditions, Ach caused a concentration-dependent increase in α7 nAChRs-mediated HIF-1α expression, enhancing expression of HIF-1α target genes, and promoting invasion and migration of human pancreatic cancer cells. We further found that Ach increased HIF-1α protein stability in pancreatic cancer cells under hypoxic conditions. This effect was mediated by YAP signaling, as evidenced by the fact that Ach-induced increases in HIF-1α protein stability were blocked by siRNA-mediated knockdown of YAP. Consistent with these findings, an investigation of the relationship between α7 nAChRs and clinicopathological features in pancreatic cancer patients using publicly available datasets showed that high levels of α7 nAChRs are correlated with a poorer prognosis in pancreatic cancer patients. Collectively, these findings identify novel mechanisms by which Ach potentiates invasion and migration of pancreatic cancer cells and provide further evidence that HIF-1α is a potential anticancer target in Ach-associated pancreatic cancer. Citation Format: Yunmi Cho, Eun-Taex Oh, Ha Gyeong Kim, Heon Joo Park. Acetylcholine potentiates invasion and migration of human pancreatic cancer cells under hypoxia through α7 nAChR-mediated increases in HIF-1α expression [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 6959.

Abstract 7281: Targeting the YAP/TAZ-TEAD pathway with TEAD inhibitors synergizes with chemotherapy and blocks diffuse gastric cancer progression

Abstract Introduction: Diffuse gastric cancer (DGC) accounts for one third of gastric cancers and is associated with poor differentiation, discohesive growth, chemoresistance, and worse survival compared to intestinal-type tumors. DGC is typically genomically stable, and there are no approved DGC-targeted therapies. Recent studies have identified a potential tumor-promoting role for Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif (YAP/TAZ), which interact with TEAD transcription factors to regulate gene expression. Therefore, we evaluated the activity and mechanism of TEAD inhibitors in DGC preclinical models. Methods: Gastric cancer patient-derived organoids (PDOs) and cell lines were treated with TEAD inhibitors (K-975, VT103, VT104, VT107) and/or 5-fluorouracil (5-FU) for in vitro assays evaluating cell proliferation or invasion. Additional experiments were conducted to test the in vivo activity of TEAD inhibitors or shRNA-mediated knockdown of YAP in subcutaneous xenograft, orthotopic xenograft, and lung metastasis models using a genetically engineered mouse model of gastric cancer driven by oncogenic KrasG12D and loss of Trp53/Cdh1. Results: TEAD inhibitors K-975, VT103, VT104, and VT107 reduced proliferation, colony formation, and invasion in DGC cell lines and PDOs. We observed no effect of TEAD inhibitors on the viability of normal gastric organoids. Combining the TEAD1-selective inhibitor VT103 with 5-FU chemotherapy resulted in further reduction of DGC PDO viability relative to monotherapy, including in a PDO derived from a patient with locally advanced DGC that demonstrated intrinsic resistance to neoadjuvant chemotherapy. In flank tumors and orthotopic models, inhibition of the YAP/TAZ-TEAD pathway using YAP shRNA and VT103, either alone or in combination with 5-FU, abrogated primary tumor formation, with the strongest inhibition observed with combination treatment. Loss of YAP or VT103 treatment also significantly reduced lung metastasis after tail vein injection of mouse gastric cancer cells. We hypothesized that TEAD inhibition blocks an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Consistent with this hypothesis, TEAD1 inhibition with VT103 or YAP knockdown reduced expression of the CSC marker CD44 in vitro and decreased expression of CD44 and the EMT marker Slug in tumor xenografts. Conclusions: The YAP/TAZ-TEAD pathway is an important driver of gastric cancer growth and metastasis, particularly in DGC where it may promote EMT and cancer stemness. TEAD inhibition reduces gastric cancer growth and invasion in vitro and in vivo in preclinical models and demonstrates synergistic activity with 5-FU chemotherapy. These results suggest a potential role for evaluating the efficacy of TEAD inhibitors in patients with DGC. Citation Format: Anastasiia Bulakhova, Jin Sun Cho, Jack T. Rifkin, Jaewon Kim, Thomas J. Ryan, Tracy T. Tang, Ryan H. Moy. Targeting the YAP/TAZ-TEAD pathway with TEAD inhibitors synergizes with chemotherapy and blocks diffuse gastric cancer progression [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 7281.

Abstract 7201: Targeting TAZ-TEAD in minimal residual disease enhances the duration of targeted therapy in melanoma models

Abstract Targeted therapies in cancer are limited by drug tolerance. In melanoma, tolerance to MAPK pathway inhibitors is associated with loss of SOX10 and enhanced YAP1/TAZ-TEAD activity. We show that loss of SOX10 is sufficient to up-regulate a TEAD transcriptional program with a strong dependence on TAZ. We developed a unique gene signature based on the transcriptomic changes following TAZ or YAP1 depletion. Expression of active TAZ was sufficient to mediate tolerance to BRAF inhibitors and MEK inhibitors. In WM983B cells, depletion of TAZ significantly reduced cell growth, whereas YAP1 knockdown resulted in little to no effect. These studies demonstrate that TAZ-TEAD activity plays an important role in melanoma drug tolerance and the development of acquired resistance. Citation Format: Connor A. Ott, Timothy Purwin, Manoela Tiago, Kristine Lou, Pan-Yu Chen, Somaneth Chowdhury, Glenn Mersky, Nir Hacohen, John Lamar, Claudia Capparelli, Gideon Bollag, Andrew Aplin. Targeting TAZ-TEAD in minimal residual disease enhances the duration of targeted therapy in melanoma models [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 7201.

Abstract 7207: Targeting hippo signaling pathway to overcome chemoresistance in glioblastoma

Abstract The study aims to investigate whether the Hippo signaling pathway contributes to temozolomide chemoresistance in glioblastoma, and to validate whether the combinational treatment of temozolomide and YAP inhibitor is effective against glioblastoma. Temozolomide-sensitive and temozolomide-resistant U87 and U251 GBM human cell lines were previously established. Temozolomide-resistant cells were maintained in low-dose temozolomide. Here, we investigate whether Hippo signaling contributes to temozolomide chemoresistance. By modulating the Hippo signaling pathway through YAP gene knockdown, the outcome on cell proliferation was studied by cell viability assay (in vitro) and mouse orthotopic xenograft (in vivo). Finally, the therapeutic effect of YAP inhibitor is studied in vitro and in vivo respectively. Our data showed that YAP is overexpressed in temozolomide-resistant glioblastoma cells. Nuclear translocation of YAP (which becomes its active state) is increased in temozolomide-resistant cells. In fact, YAP inhibition show decreased cell viability and higher susceptibility to temozolomide. In addition, mice with tumor injection of the YAP knockdown cells reduced tumor size with temozolomide treatment. Similarly, YAP inhibitor resensitizes temozolomide-resistant cells to temozolomide. Combinational treatment of YAP inhibitor and temozolomide is effective in shrinking tumor size in mouse xenograft models. In conclusion, our data suggest dysregulated Hippo signaling pathway contributes to temozolomide chemoresistance, thus targeting the Hippo signaling pathway resensitizes GBM cells to temozolomide. This research provides pre-clinical evidence on using YAP inhibitor for combinational therapy with temozolomide for the treatment of glioblastoma. Citation Format: Cheuk Lun Ethan Wong, Mei Yee Karrie Kiang, Ka Kit Gilberto Leung. Targeting hippo signaling pathway to overcome chemoresistance in glioblastoma [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 7207.

Abstract 614: Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma

Abstract Background: Cholangiocarcinoma (CCA) is a lethal and heterogenous malignancy of the biliary tree characterized by perineural invasion. About 85% of CCA show activation of YAP signaling, which promotes proliferation, anti-apoptosis, and therapeutic resistance. YAP signaling is mediated by Src family kinases (SFK) by phosphorylation of YAP (pYAPy357). NXP900 is a first-in-class, highly selective, SFK inhibitor with a novel mechanism of action which locks SFK in its closed and inhibited conformation, in contrast to other Src inhibitors such as dasatinib. A phase 1a study of NXP900 in patients with advanced solid tumors was recently initiated. Here, we examined treatment responses of CCA to NXP900 in vitro and in vivo. Methods: Cell viability was determined in seven CCA cell lines by CellTiter-Glo. Cell death and apoptosis were assayed by PI/Hoechst and Caspase-Glo 3/7 assay. CalcuSyn software was used to determine synergistic drug effects. Immunoblot analysis, RT-PCR, and IF staining of YAP localization were used to evaluate YAP knockdown. Clones of human CCA cell lines resistant to NXP900 were generated through escalating exposure to NXP900 over 6 months. Five patient-derived xenograft (PDX) tumors were expanded into flanks of NOD/SCID mice. Tumor bearing mice were randomized 1:1 with 5 mice per arm and treated with vehicle or NXP900 (40 mg/kg) once daily for up to 4 weeks. To determine drivers of sensitivity and resistance, predicted sensitivity scores based on tumor growth were linked to multi-omics (RNA seq and [phospho-]proteomics) of our PDX models. Results: All cell lines were sensitive to NXP900 with IC50 values between 7nM-15µM; IC50’s of resistant clones were 1000 times higher. NXP900 induced more cell death compared to vehicle, which appeared to be through apoptosis. NXP900 inhibited pSrc, decreased pYAPy357, and upregulated inactive pYAPs127. Correspondingly, decreased YAP target gene (Cyr61, NUAK, and CTGF) levels and a nuclear-to-cytoplasmic translocation were observed. NXP900/GemCis combination therapy increased cell death demonstrated a synergistic effect (Combination Indices &amp;lt;1) at all concentrations. In our PDX models, treatment was associated with a significant decrease in tumor growth in 3 models (mean fold change 3.1 vs. 14.2; 1.0 vs. 7.6; 1.4 vs. 2.6). The major resistant signature was the TRK signaling network, involved in nerve growth factor binding activity, while drivers of sensitivity included the SRC network. Conclusion: NXP900 demonstrated therapeutic activity in vitro and in human PDX models. We are currently performing multi-omic approaches in NXP900 sensitive and resistant cell lines to unravel determinants of activity and resistance. Additional in vivo studies will be performed to determine effects of NXP900/GemCis and NXP900/anti-PDL-1 combination therapy. Citation Format: Hendrien Kuipers, Jennifer L. Tomlinson, Danielle M. Carlson, Amro M. Abdelrahman, Erik Jessen, Jack W. Sample, Nathan W. Werneburg, Hannah E. Stumpf, Mark J. Truty, Sumera I. Ilyas, Gregory J. Gores, Rory L. Smoot. Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma [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 614.

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.

Inhibition of YAP1 Rescues Erectile Dysfunction by Inhibiting Phenotypic Modulation through Myocardin in Diabetic Rats

Background. Phenotypic modulation was shown to be related to the pathological mechanism of diabetes mellitus erectile dysfunction (DMED), and excessive expression of YAP1 was measured in the cavernous tissues of rats with DMED. Objectives. This investigation was performed to explore the efficiency and mechanism of gene therapy through the knockdown of YAP1 in the field of ED treatment. Materials and Methods. Corpus cavernosum smooth muscle cells were obtained and cultivated in vitro employing the explant technique. EdU, CCK-8, and collagen gel lattice contraction assays were subsequently performed to assess cell proliferation and contractility, which indicate smooth muscle phenotypes. Results. Initially, overexpression of YAP1 enhanced the proliferation and expression of YAP1 and osteopontin (OPN) while reducing cell contractility and the expression of myocardin, α-SMA, and calpontin, which is consistent with phenotypic modulation. Moreover, knocking down YAP1 resulted in the opposite effects, and the blockade of myocardin attenuated the reversal of phenotypic transformation. Then, 45 screened DMED rats were randomly divided into three equal groups, named streptozotocin (STZ)-induced DMED with intracavernosal injection of Ad-myocardin (DMED + myocardin), DMED with lenti-shYAP1 (DMED + shYAP1) and DMED with PBS (DMED + PBS), and 10 rats were in the negative control group. Staining with H&amp;E and Masson’s trichrome, immunochemistry, and determination of the ratio of the intracavernosal pressure/mean arterial pressure were performed to evaluate penile erection and histological changes. Western blotting was conducted to determine the molecular protein expression levels. In vivo, similar to myocardin overexpression, YAP1 inhibition rescued erectile function, repaired morphology, and maintained the contractile phenotype of smooth muscle tissues 14 days after treatment. Discussion and Conclusion. In conclusion, our study demonstrated the validity of gene therapy by downregulating excessive YAP1 expression to ameliorate ED in diabetic rats, and the ability of YAP1 inhibition to reverse phenotypic modulation was mediated through interaction with myocardin.

LATS1 Promotes B-ALL Tumorigenesis by Regulating YAP1 Phosphorylation and Subcellular Localization.

YAP1 plays a dual role as an oncogene and tumor suppressor gene in several tumors; differentiating between these roles may depend on the YAP1 phosphorylation pattern. The specific function of YAP1 in B cell acute lymphoblastic leukemia (B-ALL), however, is currently unclear. Thus, in the present study, the role of YAP1 in B-ALL was investigated using relevant cell lines and patient datasets. The effects of shRNA-mediated knockdown on YAP1 and LATS1 levels in the NALM6 and MOLT-4 cell lines were examined using Western blotting, quantitative real-time polymerase chain reaction, flow cytometry, immunostaining, and nude mouse subcutaneous tumorigenesis experiments. Gene expression levels of Hippo pathway-related molecules before and after verteporfin (VP) treatment were compared using RNA-Seq to identify significant Hippo pathway-related genes in NALM6 cells. Patients with ALL showing high YAP1 expression and low YAP1-Ser127 phosphorylation levels had worse prognoses than those with low YAP1 protein expression and high YAP1-Ser127 phosphorylation levels. YAP1-Ser127 phosphorylation levels were lower in NALM6 cells than in MOLT-4 and control cells; YAP1 was distributed in the nuclei in NALM6 cells. Knockdown of YAP1 inhibited MOLT-4 and NALM6 cell proliferation and arrested the NALM6 cell cycle in the G0/G1 phase. Before and after VP treatment, the expression of the upstream gene LATS1 was upregulated; its overexpression promoted YAP1-Ser127 phosphorylation. Further, YAP1 was distributed in the plasma. LATS1 may downregulate YAP1-Ser127 phosphorylation and maintain B-ALL cell function; thus, VP, which targets this axis, may serve as a new therapeutic method for improving the outcomes for B-ALL patients.

YAP represses intestinal inflammation through epigenetic silencing of JMJD3

BackgroundEpigenetics plays an important role in the pathogenesis of inflammatory bowel disease (IBD). Some studies have reported that YAP is involved in inflammatory response and can regulate target genes through epigenetic modifications. JMJD3, a histone H3K27me3 demethylase, is associated with some inflammatory diseases. In this study, we investigated the role of YAP in the development of IBD and the underlying epigenetic mechanisms.ResultsYAP expression was significantly increased in both in vitro and in vivo colitis models as well as in patients with IBD. Epithelial-specific knockout of YAP aggravates disease progression in dextran sodium sulfate (DSS)-induced murine colitis. In the TNF-α-activated cellular inflammation model, YAP knockdown significantly increased JMJD3 expression. Coimmunoprecipitation experiments showed that YAP and EZH2 bind to each other, and chromatin immunoprecipitation-PCR (ChIP-PCR) assay indicated that silencing of YAP or EZH2 decreases H3K27me3 enrichment on the promoter of JMJD3. Finally, administration of the JMJD3 pharmacological inhibitor GSK-J4 alleviated the progression of DSS-induced murine colitis.ConclusionOur findings elucidate an epigenetic mechanism by which YAP inhibits the inflammatory response in colitis through epigenetic silencing of JMJD3 by recruiting EZH2.

Chlamydial YAP activation in host endocervical epithelial cells mediates pro-fibrotic paracrine stimulation of fibroblasts.

Chronic or repeated infection of the female upper genital tract by C. trachomatis can lead to severe fibrotic sequelae, including tubal factor infertility and ectopic pregnancy. However, the molecular mechanisms underlying this effect are unclear. In this report, we define a transcriptional program specific to C. trachomatis infection of the upper genital tract, identifying tissue-specific induction of host YAP-a pro-fibrotic transcriptional cofactor-as a potential driver of infection-mediated fibrotic gene expression. Furthermore, we show that infected endocervical epithelial cells stimulate collagen production by fibroblasts and implicate chlamydial induction of YAP in this effect. Our results define a mechanism by which infection mediates tissue-level fibrotic pathology via paracrine signaling and identify YAP as a potential therapeutic target for the prevention of Chlamydia-associated scarring of the female genital tract.