YAP Nuclear Localization Research Articles

Abstract 13987: RBX1-CRLs Are Essential for Perinatal Heart Development by Regulating Hippo-YAP Signaling

Defects in cardiac development can lead to congenital heart diseases (CHD) or predispose the heart to cardiomyopathies in adult life. The molecular mechanisms that regulate this process remain elusive. Cullin-RING E3 ligases (CRLs) are multi-subunit complexes that are critical to maintain protein homeostasis by targeting a variety of protein substrates for proteasomal degradation. In the CRLs, RBX1 serves as the catalytic subunit that promotes the transfer of ubiquitin to the target protein. Previous animal studies uncovered a crucial role for RBX1 in development. However, its importance in the developing heart has not been explored. Here, we report for the first time the indispensable role of RBX1 in perinatal cardiac development. We created mice with cardiac-specific deletion of RBX1 driven by αMHC Cre . Compared with littermate control mice, RBX1-deficient mouse embryos exhibited no gross morphological abnormalities at E12.5 and E14.5, displayed peripheral edema at E16.5 indicative of heart failure, and eventually died around E18.5. Histological analysis revealed myocardial hypoplasia and a substantially thinner compact layer in E16.5 mutant hearts. This event was preceded by a remarkable decrease in cardiomyocyte proliferation and diminished nuclear localization of YAP, a powerful regulator of cardiomyocyte proliferation, at E14.5. In cultured cardiomyocytes, depletion of RBX1 inhibited proliferation and induced profound transcriptomic changes like the upregulation of cardiomyopathy-related genes. Mechanistically, RBX1 deficiency destabilized multiple CRLs and resulted in the accumulation of Hippo kinases and adaptor proteins such as SAV1, LATS1/2, and MOB1 leading to the inactivation of YAP by phosphorylation. We further demonstrated that RBX1 degrades the Hippo adaptors SAV1 and MOB1. Collectively, we propose that the fine-tuned, RBX1-mediated removal of Hippo components serves as a critical checkpoint to activate YAP signaling ensuring cardiac ventricular chamber development and morphogenesis.

GALNT12 promotes fibrosarcoma growth by accelerating YAP1 nuclear localization.

Fibrosarcoma is a highly malignant type of soft tissue sarcoma that currently lacks effective treatment options. Polypeptide N-acetylgalactosaminyltransferase 12 (GALNT12) belongs to the uridine diphosphate N-acetylgalactosamine gene family, which is involved in numerous biological processes of diseases, such as tumor progression. Its upregulated expression is closely associated with the development of colorectal cancer. However, research on the role of GALNT12 in fibrosarcoma is currently limited. The present study aimed to assess the expression and biological function of GALNT12 in fibrosarcoma. Patient data and tissue samples were collected and public datasets were obtained from the Gene Expression Omnibus (GSE24369 and GSE21124). Immunofluorescence assays were performed to observe the cellular localization of GALNT12. GALNT12 expression was measured using reverse transcription-quantitative PCR, western blotting and immunohistochemistry. Small interfering RNAs were constructed to knock down GALNT12 expression in HT-1080 cells. Cell Counting Kit-8 and EdU assays were used to assess fibrosarcoma cell proliferation. Wound healing and Transwell assays were used to detect migration. Gene set enrichment analysis was performed to identify key pathways. Paired and unpaired Student's t-test, Fisher's exact test and one-way ANOVA (followed by Tukey's Honest Significant Difference test) were used to analyze the data. It was demonstrated that GALNT12 expression was upregulated in both fibrosarcoma cell lines and tissue samples and predicted poor patient prognosis. In vitro experiments demonstrated that high GALNT12 expression levels significantly increased HT-1080 cell proliferation and migration. Furthermore, it was demonstrated that high GALNT12 expression levels were closely associated with the yes1 associated transcriptional regulator (YAP1) signaling pathway. Knockdown of GALNT12 inhibited YAP1 nuclear translocation, which affected activation of key downstream genes including AMOTL2, BIRC5 and CYR61. Therefore, the present study demonstrated that GALNT12 promoted fibrosarcoma progression. GALNT12 could be a potential biomarker for this disease and may potentially provide new ideas for targeted therapy of fibrosarcoma in the future.

Differential Sensitivity to Ionizing Radiation in Gemcitabine-Resistant and Paclitaxel-Resistant Pancreatic Cancer Cells

IntroductionChemoresistance remains a major challenge in treating pancreatic ductal adenocarcinoma (PDAC). While chemoradiation has proven effective in other tumor types, such as head-and-neck squamous cell carcinoma, its role in PDAC and its impact on acquired chemoresistance have yet to be fully explored. In this study, we investigated the sensitivity of gemcitabine- and paclitaxel-resistant PDAC cells to ionizing radiation (IR) and their underlying mechanisms. Materials and MethodsGemcitabine-resistant (GR) and paclitaxel-resistant (PR) clones were generated from PANC-1, PATU-T and SUIT2-007 pancreatic cancer cell lines. Cell survival after radiation was assessed using clonogenic assay, SRB assay, apoptosis and spheroid growth by bioluminescence while radiation-induced DNA damage was evaluated with Western blot, XL-PCR, ROS production and immunofluorescence. Autophagy and modulation of Hippo pathway were investigated using proteomics, Western blot, immunofluorescence and RT-qPCR. ResultsIn both 2D and 3D settings, PR cells were more sensitive to IR and showed decreased β-globin amplification indicating more DNA damage accumulation as compared to GR or wild-type (WT) cells after 24 hours. Proteomics analysis of PATU-T PR revealed that the protein MST4, a kinase involved in autophagy and Hippo signaling pathway, was highly downregulated. Differential association was found between autophagy and radiation treatment depending on the cell model. Interestingly, increased YAP nuclear localization and downstream Hippo pathway target gene expression were observed in in SUIT2-007 PR cells in response to IR, as compared to WT and GR. ConclusionThis is the first study investigating the potential of IR in targeting PDAC cells with acquired chemoresistance. Our results demonstrate that PR cells exhibit enhanced sensitivity to IR due to greater accumulation of DNA damage. Additionally, depending on the specific cellular context, radiation-induced modulation of autophagy and the Hippo pathway emerged as potential underlying mechanisms, and these findings have the potential to inform personalized treatment strategies for patients with acquired chemoresistance.

YAP1 Expression in HR+HER2- Breast Cancer: 21-Gene Recurrence Score Analysis and Public Dataset Validation.

YAP1, an oncogene in numerous cancers, is a downstream transcription factor of the Hippo pathway. This study focuses on its relationship with the Oncotype Dx (ODX) test risk score (RS) in patients with hormone-receptor-positive, HER2-negative (HR+HER2-) breast cancer. We retrospectively analyzed 401 HR+HER2- breast cancer patients from Gangnam Severance Hospital who underwent ODX tests (May 2014-April 2020). YAP1 nuclear localization was evaluated via immunohistochemical staining and its clinical correlation with clinicopathological parameters, including RS, was analyzed. Public datasets TCGA-BRCA and METABRIC validated clinical outcomes. YAP1 expression negatively correlated with ODX RS (OR 0.373, p = 0.002). Elevated YAP1 mRNA levels corresponded to better clinical outcomes, specifically in ER-positive patients, with significant results in METABRIC and TCGA-BRCA datasets (p < 0.0001 OS in METABRIC, p = 0.00085 RFS in METABRIC, p = 0.040 DFS in TCGA-BRCA). In subsets with varying ESR1 mRNA expression and pronounced YAP1 expression, superior survival outcomes were consistently observed. YAP1 may be a valuable prognostic marker and potential therapeutic target in HR+HER2- breast cancer patients.

The laminin-keratin link shields the nucleus from mechanical deformation and signalling.

The mechanical properties of the extracellular matrix dictate tissue behaviour. In epithelial tissues, laminin is a very abundant extracellular matrix component and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111-unlike fibronectin or collagen I-impairs cell response to substrate rigidity and YAP nuclear localization. Blocking the laminin-specific integrin β4 increases nuclear YAP ratios in a rigidity-dependent manner without affecting the cell forces or focal adhesions. By combining mechanical perturbations and mathematical modelling, we show that β4 integrins establish a mechanical linkage between the substrate and keratin cytoskeleton, which stiffens the network and shields the nucleus from actomyosin-mediated mechanical deformation. In turn, this affects the nuclear YAP mechanoresponses, chromatin methylation and cell invasion in three dimensions. Our results demonstrate a mechanism by which tissues can regulate their sensitivity to mechanical signals.

YAP Activation Is Associated with a Worse Prognosis of Poorly Cohesive Gastric Cancer.

Poorly cohesive (PC) gastric cancer (GC) is extremely aggressive in progression, and there is an urgent need to identify the molecular pathways involved. We hypothesized the essential role of the RhoA-YAP axis in these mechanisms. The present observational multicenter retrospective study included 133 patients with PC GC treated at two dedicated European surgical centers between 2004 and 2014. YAP nuclear localization was measured by immunohistochemical (IHC) analysis of tissue biopsies. The complete absence of nuclear reactivity was coded as negative expression; we considered "any positive" as low nuclear expression (>0% but <10% of cells) and high nuclear expression (≥10% of cells). Women represented about half of the present series (52%), and the median age was 64 years (p25-p75 range: 53-75). Neoadjuvant and adjuvant treatments were administered to 10% and 54% of the cases, respectively. Extended systemic lymphadenectomy (D2) was the most common (54%). In nearly all cases, the number of retrieved nodes was ≥15, i.e., adequate for tumor staging (94%). An R0 resection was achieved in 80% of the cases. Most patients were pathological T stage 3 and 4 (pT3/pT4 = 79.0%) and pathological N stage 2, 3a, and 3b (pN2/pN3a/pN3b = 47.0%) at the pathological examination. Twenty patients (15%) presented distant metastases. Five-year overall survival (OS) was significantly higher (p = 0.029) in patients with negative YAP (46%, 95% CI 31.1-60.0%) than in the other patients (27%, 17.5-38.1%). Moreover, when controlling for sex, age, pT, pN, and percentage of signet ring cells in the multivariable analysis, YAP expression was a significant predictor of OS (HR 2.03, 95% CI: 1.18-3.51, p = 0.011). Our results provide new insights into the role of the YAP signaling cascade, as its activation was associated with a worse prognosis in PC GC.

The role of the FSGS disease gene product and nuclear pore protein NUP205 in regulating nuclear localization and activity of transcriptional regulators YAP and TAZ.

Mutations in genes encoding nuclear pore proteins (NUPs) lead to the development of steroid-resistant nephrotic syndrome and focal segmental glomerulosclerosis (FSGS). However, the precise molecular mechanisms by which NUP dysfunction contributes to podocyte injury preceding FSGS remain unclear. The tightly regulated activity of Yes-associated protein (YAP) and WW-domain-containing transcription regulator 1 (TAZ), the transcriptional effectors of the Hippo pathway, is crucial for podocytes and the maintenance of the glomerular filter. In this study, we investigate the impact of NUPs on the regulation of YAP/TAZ nuclear import and activity in podocytes. In unbiased interactome studies using quantitative label-free mass spectrometry, we identify the FSGS disease gene products NUP107, NUP133, NUP205, and Exportin-5 (XPO5) as components of YAP and TAZ protein complexes in podocytes. Moreover, we demonstrate that NUP205 is essential for YAP/TAZ nuclear import. Consistently, both the nuclear interaction of YAP/TAZ with TEA domain transcription factor 1 and their transcriptional activity were dependent on NUP205 expression. Additionally, we elucidate a regulatory feedback mechanism whereby YAP activity is modulated in response to TAZ-mediated NUP205 expression. In conclusion, this study establishes a connection between the FSGS disease protein NUP205 and the activity of the transcriptional regulators and Hippo effectors YAP and TAZ and it proposes a potential pathological role of YAP/TAZ dysregulation in podocytes of patients with pathogenic NUP205 variants.

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.

Daughter cells inherit YAP localization from mother cells in early preimplantation embryos.

The first stage of cell differentiation during mouse development is the differentiation into the trophectoderm and inner cell mass, which occurs during the 8-32-cell stages of preimplantation embryos. This differentiation is regulated by the Hippo signaling pathway. At the 32-cell stage, embryos establish a position-dependent distribution of the Hippo pathway coactivator, Yes-associated protein 1 (YAP, encoded by Yap1). The outer and inner cells showed nuclear and cytoplasmic localization of YAP, respectively. However, the process by which embryos establish position-dependent YAP localization remains elusive. Here, we established a YAP-reporter mouse line, Yap1mScarlet , and examined YAP-mScarlet protein dynamics during the 8-32-cell stages using live imaging. During mitosis, YAP-mScarlet diffused throughout the cells. YAP-mScarlet dynamics in daughter cells varied depending on the cell division patterns. YAP-mScarlet localization in daughter cells at the completion of cell division coincided with that in mother cells. Experimental manipulation of YAP-mScarlet localization in mother cells also altered its localization in daughter cells upon completion of cell division. In daughter cells, YAP-mScarlet localization gradually changed to the final pattern. In some divisions during the 8-16-cell stages, the cytoplasmic YAP-mScarlet localization preceded cell internalization. These results suggest that cell position is not a primary determinant of YAP localization and that the Hippo signaling status of the mother cell is inherited by the daughter cells, which likely contributes to the stabilization of the cell fate specification process beyond cell division.

Mechano-responsive hydrogel for direct stem cell manufacturing to therapy.

Bone marrow-derived mesenchymal stem cell (MSC) is one of the most actively studied cell types due to its regenerative potential and immunomodulatory properties. Conventional cell expansion methods using 2D tissue culture plates and 2.5D microcarriers in bioreactors can generate large cell numbers, but they compromise stem cell potency and lack mechanical preconditioning to prepare MSC for physiological loading expected in vivo. To overcome these challenges, in this work, we describe a 3D dynamic hydrogel using magneto-stimulation for direct MSC manufacturing to therapy. With our technology, we found that dynamic mechanical stimulation (DMS) enhanced matrix-integrin β1 interactions which induced MSCs spreading and proliferation. In addition, DMS could modulate MSC biofunctions including directing MSC differentiation into specific lineages and boosting paracrine activities (e.g., growth factor secretion) through YAP nuclear localization and FAK-ERK pathway. With our magnetic hydrogel, complex procedures from MSC manufacturing to final clinical use, can be integrated into one single platform, and we believe this 'all-in-one' technology could offer a paradigm shift to existing standards in MSC therapy.

P150 The Triptryium wilfordii derivative celastrol has anti-fibrotic effects in systemic sclerosis

Abstract Background/Aims Scleroderma (systemic sclerosis, SSc) is an autoimmune inflammatory-fibrosis syndrome that damages the skin and internal organs and is considered a prototypic complex fibrotic disease. In SSc, persistently activated myofibroblasts are maintained by an excessive, autocrine mechanotranductive/pro-adhesive signaling loop. Drugs targeting this pathway are therefore of likely therapeutic benefit in SSc. The mechanosensitive transcriptional co-activator, yes activated protein-1 (YAP1), is activated in SSc fibroblasts. The terpenoid plant-derivative celastrol has recently been identified as a YAP inhibitor; however, if celastrol can alleviate SSc fibrosis, and its underlying mechanism of action, is as yet unclear. Methods We cultured age-, sex-, and site-matched human dermal fibroblasts sampled from healthy individuals and patients with early onset (&amp;lt;18 month after first diagnosis) diffuse cutaneous scleroderma (systemic sclerosis, dcSSc) and treated with or without transforming growth factor β1 (TGFβ1, 4ng/ml) in the presence or absence of celastrol (500nM). We also subjected C57BL6J mice to the inflammatory-driven bleomycin-induced model of skin SSc, in the presence or absence of celastrol (28days, every day, 0.1U bleomycin, 1mg/kg celastrol). RNA expression was assessed using RNAseq and real-time polymerase chain reaction analysis. Protein expression was determined using Western blot and enzyme-linked immunosorbent assay. Fibrosis was monitored by hematoxylin and eosin and trichrome staining of tissue sections, and by using indirect immunofluorescence analysis with anti-alpha-smooth muscle actin (SMA) antibodies to detect myofibroblasts. Results In dermal fibroblasts, celastrol impaired the ability of TGFβ1 to induce an SSc-like pattern of gene expression, including the induction of cellular communication network factor 2 (CCN2), collagen I and TGFβ1 protein (N = 6, all p &amp;lt; 0.01). Celastrol also alleviated the persistent fibrotic phenotype of dermal fibroblasts cultured from lesions of SSc patients, including the overproduction of CCN2 and collagen (N = 3, p &amp;lt; 0.05). Finally, in the bleomycin-induced model of SSc dermatopathology, celastrol inhibited skin thickness/collagen deposition in vivo and blocked nuclear localization of YAP in SMA-positive myofibroblasts (all N = 6, p &amp;lt; 0.01). Conclusion Our data are consistent with the hypothesis that compounds, such as celastrol, that antagonize the YAP pathway warrant further consideration as potential treatments for SSc skin fibrosis. Disclosure A. Leask: Grants/research support; CIHR, NSERC, Arthritis Society of Canada. R.J. Stratton: None. S. Xu: None. P. Chitturi: None.

Abstract 638: EPB-53 prevents NASH-HCC transition via regulation of SPHK1-S1P-HIPPO signaling and immune modulation in a murine model

Abstract Background: Non-alcoholic fatty liver disease (NAFLD) encompasses a broad spectrum of disorders, ranging from non-progressive bland steatosis to non-alcoholic hepatitis (NASH), cirrhosis, and malignant transformation into hepatocellular carcinoma (HCC). Fibroblast growth factor 21 (FGF21) exerts a key role in lipid homeostasis and play a promising therapeutic candidate for the treatment of obesity, diabetes, and NASH. Small molecules like EPB-53 that are activators of heme-regulated inhibitor (HRI) of eukaryotic initiation factor 2α (eIF2α) kinase have been synthesized to induce FGF21. Previous study has shown that oral administration of EPB-53 alleviates NASH through increased FGF21 to prevent hepatic steatosis. However, it is unknown whether EPB-53 can prevent NASH-HCC transition. Aim: To investigate the effect of EPB-53 on NASH-HCC transition and the therapeutic mechanism, focusing on the lipid metabolic reprogramming and tumor immune microenvironment (TIM). Methods: NASH-HCC transition model was established in two-week-old male FGF21 knockout (KO) mice and wild type controls (C57BL/6J mice) by diethylnitrosamine (DEN) administration at 40 mg/kg body weight (i.p.). Four weeks after DEN administration, the mice were fed with western-style high fat diet (WSHFD, 60% kcal% fat and 19% kcal% fructose) for 3 months, and gavaged daily with either vehicle, 2% w/v, (2-hydroxypropyl)-β-cyclodextrin or EPB-53 (300 mg/kg/day) in vehicle for 4 weeks before sacrifice. The tumor incidence, lipid metabolism, and carcinogenesis signaling pathways were determined. Mass cytometry (CyTOF) analysis was performed to detect the cell subpopulations in the NASH microenvironment and NASH-HCC TIM. Results: Severe NASH with tumor cell detection, aberrant lipid components and HIPPO-YAP signaling were found in liver tissues of FGF21-KO mice compared to the WT controls. The upregulated sphingosine Kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) in liver subsequently induced YAP dephosphorylation and nuclear localization. EPB-53 treatment prevents NASH-HCC transition evident as decreased HCC incidence in WT mice but not in FGFKO mice. Continuously increased FGF21 production in WT mice contributes to down-regulation of SPHK1-S1P-HIPPO-YAP signaling. Interestingly, EPB-53 treatment can alleviate the compromised function of macrophages/Kupffer cells which render a TIM in the NASH-HCC mice. Conclusion: 1) Lack of FGF21 played a key role in lipid metabolic disorder and SPHK1-S1P-HIPPO signaling to promote NASH-HCC transition; 2) Lack of FGF21 rendered a TIM in NASH liver contributing to the NASH-HCC transition; 3) EPB-53 treatment can not only alleviate NASH, but also prevent NASH-HCC transition via lipid metabolic reprogramming, down-regulation of the SPHK1-S1P-HIPPO axis, and modulation of TIM. Citation Format: Min Tan, Eugenia Pujol, Santiago Vazquez, Jingwen Zhang, Jun Yan, Robert C. Martin, Manuel Vazquez-Carrera, Yan Li. EPB-53 prevents NASH-HCC transition via regulation of SPHK1-S1P-HIPPO signaling and immune modulation in a murine model [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 638.

Angelicin impedes the progression of glioblastoma via inactivation of YAP signaling pathway

Glioblastoma (GBM) is a human malignant tumor with low survival and high recurrence rate. Angelicin, an active furanocoumarin compound, has been reported to possess potential antitumor activity towards various malignancies. However, the effect of angelicin on GBM cells and its mechanism are still unclear. In this study, we found that angelicin inhibited the proliferation of GBM by inducing the cell cycle arrested in G1 phase and suppressed the migration of GBM cells in vitro. Mechanically, we found that angelicin downregulated the expression of YAP and decreased the nuclear localization of YAP, and suppressed the expression of β-catenin. Furthermore, overexpression of YAP partially restored the inhibitory effect of angelicin on GBM cells in vitro. Finally, we found that angelicin could inhibit the growth of tumor and reduce the expression of YAP in the subcutaneous xenograft model of GBM in nude mice and the syngeneic intracranial orthotopic model of GBM in C57BL/6 mice. Taken together, our results suggest that the natural product angelicin exerts its anticancer effects on GBM via YAP signaling pathway, and is expected to be a promising compound for the treatment of GBM.

Nuclear stiffness regulates cellular senescence via YAP dependent mechanotransduction.

The cellular nucleus is a critical mechanosensing organelle subjected to and integrating cytoskeletal forces for biochemical responses. Changes in nuclear stiffness influence this mechanotransduction, and pathologies involving accelerated cellular senescence such as Hutchison Gilford Progeria Syndrome (HGPS) have previously been shown to increase nuclear stiffness, but if this plays a causal role in cellular senescence is unclear. Here, we demonstrate that changes in nuclear stiffness directly influence accelerated or delayed cellular senescence through YAP-mediated modulation of human telomerase (hTERT) expression. To study how nuclear stiffness regulates hTERT expression, first, we established that the nuclear stiffness increases with culture age, observing that nuclear stiffening in time for progeria cells was approximately 40% faster than in wildtype (WT) human dermal fibroblast cells. hTERT is a downstream protein of YAP, and we hypothesized that nuclear stiffness regulates hTERT expression via YAP dependent mechanotransduction. By modulating nuclear stiffness via lamin A/C expression in WT and progeria cells, we then identified that nuclear stiffness directly influences YAP nuclear localization. Using both nuclear mechanics and chemical compounds to manipulate YAP shuttling, we found that YAP nuclear localization inversely influences the cellular senescence biomarker senescence-associated β-galactosidase (SA β-gal) and that in turn telomerase expression is inversely related to SA β-gal expression. These results identify new strategies for diagnosing aging disorders via nuclear mechanics and suggest possible future strategies that could mechanically correct dysfunctional nuclear mechanics.

Plpp3, a novel regulator of pluripotency exit and endodermal differentiation of mouse embryonic stem cells.

In recent decades the study of the actions of bioactive lipids such as Lysophosphatidic Acid (LPA) and Sphingosine-1-Phosphate (S1P) has importantly increased since they are involved in regulating many processes, including self-renewal of embryonic stem cells, embryo development and cancer. The Phospholipid Phosphatase type 3 (PLPP3) has been shown key player in regulating the balance of these lipids, and in consequence their signaling. Different lines of evidence suggest PLPP3 could play a role in endoderm development, to approach this hypothesis we use mouse embryonic stem cells (ESC) as a model to study Plpp3 function in self-renewal and the transition towards differentiation. We found that lack of PLPP3 mainly affects endoderm formation during suspension embryoid bodies differentiation. PLPP3-deficient ESC strongly decreases the amount of FOXA2 expressing cells and fail to properly downregulate the expression of pluripotency factors when subjected to an endoderm directed differentiation protocol. Impaired endoderm differentiation correlated with a transient reduction in nuclear localization of YAP1. These phenotypes were rescued by transiently restoring the expression of catalytically active hPLPP3. In conclusion, PLPP3 plays a role in downregulating pluripotency-associated factors and in endodermal differentiation. PLPP3 regulates proper lipid/YAP1 signaling required for endodermal differentiation.

Yap governs a lineage-specific neuregulin1 pathway-driven adaptive resistance to RAF kinase inhibitors

BackgroundInactivation of the Hippo pathway promotes Yap nuclear translocation, enabling execution of a transcriptional program that induces tissue growth. Genetic lesions of Hippo intermediates only identify a minority of cancers with illegitimate YAP activation. Yap has been implicated in resistance to targeted therapies, but the mechanisms by which YAP may impact adaptive resistance to MAPK inhibitors are unknown.MethodsWe screened 52 thyroid cancer cell lines for illegitimate nuclear YAP localization by immunofluorescence and fractionation of cell lysates. We engineered a doxycycline (dox)-inducible thyroid-specific mouse model expressing constitutively nuclear YAPS127A, alone or in combination with endogenous expression of either HrasG12V or BrafV600E. We also generated cell lines expressing dox-inducible sh-miR-E-YAP and/or YAPS127A. We used cell viability, invasion assays, immunofluorescence, Western blotting, qRT-PCRs, flow cytometry and cell sorting, high-throughput bulk RNA sequencing and in vivo tumorigenesis to investigate YAP dependency and response of BRAF-mutant cells to vemurafenib.ResultsWe found that 27/52 thyroid cancer cell lines had constitutively aberrant YAP nuclear localization when cultured at high density (NU-YAP), which rendered them dependent on YAP for viability, invasiveness and sensitivity to the YAP-TEAD complex inhibitor verteporfin, whereas cells with confluency-driven nuclear exclusion of YAP (CYT-YAP) were not. Treatment of BRAF-mutant thyroid cancer cells with RAF kinase inhibitors resulted in YAP nuclear translocation and activation of its transcriptional output. Resistance to vemurafenib in BRAF-mutant thyroid cells was driven by YAP-dependent NRG1, HER2 and HER3 activation across all isogenic human and mouse thyroid cell lines tested, which was abrogated by silencing YAP and relieved by pan-HER kinase inhibitors. YAP activation induced analogous changes in BRAF melanoma, but not colorectal cells.ConclusionsYAP activation in thyroid cancer generates a dependency on this transcription factor. YAP governs adaptive resistance to RAF kinase inhibitors and induces a gene expression program in BRAFV600E-mutant cells encompassing effectors in the NRG1 signaling pathway, which play a central role in the insensitivity to MAPK inhibitors in a lineage-dependent manner. HIPPO pathway inactivation serves as a lineage-dependent rheostat controlling the magnitude of the adaptive relief of feedback responses to MAPK inhibitors in BRAF-V600E cancers.

CSIG-25. DIFFERENTIAL YAP ACTIVITY IN HUMAN GLIOBLASTOMA TUMORSPHERES AS A POTENTIAL BIOMARKER

Abstract BACKGROUND Hippo/YAP signaling pathway has emerged as an important driver of GBM. However, the clinical significance and expression correlation of YAP in GBM is still unknown. The purpose of this study is to elucidate the regulatory functions of YAP in response to tumorspheres (TS) in GBM. Material and METHODS In GBM-tumorspheres (TS), mRNA levels of Yap1 and TAZ were determined by RNA-Seq, and nuclear YAP1 expression level and its correlation with tumor aggressiveness were assessed through nucleus cytosolic fractionation, quantitative confocal microscopy, western blot, and TEAD4 reporter assay. Cell proliferation, stemness, and invasive properties were also measured using YAP siRNA and its inhibitor Verteporfin to reveal the effect of YAP on glioma progression. In orthotopic xenograft models, the inhibition of YAP function were tested. RESULTS All GBM-TS exhibited various phosphorylation states of YAP but no significant differences regarding the expression of YAP. Among them, TS15-88 cell line showed relatively low YAP phosphorylation in the quantified immunoblot results. Indeed, TS with reduced YAP phosphorylation was associated with aggressive cancer phenotypes. Predominant nuclear localization of YAP and binding of YAP/TAZ to TEAD in the nucleus was evident in the TS15-88 cells. The YAP knockdown (KD) by siRNA significantly resulted decrease in TS proliferation through WST and ATP assay and values were validated again through YAP inhibitor, Verteporfin (VP). YAP KD attenuated not only the invasion and stemness of TS but also caused a reduction in tumor volume and prolonged the overall survival of mice. CONCLUSION We showed alteration of gene activities by YAP and nuclear localization of YAP/TAZ complex contributes to GBM progression. Moreover, thus tuning of Yap activity results a marked suppression in aggressive phenotypes of cancer. These associations emphasize that the YAP signaling network highly suggests new therapeutic opportunities in diagnosing and treating GBM.

Programmable integrin and N-cadherin adhesive interactions modulate mechanosensing of mesenchymal stem cells by cofilin phosphorylation

During mesenchymal development, the sources of mechanical forces transduced by cells transition over time from predominantly cell-cell interactions to predominantly cell-extracellular matrix (ECM) interactions. Transduction of the associated mechanical signals is critical for development, but how these signals converge to regulate human mesenchymal stem cells (hMSCs) mechanosensing is not fully understood, in part because time-evolving mechanical signals cannot readily be presented in vitro. Here, we established a DNA-driven cell culture platform that could be programmed to present the RGD peptide from fibronectin, mimicking cell-ECM interactions, and the HAVDI peptide from N-cadherin, mimicking cell-cell interactions, through DNA hybridization and toehold-mediated strand displacement reactions. The platform could be programmed to mimic the evolving cell-ECM and cell-cell interactions during mesenchymal development. We applied this platform to reveal that RGD/integrin ligation promoted cofilin phosphorylation, while HAVDI/N-cadherin ligation inhibited cofilin phosphorylation. Cofilin phosphorylation upregulated perinuclear apical actin fibers, which deformed the nucleus and thereby induced YAP nuclear localization in hMSCs, resulting in subsequent osteogenic differentiation. Our programmable culture platform is broadly applicable to the study of dynamic, integrated mechanobiological signals in development, healing, and tissue engineering.

Emerging Role of YAP and the Hippo Pathway in Prostate Cancer.

The Hippo pathway regulates and contributes to several hallmarks of prostate cancer (PCa). Although the elucidation of YAP function in PCa is in its infancy, emerging studies have shed light on the role of aberrant Hippo pathway signaling in PCa development and progression. YAP overexpression and nuclear localization has been linked to poor prognosis and resistance to treatment, highlighting a therapeutic potential that may suggest innovative strategies to treat cancer. This review aimed to summarize available data on the biological function of the dysregulated Hippo pathway in PCa and identify knowledge gaps that need to be addressed for optimizing the development of YAP-targeted treatment strategies in patients likely to benefit.

Ferroptosis Induction and YAP Inhibition as New Therapeutic Targets in Gastrointestinal Stromal Tumors (GISTs).

Simple SummaryDespite targeted therapy with tyrosine kinase inhibitors (TKIs), unresectable or metastatic human GISTs generally relapse under treatment. Thus, alternative therapeutic approaches are needed to overcome GIST resistance. Ferroptosis, a cell death driven by iron-dependent phospholipid peroxidation, is an emerging therapeutic approach in cancers. However, the potential of ferroptosis induction in GISTs remains largely unknown. This study shows that GIST cell lines are highly sensitive to the type II ferroptosis inducer, RSL3. We also provide evidence that inhibition of YAP by verteporfin (VP) promotes ferroptosis in GIST cells while, surprisingly, CA3 mediates ferroptosis independently of YAP inhibition. Finally, we highlight a positive correlation between the highly expressed transferrin receptor protein 1 (TFRC), GISTs with elevated mitotic counts or higher risk, and with YAP expression/activation in human GIST tissue microarrays (TMA). Taken together, our results suggest that induction of ferroptosis and/or modulation of YAP activity offer promising perspectives for GIST treatment.GISTs are sarcomas of the gastrointestinal tract often associated with gain-of-function mutations in KIT or PDGFRA receptor genes. While most GISTs initially respond to tyrosine kinase inhibitors, relapses due to acquired resistance frequently occur. The induction of ferroptosis, an iron-dependent form of non-apoptotic cell death, emerged as a novel therapeutic approach in cancers and remains poorly characterized in GISTs. We studied hallmarks of ferroptosis, i.e., lipid peroxidation, iron and glutathione content, and GPX4 protein expression in imatinib-sensitive (GIST882) and -resistant (GIST48) GIST cell lines. GIST cells were highly sensitive to the induction of ferroptosis by RSL3, which was reversed by liproxstatin and deferoxamine. Lipid peroxidation and ferroptosis were mediated by VP and CA3 in GIST cells through a significant decrease in antioxidant defenses. Moreover, VP, but surprisingly not CA3, inhibited a series of target genes downstream of YAP in GIST cells. The ferroptosis marker TFRC was also investigated by immunohistochemistry in GIST tissue arrays. TFRC expression was observed in all samples. High TFRC expression was positively correlated with high-risk GISTs, elevated mitotic count, and YAP nuclear localization, reflecting YAP activation. This study highlights ferroptosis as a novel cell death mechanism in GISTs, and a potential therapeutic target to overcome resistance to tyrosine kinase inhibitors.