Accumulation Of YAP Research Articles

Fate specification triggers a positive feedback loop of TEAD-YAP and NANOG to promote epiblast formation in preimplantation embryos.

In preimplantation embryos, epiblast (EPI) fate specification from the inner cell mass is controlled by the segregation of NANOG and GATA6 expression. TEAD-YAP interaction is activated during EPI formation, and is required for pluripotency factor expression. These events occur asynchronously with similar timing during EPI formation, and their relationship remains elusive. Here, we examined the relationship between NANOG-GATA6 and TEAD-YAP. The nuclear accumulation of YAP takes place only in EPI-specified cells, and a positive feedback loop operates between NANOG and TEAD-YAP. The effects of TEAD-YAP on SOX2 upregulation in EPI-specified cells are likely indirect. EPI fate specification also alters the response of Nanog, Sox2 and Cdx2 to TEAD-YAP. These results suggest that EPI-fate specification alters the transcriptional network from the morula-like to the EPI-specified state and activates TEAD-YAP to trigger a positive feedback loop with NANOG, which stabilizes the EPI fate. The coordinated occurrence of these processes in individual cells likely supports proper EPI formation under the condition of asynchronous EPI-fate specification.

YAP signaling orchestrates the endothelin-1-guided invadopodia formation in high-grade serous ovarian cancer.

The high-grade serous ovarian cancer (HG-SOC) is a notoriously challenging disease, characterized by a rapid peritoneal dissemination. HG-SOC cells leverage actin-rich membrane protrusions, known as invadopodia, to degrade the surrounding extracellular matrix (ECM) and invade, initiating the metastatic cascade. In HG-SOC, the endothelin-1 (ET-1)/endothelin A receptor (ETAR)-driven signaling coordinates invadopodia activity, however how this axis integrates pro-oncogenic signaling routes, as YAP-driven one, impacting on the invadopodia-mediated ECM degradation and metastatic progression, deserves a deeper investigation. Herein, we observed that downstream of the ET-1/ET-1R axis, the RhoC and Rac1 GTPases, acting as signaling intermediaries, promote the de-phosphorylation and nuclear accumulation of YAP. Conversely, the treatment with the dual ETA/ETB receptor antagonist, macitentan, inhibits the ET-1-driven YAP activity. Similarly, RhoC silencing, or cell transfection with a dominant inactive form of Rac1, restore the YAP phosphorylated and inhibited state. Mechanistically, the ET-1R/YAP signal alliance coordinates invadopodia maturation into ECM-degrading structures, indicating how such ET-1R-guided protein network represents a route able to enhance the HG-SOC invasive potential. At functional level, we found that the interconnection between the ET-1R/RhoC and YAP signals is required for MMP-2 and MMP-9 proteolytic functions, cell invasion, and cytoskeleton architecture changes, supporting the HG-SOC metastatic strength. In HG-SOC patient-derived xenografts (PDX) macitentan, turning-off the invadopodia regulators RhoC/YAP, halt the metastatic colonization. ET-1R targeting, hindering the YAP activity, weakens the invadopodia machinery, embodying a promising therapeutic avenue to prevent peritoneal dissemination in HG-SOC.

Adaptor protein CEMIP reduces the chemosensitivity of small cell lung cancer via activation of an SRC-YAP oncogenic module.

Small cell lung cancer (SCLC) is a recalcitrant malignancy with dismal prognosis due to rapid relapse after an initial treatment response. More effective treatments for SCLC are desperately needed. Our previous studies showed that cell migration-inducing hyaluronan binding protein (CEMIP) functionally promotes SCLC cell proliferation and metastasis. In this study, we investigated whether and how CEMIP regulates the chemosensitivity of SCLC. Through the GDSC database, we found that CEMIP expression levels were positively correlated with the IC50 values of several commonly used chemotherapeutic drugs in SCLC cells (cisplatin, gemcitabine, 5-fluorouracil and cyclophosphamide). We demonstrated that overexpression or knockdown of CEMIP in SCLC cells resulted in a notable increase or reduction in the IC50 value of cisplatin or etoposide, respectively. We further revealed that CEMIP functions as an adaptor protein in SCLC cells to interact with SRC and YAP through the 1-177 aa domain and 820-1361 aa domain, respectively, allowing the autophosphorylation of Y416 and activation of SRC, thus facilitating the interaction between YAP and activated SRC, and resulting in increased phosphorylation of Y357, protein stability, nuclear accumulation and transcriptional activation of YAP. Overexpressing SRC or YAP counteracted the CEMIP knockdown-mediated increase in the sensitivity of SCLC cells to cisplatin and etoposide. The combination of the SRC inhibitor dasatinib or the YAP inhibitor verteporfin and cisplatin/etoposide (EP regimen) displayed excellent synergistic antitumor effects on SCLC both in vitro and in vivo. This study demonstrated that targeted therapy against the CEMIP/SRC/YAP complex is a potential strategy for SCLC and provides a rationale for the development of future clinical trials with translational prospects.

Sag deletion promotes DMBA/TPA-induced skin carcinogenesis via YAP accumulation.

Sag deletion promotes DMBA/TPA-induced skin carcinogenesis via YAP accumulation.

The histone lysine acetyltransferase KAT2B inhibits cholangiocarcinoma growth: evidence for interaction with SP1 to regulate NF2-YAP signaling

BackgroundCholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tract with poor prognosis. Further mechanistic insights into the molecular mechanisms of CCA are needed to develop more effective target therapy.MethodsThe expression of the histone lysine acetyltransferase KAT2B in human CCA was analyzed in human CCA tissues. CCA xenograft was developed by inoculation of human CCA cells with or without KAT2B overexpression into SCID mice. Western blotting, ChIP-qPCR, qRT-PCR, protein immunoprecipitation, GST pull-down and RNA-seq were performed to delineate KAT2B mechanisms of action in CCA.ResultsWe identified KAT2B as a frequently downregulated histone acetyltransferase in human CCA. Downregulation of KAT2B was significantly associated with CCA disease progression and poor prognosis of CCA patients. The reduction of KAT2B expression in human CCA was attributed to gene copy number loss. In experimental systems, we demonstrated that overexpression of KAT2B suppressed CCA cell proliferation and colony formation in vitro and inhibits CCA growth in mice. Mechanistically, forced overexpression of KAT2B enhanced the expression of the tumor suppressor gene NF2, which is independent of its histone acetyltransferase activity. We showed that KAT2B was recruited to the promoter region of the NF2 gene via interaction with the transcription factor SP1, which led to enhanced transcription of the NF2 gene. KAT2B-induced NF2 resulted in subsequent inhibition of YAP activity, as reflected by reduced nuclear accumulation of oncogenic YAP and inhibition of YAP downstream genes. Depletion of NF2 was able to reverse KAT2B-induced reduction of nuclear YAP and subvert KAT2B-induced inhibition of CCA cell growth.ConclusionsThis study provides the first evidence for an important tumor inhibitory effect of KAT2B in CCA through regulation of NF2-YAP signaling and suggests that this signaling cascade may be therapeutically targeted for CCA treatment.

The Role of ZO-2 in Modulating JAM-A and γ-Actin Junctional Recruitment, Apical Membrane and Tight Junction Tension, and Cell Response to Substrate Stiffness and Topography.

This work analyzes the role of the tight junction (TJ) protein ZO-2 on mechanosensation. We found that the lack of ZO-2 reduced apical membrane rigidity measured with atomic force microscopy, inhibited the association of γ-actin and JAM-A to the cell border, and instead facilitated p114RhoGEF and afadin accumulation at the junction, leading to an enhanced mechanical tension at the TJ measured by FRET, with a ZO-1 tension probe, and increased tricellular TJ tension. Simultaneously, adherens junction tension measured with an E-cadherin probe was unaltered. The stability of JAM-A and ZO-2 binding was assessed by a collaborative in silico study. The absence of ZO-2 also impacted the cell response to the substrate, as monolayers plated in 20 kPa hydrogels developed holes not seen in parental cultures and displayed a retarded elongation and formation of cell aggregates. The absence of ZO-2 was sufficient to induce YAP and Snail nuclear accumulation in cells cultured over glass, but when ZO-2 KD cells were plated in nanostructured ridge arrays, they displayed an increased abundance of nuclear Snail and conspicuous internalization of claudin-4. These results indicate that the absence of ZO-2 also impairs the response of cells to substrate stiffness and exacerbates transformation triggered by substrate topography.

α-Catenin promotes dermal fibroblasts proliferation and migration during wound healing via FAK/YAP activation.

Skin wound healing is a complex and organized biological process, and the dermal fibroblasts play a crucial role. α-Catenin is known to be involved in regulating various cellular signals, and its role in wound healing remains unclear. Here, we have identified the pivotal role of the α-catenin/FAK/YAP signaling axis in the proliferation and migration of dermal fibroblasts, which contributes to the process of skin wound healing. Briefly, when α-catenin was knocked down specifically in dermal fibroblasts, the wound healing rate is significantly delayed. Moreover, interfering with α-catenin can impede the proliferation and migration of dermal fibroblasts both invitro and invivo. Mechanistically, the overexpression of α-catenin upregulates the nuclear accumulation of YAP and transcription of downstream target genes, resulting in enhanced the proliferation and migration of dermal fibroblasts. Furthermore, the FAK Tyr397 phosphorylation inhibitor blocked the promoting effects of α-catenin on YAP activation. Importantly, the continuous phosphorylation mutation of FAK Tyr397 reversed the retardatory effects of α-catenin knockdown on wound healing, by increasing the vitality of fibroblasts. Likewise, α-catenin/FAK was validated as a therapeutic target for wound healing in the db/db chronic trauma model. In summary, our findings have revealed a novel mechanism by which α-catenin facilitates the function of fibroblasts through the activity of the FAK/YAP signaling axis. These findings define a promising therapeutic strategy for accelerating the wound healing process.

Piezo1-ERK1/2-YAP Signaling Cascade Regulates the Proliferation of Urine-derived Stem Cells on Collagen Gels.

Urine-derived stem cells (USCs) were considered to be an ideal source of stem cells for repairing urological diseases. However, the proliferative ability of USCs significantly decreased when cultured on plastic dishes, which limited their clinical application. It was found that collagen gels could promote the proliferation of USCs, but the underlying molecular mechanisms were unclear. The study aims to investigate the role of the mechanically activated cation channel Piezo1 and the transcriptional coactivator YAP in the regulation of proliferation of USCs on collagen gels. USCs were cultured on collagen gels (group COL), or plastic dishes (group NON). MTT assay, Scratch assay, EDU staining, and immunofluorescence (IF) of Ki67 were performed to evaluate the proliferation of USCs; IF of YAP was conducted to observe its nuclear localization; calcium imaging experiment was executed to evaluate the function of Piezo1; western blot was used to compare changes in protein expression of YAP, LATS1, ERK1/2, and p-ERK1/2. In addition, the regulatory effect of YAP on the proliferative capacity of USCs was confirmed by intervening YAP with its inhibitor verteporfin (VP); and the inhibitor or activator of Piezo1, GsMTx4 or Yoda1 was used to explore the effect of Piezo1 on the nuclear localization of YAP, the proliferation of USCs and the regeneration of injured bladder. The results showed that cell proliferation was significantly enhanced in USCs in the COL group with the nuclear accumulation of YAP compared with the NON group and VP attenuated these effects. The expression and function of Piezo1 were higher in the COL group compared with the NON group. Blockage of Piezo1 by GsMTx4 decreased nuclear localization of YAP, the proliferation of USCs, and caused the failure of bladder reconstruction. Activation of Piezo1 by Yoda1 increased the nuclear expression of YAP, and the proliferation of USCs, which further improved the regeneration of the injured bladder. Finally, the ERK1/2 rather than LATS1 was revealed to participate in the Piezo1/YAP signal cascades of USCs proliferation. Taken together, Piezo1-ERK1/2-YAP signal cascades were involved in regulating the proliferation ability of USCs in collagen gels which would be beneficial for the regeneration of the bladder.

Piezo1 activates noncanonical EGFR endocytosis and signaling.

EGFR-ERK signaling controls cell cycle progression during development, homeostasis, and disease. While EGF ligand and mechanical inputs can activate EGFR-ERK signaling, the molecules linking mechanical force to this axis have remained mysterious. We previously found that stretch promotes mitosis via the stretch-activated ion channel Piezo1 and ERK signaling. Here, we show that Piezo1 provides the missing link between mechanical signals and EGFR-ERK activation. While both EGF- and Piezo1-dependent activation trigger clathrin-mediated EGFR endocytosis and ERK activation, EGF relies on canonical tyrosine autophosphorylation, whereas Piezo1 involves Src-p38 kinase-dependent serine phosphorylation. In addition, unlike EGF, ex vivo lung slices treated with Piezo1 agonist promoted cell cycle re-entry via nuclear ERK, AP-1 (FOS and JUN), and YAP accumulation, typical of regenerative and malignant signaling. Our results suggest that mechanical activation via Piezo1, Src, and p38 may be more relevant to controlling repair, regeneration, and cancer growth than tyrosine kinase signaling via canonical EGF signaling, suggesting an alternative therapeutic approach.

Abstract P2006: Cardiac Cytoskeleton Sequesters Acetylated Yap In The Cytoplasm To Inhibit Cardiomyocyte Renewal After Myocardial Infarction

The Hippo pathway effector YAP is essential in cardiomyocyte proliferation and heart regeneration. How YAP responds to physiological conditions such as myocardial infarction (MI) is unclear. Here we show YAP transiently localizes to the nucleus of border zone CMs after MI. Nuclear YAP is acetylated by P300/CBP, and SIRTs deacetylation activity is decreased due to metabolic stress resulting from the injury. These mechanisms in conjunction results in increased acetylated YAP accumulation in the cytoplasm. After MI, the cardiac cytoskeleton is stabilized and sequesters cytoplasmic acetylated YAP, which reduces YAP activity and inhibits cardiomyocyte renewal. In a genetically engineered mouse model with a mutation that abolishes YAP acetylation, the mutant heart exhibits improved cardiac regeneration ability compared to the wild-type heart due to increased nuclear YAP localization. Together, we demonstrate that the injury-induced metabolic shift results in YAP acetylation, which translocates YAP to the cytoplasm in a manner independent of the Hippo pathway, and is detrimental to cardiac regeneration after MI.

MicroRNA-205 promotes hair regeneration by modulating mechanical properties of hair follicle stem cells

Stiffness and actomyosin contractility are intrinsic mechanical properties of animal cells required for the shaping of tissues. However, whether tissue stem cells (SCs) and progenitors located within SC niche have different mechanical properties that modulate their size and function remains unclear. Here, we show that hair follicle SCs in the bulge are stiff with high actomyosin contractility and resistant to size change, whereas hair germ (HG) progenitors are soft and periodically enlarge and contract during quiescence. During activation of hair follicle growth, HGs reduce contraction and more frequently enlarge, a process that is associated with weakening of the actomyosin network, nuclear YAP accumulation, and cell cycle reentry. Induction of miR-205, a novel regulator of the actomyosin cytoskeleton, reduces actomyosin contractility and activates hair regeneration in young and old mice. This study reveals the control of tissue SC size and activities by spatiotemporally compartmentalized mechanical properties and demonstrates the possibility to stimulate tissue regeneration by fine-tuning cell mechanics.

Abstract 5403: Predicting cancer cell response to TEAD auto-palmitoylation inhibitor using bulk RNA-seq data and a random-forest based algorithm

Abstract TEAD transcription factors are the major effectors of the Hippo-YAP/TAZ pathway essential in controlling organ size and maintaining tissue homeostasis. Upon coactivator YAP/TAZ binding, TEAD transcription factors are activated to induce expression of genes involved in cell proliferation and survival. It has been shown that auto-palmitoylation is required for TEAD interaction with YAP/TAZ and hence activation of transcriptional activity. Potent small molecule TEAD auto-palmitoylation inhibitors have been reported (Tang et al, 2021, Mol Cancer Ther.). These TEAD inhibitors disrupt YAP/TAZ-TEAD protein interaction, suppress TEAD transcriptional activity, and selectively block proliferation of NF2-deficient mesothelioma in vitro and inhibit NF2 mutant xenograft tumor growth in vivo. Although genetic alterations of pathway components (such as, NF2) leading to YAP/TAZ constitutive nuclear localization and TEAD activation have been reported in a variety of human malignancies, these alterations are infrequent in most cancers, and increased accumulation of nuclear YAP has been reported in cancers (e.g., hepatocellular carcinoma) that do not harbor pathway mutations. Utilizing in vitro efficacy data from cell line screens, gene expression (RNA-Seq) datasets, and a custom bioinformatics data-processing and normalization pipeline, we sought to elucidate gene expression patterns by using a random-forest based classifier. Our classifier, which aims to predict the response (efficacy) of treatment using normalized gene expression of selected genes, was originally trained on the public bulk RNA-seq pan-cancer in vitro dataset available in the Cancer Cell Line Encyclopedia (CCLE) from the Broad Institute, as well as a privately obtained TEADi response data for corresponding CCLE cell lines. Binary response labels (responder/non responder) for each cell line were obtained by thresholding efficacy measures. The classifier - evaluated using cross-validation - achieved an average AUC of 0.80. Then from our screen of 50 patient-derived Chinese liver cancer (CLC) cell models (Qiu et al, 2019, Cancer Cell), both as single agent and in combination with mTOR inhibitor everolimus, we found that TEAD inhibitors (TEADi) were efficacious in several of these liver cancer models. This allowed us to verify the classifier without any re-training on an independent dataset (CLC). In this independent validation scenario, the classifier achieved the AUC of 0.82. Having established efficacy of predictions on both in vitro datasets, we re-trained the classifier using both CCLE and CLC datasets as input to achieve maximum predictive performance, and we predicted, post-hoc, response status of 99 responders out of 2056 patient-derived xenograft models. The algorithm showed early promise by retrospectively classifying a model with in vivo efficacy as the most likely to respond. Citation Format: Adam Kurkiewicz, Sebastian Y. Müller, Oliver Gibson, Sara Castellano, Germán Stark, Pol A. Vecino, Shuirong Zhou, Tracy T. Tang. Predicting cancer cell response to TEAD auto-palmitoylation inhibitor using bulk RNA-seq data and a random-forest based algorithm. [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 5403.

YAP and β-catenin cooperate to drive H. pylori-induced gastric tumorigenesis

ABSTRACT H. pylori infection is the strongest known risk factor for gastric carcinoma. The activation of the yes-associated protein 1 (YAP) and β-catenin pathways has been associated with multiple tumor types. In this study, we investigated the crosstalk between the YAP and β-catenin pathways in H. pylori-associated gastric tumorigenesis. Immunohistochemical analysis of YAP and β-catenin expression was performed in human gastric cancer tissues. The small molecules Super-TDU and KYA1797K, pharmacological inhibitors of YAP and β-catenin, respectively, were used to investigate the role of these signaling pathways in H. pylori-induced gastric carcinogenesis in murine models of infection. The common downstream targets of YAP and β-catenin signaling were evaluated by RNA sequencing (RNA-seq). Western blot, immunofluorescence, luciferase, RT-PCR, immunoprecipitation, cell counting kit-8 (CCK8), EdU and spheroid assays were used. H. pylori infection promoted YAP and β-catenin nuclear accumulation and transcriptional activity in gastric epithelial cells and transgenic insulin–gastrin (INS-GAS) mice, whereas silencing of both YAP and β-catenin synergistically inhibited H. pylori-induced cell proliferation and expansion. In addition, YAP was found to directly interact with β-catenin and knockdown of YAP suppressed H. pylori-induced nuclear translocation of β-catenin. Moreover, downstream genes caudal-type homeobox 2 (CDX2), leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) and RuvB like AAA ATPase 1 (RUVBL1) were shared by both YAP and β-catenin signaling. Furthermore, treatment with the YAP inhibitor Super-TDU or β-catenin inhibitor KYA1797A significantly alleviated gastric inflammation and epithelial DNA damage in H. pylori-infected mice. Finally, the elevation of gastric YAP was positively correlated with β-catenin expression in human gastric cancer tissues. These findings indicate that YAP and β-catenin synergistically promote H. pylori-induced gastric carcinogenesis via their physical interaction and reveal that CDX2, LGR5 and RUVBL1 are the downstream genes shared by both the YAP and β-catenin signaling pathways, and potentially contribute to H. pylori pathogenesis.

A26 BMP-SIGNALING IMPAIRED TELOCYTES CREATE A DISRUPTED NICHE GRADIENT FOSTERING COLITIS-ASSOCIATED CANCER

Abstract Background The colonic stem cell niche is established by a gradient of WNT, R-spondin, BMP factors and their antagonists along the colonic epithelial vertical axis. Telocytes (TCFoxL1+) are mesenchymal cell forming a 3D hub underneath the epithelium, identified as an important source of niche factors. Specifically, they express non-canonical (nc) WNT factors and are the richest source of BMPs. Disruption of the BMPs gradient has been shown to be related to the development of several gastrointestinal diseases like Inflammatory Bowel Diseases (IBD). Such chronic inflammation drives the onset of Colitis-Associated Cancer (CAC) in about 60% of IBD patients. We previously showed that following a chronic inflammatory stress, 50% of the KO mouse for the BMP receptor 1a in colon telocytes (Bmpr1a△FoxL1+) presented malignant epithelial transformations. These cancer-like regions showed an aberrant epithelial b-catenin localization and an enlargement of the double positive α-SMA+/Vimentin+ mesenchymal population. Purpose: Loss of BMP signaling in TCFoxL1+ affects the mesenchymal-epithelial crosstalk and makes the colonic epithelium vulnerable to injuries, promoting/perpetuating inflammation fostering CAC onset. Method Following a DSS-based chronic inflammatory challenge in mutant and control mice, TCFoxL1+ ultrastructure was analyzed using transmission electron microscopy. Expression levels of members of the WNT-BMP axis (BMPs, WNTs and associated antagonists) were evaluated by qPCR in tumor-like areas and adjacent tissue. YAP cellular localization was evaluated by immunofluorescence in colon after chronic DSS challenge in Bmpr1a△FoxL1+ mice and controls. To differentiate cancer-associated fibroblasts (CAFs) subtypes, myCAF (myofibroblastic) and iCAF (inflammatory), in tumor-like region and adjacent tissue, we used co-staining against gp38, ICAM, Tagln and αSMA. Result(s) Following a chronic DSS-challenge, electron microscopy analysis demonstrated that TCFoxL1+ in the control mice exhibited a shortening and erosion in their telopodes (Tp). TCFoxL1+ in Bmpr1a△FoxL1+ mice tumor-like regions presented an expanded endoplasmic reticulum with fragmented and dilated Tp. A significant increase in BMP 4, 5 and 7 and in Wnt5 (nc) was detected in Bmpr1a△FoxL1+ mice compared to controls. Confocal analysis revealed a strong nuclear accumulation of YAP in cancer-like regions in mutant mice compared to controls. Finally, tumour-like regions presented an heterogeneous distribution of iCAF and myCAF compared to controls. Conclusion(s) These results exposed that the disruption of TCFoxL1+ associated BMP signaling disturbs the WNT-BMP gradient essential for the optimal maintenance of the SC niche and thus impacting epithelial regeneration when under stress. Thus, defective TCFoxL1+ assume a key role in the poor regeneration process of the epithelium which in the end promotes the development and progression of CAC. Please acknowledge all funding agencies by checking the applicable boxes below CIHR Disclosure of Interest None Declared

PIEZO1 activates non-canonical EGFR internalization and signaling.

EGFR-ERK signaling controls cell cycle progression during development, homeostasis, and disease. Both the soluble extracellular ligand, EGF, and mechanical inputs like matrix stiffness, cell adhesion, or stretch can activate EGFR-ERK signaling. However, the molecules transducing mechanical forces into EGFR signaling remain unidentified. We previously found that stretch promotes mitosis by mechanically activating the ion channel Piezo1 to trigger ERK signaling. Here, we show that Piezo1 provides the missing link to mechanical EGFR-ERK activation. Both EGF or the synthetic Piezo1 agonist Yoda1 triggered EGFR internalization and ERK activation, established markers of EGFR signaling. However, while EGF stimulation requires canonical EGFR tyrosine autophosphorylation, activation by Piezo1 requires non-canonical serine EGFR phosphorylation by a Src-p38 kinase axis. Additionally, Yoda1 stimulation of Piezo1 promoted cell cycle re-entry and proliferation via long-term nuclear accumulation of ERK, AP-1 (FOS and JUN), and YAP, typical of regenerative and malignant signaling. Our results suggest two modes of EGFR signaling: basal EGF-dependent signaling, requiring tyrosine autophosphorylation, and mechanically activated EGFR signaling, that results in sustained proliferation, critical to repair and regeneration, and cancer growth.

Suppression of YAP safeguards human naïve pluripotency.

Propagation of human naïve pluripotent stem cells (nPSCs) relies on the inhibition of MEK/ERK signalling. However, MEK/ERK inhibition also promotes differentiation into trophectoderm (TE). Therefore, robust self-renewal requires suppression of TE fate. Tankyrase inhibition using XAV939 has been shown to stabilise human nPSCs and is implicated in TE suppression. Here, we dissect the mechanism of this effect. Tankyrase inhibition is known to block canonical Wnt/β-catenin signalling. However, we show that nPSCs depleted of β-catenin remain dependent on XAV939. Rather than inhibiting Wnt, we found that XAV939 prevents TE induction by reducing activation of YAP, a co-factor of TE-inducing TEAD transcription factors. Tankyrase inhibition stabilises angiomotin, which limits nuclear accumulation of YAP. Upon deletion of angiomotin-family members AMOT and AMOTL2, nuclear YAP increases and XAV939 fails to prevent TE induction. Expression of constitutively active YAP similarly precipitates TE differentiation. Conversely, nPSCs lacking YAP1 or its paralog TAZ (WWTR1) resist TE differentiation and self-renewal efficiently without XAV939. These findings explain the distinct requirement for tankyrase inhibition in human but not in mouse nPSCs and highlight the pivotal role of YAP activity in human naïve pluripotency and TE differentiation. This article has an associated 'The people behind the papers' interview.

Inhibition of 14-3-3ε by K50 acetylation activates YAP1 to promote cholangiocarcinoma growth

14-3-3 proteins are ubiquitous adapters combining with phosphorylated serine/threonine motifs to regulate multiple cellular processes. As a negative regulator, 14-3-3 proteins could sequester the phosphorylated YAP1 in cytoplasm to inhibit its activity. In this study, we identified the K50 acetylation (K50ac) of 14-3-3ε protein and investigated its roles and mechanism in cholangiocarcinoma progression. The NAD (+)-dependent protein deacetylases inhibitor, NAM treatment significantly up-regulated the K50ac of 14-3-3ε. K50R mutation resulted in the decrease of K50ac of 14-3-3ε. The K50ac of 14-3-3ε was reversibly mediated by PCAF acetyltransferase and sirt1 deacetylases. K50ac had no obvious effect on the protein stability of 14-3-3ε, but inhibited the combination of 14-3-3ε with phosphorylated YAP1, which resulted in the activation of YAP1 in cholangiocarcinoma. K50R significantly decreased cholangiocarcinoma cell proliferation in vitro and the growth of tumor xenograft in vivo compared with WT (wild type) 14-3-3ε. The level of K50ac were higher in cholangiocarcinoma tissues accompanied by the accumulation of YAP1 in nuclear than para-carcinoma tissues. Our study revealed the underlying mechanism of K50ac of 14-3-3ε and its roles in cholangiocarcinoma, providing a potential targeting for cholangiocarcinoma therapy.

Salt-inducible kinase 1 deficiency promotes vascular remodeling in pulmonary arterial hypertension via enhancement of yes-associated protein-mediated proliferation

Pulmonary arterial remodeling at an early stage, including excessive proliferation and migration of smooth muscle cells, is a hallmark of pulmonary arterial hypertension (PAH). Salt-inducible kinases (SIKs) have been increasingly reported to play a key role in smooth muscle cell proliferation and phenotype switching, which may be associated with arterial remodeling. However, the potential effects of SIK1 in PAH and the underlying mechanisms have not been explored. The aim of this study was to determine whether reduced expression or inactivation of SIK1 is associated with pulmonary arterial remodeling in PAH and to elucidate whether it is related to the Hippo/Yes-associated protein (YAP) pathway. Using mouse models of PAH and hypoxia-stimulated hPASMCs, we observed that SIK1 expression was robustly reduced in lung tissues of PAH mice and hPASMCs cultured under hypoxia. In hypoxia-induced PAH mice, pharmacological SIK inhibition or AAV9-mediated specific smooth muscle SIK1 knockdown strongly aggravated pathological changes caused by hypoxia, including right ventricular hypertrophy and small pulmonary arterial remodeling. Meanwhile, in hypoxia-stimulated hPASMCs, SIK1 knockdown or inhibition promoted proliferation and migration under hypoxia, accompanied by decreased phosphorylation and increased nuclear accumulation of YAP, while SIK1 overexpression inhibited hypoxia-induced proliferation, migration and nuclear translocation of YAP in hPASMCs. YAP knockdown attenuated the increase in cell proliferation induced by HG-9-91-01 treatment or SIK1 siRNA transfection under hypoxia in hPASMCs. Here, we identified SIK1 as an antiproliferative factor in hypoxia-induced pulmonary arterial remodeling via YAP-mediated mechanisms. These results show that targeting SIK1 may be a promising therapeutic strategy for the treatment of PAH.

Abstract 5364: The TEAD autopalmitoylation inhibitor VT3989 improves efficacy and increases durability of efficacy of osimertinib in preclinical EGFR mutant tumor models

Abstract The Hippo-YAP/TAZ pathway is involved in the regulation of cell proliferation, survival, and cell migration. The major effectors of the Hippo pathway are the TEAD transcription factors, which become transcriptionally activated upon YAP/TAZ binding. Genetic alterations of pathway components have been reported in a variety of human malignancies, resulting in YAP/TAZ constitutive nuclear localization and TEAD activation. The TEAD transcription factors have been shown to autopalmitoylate, and palmitoylation is required for its interaction with YAP/TAZ and hence activation of transcriptional activity. We have identified potent, selective, and orally available small molecule compounds that directly interact with TEAD and block its autopalmitoylation (Tang et al, 2021, Mol Cancer Ther. 20(6):986-998). These TEAD inhibitors disrupt YAP/TAZ-TEAD protein interaction, suppress TEAD transcriptional activity, and selectively block proliferation of NF2-deficient mesothelioma in vitro and inhibit NF2 mutant xenograft tumor growth in vivo. Nuclear YAP accumulation and functional requirement have also been linked to resistance to targeted therapies and cancer relapse in BRAF-mutant, KRAS-mutant, EGFR-mutant, and ALK-rearranged non-small cell lung cancers (NSCLC). Hence, as TEAD transcription factors are the main drivers for YAP/TAZ recruitment to chromatin, we evaluated the effect of our TEAD inhibitor VT3989, currently in clinical testing, on emerging drug tolerant persistent cancer cells and drug resistance during EGFR-tyrosine kinase inhibitor osimertinib treatment in combination studies. In in vitro cell proliferation assays, VT3989 showed strong to very strong synergy in combination with osimertinib in several EGFR mutant NSCLC cell lines tested. In vivo, we observed strong VT3989 and osimertinib combination effect in NCI-H1975 and HCC827 cell line-derived xenograft models. We also tested the combination in several EGFR mutant NSCLC patient-derived xenograft models and demonstrated that the addition of VT3989 enhanced the efficacy of osimertinib and delayed tumor regrowth compared to osimertinib alone. Citation Format: Tracy T. Tang, Leonard Post. The TEAD autopalmitoylation inhibitor VT3989 improves efficacy and increases durability of efficacy of osimertinib in preclinical EGFR mutant tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5364.

Fusion protein-driven IGF-IR/PI3K/AKT signals deregulate Hippo pathway promoting oncogenic cooperation of YAP1 and FUS-DDIT3 in myxoid liposarcoma

Myxoid liposarcoma (MLS) represents a common subtype of liposarcoma molecularly characterized by a recurrent chromosomal translocation that generates a chimeric FUS-DDIT3 fusion gene. The FUS-DDIT3 oncoprotein has been shown to be crucial in MLS pathogenesis. Acting as a transcriptional dysregulator, FUS-DDIT3 stimulates proliferation and interferes with adipogenic differentiation. As the fusion protein represents a therapeutically challenging target, a profound understanding of MLS biology is elementary to uncover FUS-DDIT3-dependent molecular vulnerabilities. Recently, a specific reliance on the Hippo pathway effector and transcriptional co-regulator YAP1 was detected in MLS; however, details on the molecular mechanism of FUS-DDIT3-dependent YAP1 activation, and YAP1´s precise mode of action remain unclear. In elaborate in vitro studies, employing RNA interference-based approaches, small-molecule inhibitors, and stimulation experiments with IGF-II, we show that FUS-DDIT3-driven IGF-IR/PI3K/AKT signaling promotes stability and nuclear accumulation of YAP1 via deregulation of the Hippo pathway. Co-immunoprecipitation and proximity ligation assays revealed nuclear co-localization of FUS-DDIT3 and YAP1/TEAD in FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines. Transcriptome sequencing of MLS cells demonstrated that FUS-DDIT3 and YAP1 co-regulate oncogenic gene signatures related to proliferation, cell cycle progression, apoptosis, and adipogenesis. In adipogenic differentiation assays, we show that YAP1 critically contributes to FUS-DDIT3-mediated adipogenic differentiation arrest. Taken together, our study provides mechanistic insights into a complex FUS-DDIT3-driven network involving IGF-IR/PI3K/AKT signals acting on Hippo/YAP1, and uncovers substantial cooperative effects of YAP1 and FUS-DDIT3 in the pathogenesis of MLS.