YAP Protein Research Articles

Abstract 3751: Altered prostatic HOX code is associated with decreased YAP1 expression during neuroendocrine prostate cancer development

Abstract Background: Neuroendocrine (NE) phenotype contributes to 25 to 30% androgen deprivation resistant prostate cancer cases. Until now, this aggressive phenotype is untreatable. Therefore, it is urgent to study the molecular mechanism of NE development to reveal therapeutic strategies. Methods: Data were downloaded from CCLE, cBioPortal, and GEO and analyzed by R. RT-qPCR, western-blot, Immunohistochemistry, and Immunofluorescence were used to assess gene expression. Results: Neuroendocrine prostate cancer (NEPCa) develops from prostate adenocarcinoma (AdPCa). We screened and identified the consensus differentially expressed genes across all public NEPCa transcriptome datasets. We found several HOX genes are included. HOXB13 is deceased but HOXB3, HOXD8 are increased in NEPCa. Based on the HOX code (total 39 HOX genes expression) theory, different cell lineages have distinct HOX codes and HOX codes can represent tissue identities, we established 42 HOX codes for 42 cells lineages including prostate through bioinformatics analysis of 1019 cancer cell lines transcriptome. We found NEPCa samples have changed prostatic HOX code. We also found YAP1, a previously characterized oncogene, is decreased in mRNA level in NEPCa than AdPCa. We confirmed the YAP1 protein is not detected in NEPCa human/mouse samples, PDX samples, and cell line samples (NCI-H660). We used YAP1 as a model. We hypothesized that the signaling involved in silencing YAP1 expression can also contribute to NE development. We first confirmed the loss of YAP1 expression in NEPCa cells is under epigenetic control by inducing YAP1 mRNA expression in primary mouse NEPCa cells through epigenetic inhibitors treatments (HDACs/DNA methyltransferase inhibitors). We characterized the YAP1 promoter through data mining chromatin ChIP-seq data and DNA methylation-seq data. We identified and cloned a 3kb DNA fragment across the YAP1 transcription start site as YAP1 regulatory element. We then built a YAP1-promoter-luciferase reporter using this fragment. We also generated a list of transcription factors that can bind the YAP1 regulatory element. We will then confirm the roles of these transcriptional factors in both regulating YAP1 and NE development. We also found YAP1 expression is co-expressed with many HOX genes in human samples. This suggested the potential regulatory function of HOX code to YAP1 expression. Conclusion: We proposed 2 critical events during NE development, changed prostatic HOX code and loss of YAP expression. We identified HOX code change as one of the potential regulatory mechanisms of YAP1. We then proposed to use YAP1 regulatory element as a model system to study the NE development mechanism. Funding: The LSUHS Office of Research SEED awards, NIH R01 CA226285, and Feist-Weiller Cancer Center pre-doc fellowship to SC. Acknowledgment: We acknowledge Feist-Weiller Cancer Center for travel support to SC. Citation Format: Siyuan Cheng, Shu Yang, Yingli Shi, Xiuping Yu. Altered prostatic HOX code is associated with decreased YAP1 expression during neuroendocrine prostate cancer development [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 3751.

YAP plays a protective role in T-2 toxin-induced inhibition of chondrocyte proliferation and matrix degradation

Previous research has shown that T-2 toxin can damage cartilage, resulting in a disease phenotype similar to osteoarthritis. The precise molecular mechanism by which T-2 toxin causes chondrocyte injury, however, is unknown. The purpose of this study was to look into the role of YAP in T-2 toxin-induced rat chondrocyte injury. Based on research results, T-2 toxin decreased the levels of collagen II and PCNA while increasing the expression of matrix metalloproteinase MMP13. These findings supported the T-2 toxin's detrimental effect on chondrocytes. YAP's role in T-2 toxin-induced chondrocyte injury was also investigated. Total YAP and related nuclear proteins were found to decrease as the concentration of T-2 toxin increased. While PYAP expression was not significantly altered in response to T-2 toxin, the PYAP/YAP ratio decreased as the T-2 toxin concentration increased, implying that the HIPPO signaling pathway was activated. Furthermore, the YAP-specific inhibitor Verteporfin was used to investigate the role of YAP in T-2 toxin-induced chondrocyte injury. YAP inhibition increased MMP13 expression while decreasing COL2 and PCNA levels. In summary, the current study found that T-2 toxin decreased the levels of COL2 and PCNA while increasing the expression of MMP13 in chondrocytes after inhibiting YAP, providing a new insight into the mechanism of T-2 toxin-induced cartilage damage.

YAP1 is essential for malignant mesothelioma tumor maintenance

Malignant pleural mesothelioma, a tumor arising from the membrane covering the lungs and the inner side of the ribs, is a cancer in which genetic alterations of genes encoding proteins that act on or are part of the Hippo-YAP1 signaling pathway are frequent. Dysfunctional Hippo signaling may result in aberrant activation of the transcriptional coactivator protein YAP1, which binds to and activates transcription factors of the TEAD family. Recent studies have associated elevated YAP1 protein activity with a poor prognosis of malignant mesothelioma and its resistance to current therapies, but its role in tumor maintenance is unclear. In this study, we investigate the dependence of malignant mesothelioma on YAP1 signaling to maintain fully established tumors in vivo. We show that downregulation of YAP1 in a dysfunctional Hippo genetic background results in the inhibition of YAP1/TEAD-dependent gene expression, the induction of apoptosis, and the inhibition of tumor cell growth in vitro. The conditional downregulation of YAP1 in established tumor xenografts leads to the inhibition of YAP1-dependent gene transcription and eventually tumor regression. This effect is only seen in the YAP1-activated MSTO-211H mesothelioma xenograft model, but not in the Hippo-independent HCT116 colon cancer xenograft model. Our data demonstrate that, in the context of a Hippo pathway mutated background, YAP1 activity alone is enough to maintain the growth of established tumors in vivo, thus validating the concept of inhibiting the activated YAP1-TEAD complex for the treatment of malignant pleural mesothelioma patients.

Mice with Trp53 and Rb1 deficiency in chondrocytes spontaneously develop chondrosarcoma via overactivation of YAP signaling

Chondrosarcoma (CHS) is a rare type of soft sarcoma with increased production of cartilage matrix arising from soft bone tissues. Currently, surgical resection is the primary clinical treatment for chondrosarcoma due to the poor response to radiotherapy and chemotherapy. However, the therapeutic effect is not satisfactory due to the higher local recurrence rate. Thus, management and elucidation of the pathological mechanism of chondrosarcoma remain an ongoing challenge, and the development of effective chondrosarcoma mouse models and treatment options are urgently needed. Here, we generated a new transgenic chondrosarcoma model by double conditional deletions of Trp53 and Rb1 in chondrocyte lineage which spontaneously caused spinal chondrosarcoma and lung metastasis. Bioinformatic analysis of the human soft sarcoma database showed that Trp53 and Rb1 genes had higher mutations, reaching up to approximately 33.5% and 8.7%, respectively. Additionally, Trp53 and Rb1 signatures were decreased in the human and mouse chondrosarcoma tissues. Mechanistically, we found that YAP expression and activity were significantly increased in mouse Col2-Cre;Trp53f/f/Rb1f/f chondrosarcoma tissues compared to the adjacent normal cartilage. Knockdown of YAP in primary chondrosarcoma cells significantly inhibited chondrosarcoma proliferation, invasion, and tumorsphere formation. Chondrocyte lineage ablation of YAP delayed chondrosarcoma progression and lung metastasis in Col2-Cre;Trp53f/f/Rb1f/f mice. Moreover, we found that metformin served as a YAP inhibitor, which bound to the activity area of YAP protein, and inhibited chondrosarcoma cell proliferation, migration, invasion, and progression in vitro and significantly suppressed chondrosarcoma formation in vivo. Collectively, this study identifies the inhibition of YAP may be an effective therapeutic strategy for the treatment of chondrosarcoma.

227-OR: Disruption of Adipocyte YAP Improves Glucose Homeostasis in Mice and Decreases Adipose Tissue Fibrosis

Adipose tissue is a dynamic metabolic organ that is essential for the regulation of whole-body glucose homeostasis. Dysfunctional adipose tissue hypertrophy with obesity is associated with glucose intolerance and insulin resistance underlying type 2 diabetes. Yes-associated protein 1 (YAP) is a transcription cofactor important in the Hippo signaling pathway. However, the role of YAP in adipose tissue and glucose homeostasis is unknown. To understand the role of YAP in vivo under metabolic stress conditions, we assessed how increased weight and insulin resistance impact YAP protein levels. We found that YAP protein levels increase in adipose tissue in humans with type 2 diabetes and mouse models. This suggests that YAP signaling may contribute to adipocyte dysfunction and insulin resistance under metabolic stress conditions. To further investigate the role of YAP in adipose tissue and glucose homeostasis in vivo, we developed adipose tissue-specific YAP knockout (YAP-KO) mice and placed them on either chow or high fat diet (HFD) for 12-14 weeks. On an HFD, adipose tissue YAP-KO mice show improved glucose tolerance compared to controls. Perigonadal fat pad weight was also decreased in knockout animals, with smaller adipocyte size. Adipose tissue fibrosis and gene expression associated with fibrosis was decreased in vivo and in 3T3-L1 cells treated with a YAP inhibitor. Together, these data indicate that YAP is increased in adipose tissue with weight gain and insulin resistance. Disruption of YAP in adipocytes improves glucose tolerance and adipose tissue fibrosis; suggesting that YAP plays an important role in adipose tissue and modulating glucose homeostasis under metabolic stress conditions. Disclosure D.Han: None. R.Aslam: None. T.Ojha: n/a. D.Yuen: Consultant; Fibrocor Therapeutics. C.T.Luk: None. Funding NSERC Discovery Grant (RGPIN-2018-05671) BBDC Gales Family Charitable Foundation Pilot and Feasibility GrantCanadian Institutes of Health Research Project Grant (PJT 168996)

Expression patterns and prognostic relevance of subtype-specific transcription factors in surgically resected small-cell lung cancer: an international multicenter study.

The tissue distribution and prognostic relevance of subtype‐specific proteins (ASCL1, NEUROD1, POU2F3, YAP1) present an evolving area of research in small‐cell lung cancer (SCLC). The expression of subtype‐specific transcription factors and P53 and RB1 proteins were measured by immunohistochemistry (IHC) in 386 surgically resected SCLC samples. Correlations between subtype‐specific proteins and in vitro efficacy of various therapeutic agents were investigated by proteomics and cell viability assays in 26 human SCLC cell lines. Besides SCLC‐A (ASCL1‐dominant), SCLC‐AN (combined ASCL1/NEUROD1), SCLC‐N (NEUROD1‐dominant), and SCLC‐P (POU2F3‐dominant), IHC and cluster analyses identified a quadruple‐negative SCLC subtype (SCLC‐QN). No unique YAP1‐subtype was found. The highest overall survival rates were associated with non‐neuroendocrine subtypes (SCLC‐P and SCLC‐QN) and the lowest with neuroendocrine subtypes (SCLC‐A, SCLC‐N, SCLC‐AN). In univariate analyses, high ASCL1 expression was associated with poor prognosis and high POU2F3 expression with good prognosis. Notably, high ASCL1 expression influenced survival outcomes independently of other variables in a multivariate model. High POU2F3 and YAP1 protein abundances correlated with sensitivity and resistance to standard‐of‐care chemotherapeutics, respectively. Specific correlation patterns were also found between the efficacy of targeted agents and subtype‐specific protein abundances. In conclusion, we investigated the clinicopathological relevance of SCLC molecular subtypes in a large cohort of surgically resected specimens. Differential IHC expression of ASCL1, NEUROD1, and POU2F3 defines SCLC subtypes. No YAP1‐subtype can be distinguished by IHC. High POU2F3 expression is associated with improved survival in a univariate analysis, whereas elevated ASCL1 expression is an independent negative prognosticator. Proteomic and cell viability assays of human SCLC cell lines revealed distinct vulnerability profiles defined by transcription regulators. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

N-Acetyl-l-tryptophan inhibits CCl4-induced hepatic fibrogenesis via regulating TGF-β1/SMAD and Hippo/YAP1 signal

BackgroundAlthough liver fibrosis is a key pathologic process in many liver diseases, therapeutic approaches for inhibiting liver fibrosis are still very limited. N-Acetyl-l-tryptophan (l-NAT) has a hepatoprotective effect via inhibiting the destruction of liver cells, enhancing cell viability and reducing the inflammation. However, the effect of l-NAT on liver fibrosis is not determined. PurposeThe present study investigated the effect of l-NAT on liver fibrosis and explored it potential molecular mechanism. MethodsTo address this concern, this study was carried out via fibrotic mice model induced by CCl4 and many approaches such as various histological staining methods, western blot assay, etc. Resultl-NAT decreased the levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) in fibrotic mice model induced by carbon tetrachloride (CCl4). Histological staining showed that l-NAT ameliorated liver injury and fibrosis, and reduced the expression of α-smooth muscle actin (α-SMA) and Collagen I protein. l-NAT also attenuated apoptosis by down-regulating the level of pro-apoptotic protein Bax and up-regulating that of anti-apoptotic protein Bcl-2. Moreover, l-NAT inhibited the expressions of TGF-β1/SMAD and matrix metalloproteinase 9 (MMP9) proteins, and reversed the expression of YAP1 protein in CCl4-induced liver fibrosis. ConclusionThese results clearly demonstrated that l-NAT attenuated CCl4-induced liver fibrosis in mice, and this protective mechanism might relate to TGF-β1/SMAD and Hippo/YAP1 signaling pathway. Thus, this study provided data basis for the prevention and treatment of liver fibrosis.

Metformin inhibits proliferation and promotes apoptosis of HER-2 positive breast cancer cells possibly through the Hippo-YAP pathway

To investigate the effect of metformin on the proliferation and apoptosis of HER-2-positive breast cancer cell line SKBR3 and explore the possible mechanism of its action. SKBR3 cells were treated with different concentrations (20-120 μmol/L) of metformin, and the changes in cell proliferation and colony formation ability were assessed using CCK-8 assay and crystal violet staining, respectively. Flow cytometry was performed to analyze cell apoptosis and cell cycle changes. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect mRNA expressions of YAP, TAZ, EGFR, CTGF, CYR61, E-cadherin, N-cadherin, vimentin and fibronectin in the treated cells, and the protein expressions of YAP and TAZ were detected using Western blotting; immunofluorescence assay was used to observe YAP/TAZ nuclear translocation in the cells. Metformin treatment significantly inhibited the proliferation of SKBR3 cells (P < 0.05) in a concentration- and time-dependent manner. The results of flow cytometry showed that metformin significantly promoted apoptosis and caused cell cycle arrest at G1 phase in SKBR3 cells. Metformin treatment significantly down-regulated the mRNA expressions of YAP, TAZ, EGFR, CTGF and CYR61, N-cadherin, vimentin and fibronectin (P < 0.05) and up-regulated the expression of E-cadherin (P < 0.05); Western blotting results showed that YAP and TAZ protein expressions were significantly down-regulated in the cells after metformin treatment (P < 0.05). Immunofluorescence assay revealed that metformin treatment caused the concentration of YAP and TAZ in the cytoplasm, and significantly reduced their amount in the cell nucleus. Metformin can inhibit proliferation and promote apoptosis and epithelal-mesenchymal transition of HER-2 positive breast cancer cells possibly by that inhibing YAP and TAZ expression and their nuclear localization.

High MST2 expression regulates lens epithelial cell apoptosis in age-related cataracts through YAP1 targeting GLUT1

Age-related cataract (ARC) is a severe visual impairment disease and its pathogenesis remains unclear. This study investigated the relevance of MST2/YAP1/GLUT1 in ARC development in vivo and in vitro, and explored the role and possible mechanisms of this pathway in oxidative damage-mediated apoptosis of lens epithelial cells (LECs). Western blot analysis and immunohistochemistry showed that MST2 and phosphorylated (p)-YAP (Ser127) protein levels were increased, and YAP1 and GLUT1 protein levels were decreased in LECs of ARC patients and aged mice. Additionally, differential expression of MST2 and YAP1 was associated with H2O2-induced apoptosis of human lens epithelial B3 (HLE-B3) cells. CCK-8 and Hoechst 33,342 apoptosis assays showed that MST2 and YAP1 were involved in H2O2-induced apoptosis of LECs. Subsequent experiments showed that, during MST2-mediated H2O2-induced apoptosis, p-YAP (Ser127) levels were elevated and immunofluorescence revealed nucleoplasmic translocation and inhibition of YAP1 protein expression. Furthermore, GLUT1 was in turn synergistically transcriptionally regulated by YAP1-TEAD1 in dual luciferase reporter assays. In conclusion, our study indicates that the MST2/YAP1/GLUT1 pathway plays a major role in the pathogenesis of ARC and LECs apoptosis, providing a new direction for future development of targeted inhibitors that block this signaling pathway to prevent, delay, or even cure ARC.

BZIP transcription factors PcYap1 and PcRsmA link oxidative stress response to secondary metabolism and development in Penicillium chrysogenum

BackgroundReactive oxygen species (ROS) trigger different morphogenic processes in filamentous fungi and have been shown to play a role in the regulation of the biosynthesis of some secondary metabolites. Some bZIP transcription factors, such as Yap1, AtfA and AtfB, mediate resistance to oxidative stress and have a role in secondary metabolism regulation. In this work we aimed to get insight into the molecular basis of this regulation in the industrially important fungus Penicillium chrysogenum through the characterization of the role played by two effectors that mediate the oxidative stress response in development and secondary metabolism.ResultsIn P. chrysogenum, penicillin biosynthesis and conidiation are stimulated by the addition of H2O2 to the culture medium, and this effect is mediated by the bZIP transcription factors PcYap1 and PcRsmA. Silencing of expression of both proteins by RNAi resulted in similar phenotypes, characterized by increased levels of ROS in the cell, reduced conidiation, higher sensitivity of conidia to H2O2 and a decrease in penicillin production. Both PcYap1 and PcRsmA are able to sense H2O2-generated ROS in vitro and change its conformation in response to this stimulus. PcYap1 and PcRsmA positively regulate the expression of brlA, the first gene of the conidiation central regulatory pathway. PcYap1 binds in vitro to a previously identified regulatory sequence in the promoter of the penicillin gene pcbAB: TTAGTAA, and to a TTACTAA sequence in the promoter of the brlA gene, whereas PcRsmA binds to the sequences TGAGACA and TTACGTAA (CRE motif) in the promoters of the pcbAB and penDE genes, respectively.ConclusionsbZIP transcription factors PcYap1 and PcRsmA respond to the presence of H2O2-generated ROS and regulate oxidative stress response in the cell. Both proteins mediate ROS regulation of penicillin biosynthesis and conidiation by binding to specific regulatory elements in the promoters of key genes. PcYap1 is identified as the previously proposed transcription factor PTA1 (Penicillin Transcriptional Activator 1), which binds to the regulatory sequence TTAGTAA in the pcbAB gene promoter. This is the first report of a Yap1 protein directly regulating transcription of a secondary metabolism gene. A model describing the regulatory network mediated by PcYap1 and PcRsmA is proposed.

HJURP regulates cell proliferation and chemo-resistance via YAP1/NDRG1 transcriptional axis in triple-negative breast cancer

Triple-negative breast cancer is still a difficult point in clinical treatment at present, and a deep study of its pathogenesis has great clinical value. Therefore, our research mainly focuses on exploring the progression of triple-negative breast cancer and determines the important role of the HJURP/YAP1/NDRG1 transcriptional regulation axis in triple-negative breast cancer. We observed significantly increased HJURP expression levels in triple-negative breast cancer compared to other subtypes. HJURP could affect the level of ubiquitination modification of YAP1 protein and then regulate its downstream transcriptional activity. Mechanistically, we found that YAP1 positively regulates NDRG1 transcription by binding the promoter region of the NDRG1 gene. And HJURP/YAP1/NDRG1 axis could affect cell proliferation and chemotherapy sensitivity in triple-negative breast cancer. Taken together, these findings provide insights into the transcriptional regulation axis of HJURP/YAP1/NDRG1 in triple-negative breast cancer progression and therapeutic response.

Perfluorooctanoic acid promotes proliferation of the human granulosa cell line HGrC1 and alters expression of cell cycle genes and Hippo pathway effector YAP1

Perfluorooctanoic acid (PFOA) is a common environmental contaminant that belongs to a group of manmade fluorinated chemicals called per- and polyfluoroalkyl substances (PFAS). Due to the pervasive nature of PFOA, the environmental health risks of PFOA contamination and exposure on reproductive health have increasing concern. In the present study, we exposed HGrC1 cells, an immortalized human granulosa cell line, to environmentally relevant (1–10 μM) concentrations of PFOA. Results indicated that HGrC1 cells treated with PFOA had increased proliferation and migration relative to vehicle treated controls. No differences in cell apoptosis were observed with 1–10 μM PFOA. Gene expression analysis revealed increases in mRNA transcripts for cell cycle regulators CCND1, CCNA2, and CCNB1. Upregulation of YAP1 protein and downstream target CTGF protein was also observed, suggesting that the Hippo pathway is involved in the proliferation and migratory effects of PFOA on HGrC1 cells. Further, the YAP1 inhibitor Verteporfin prevented the stimulatory effects of PFOA on HGrC1 cells. Together, these findings support a role for the Hippo pathway effector YAP1 in response to PFOA exposure in human granulosa cells.

Essential roles of YAP-TEAD complex in adult stem cell development during thyroid hormone-induced intestinal remodeling of Xenopus laevis.

During amphibian metamorphosis which is triggered by thyroid hormone (TH), the small intestine is extensively remodeled from the larval to adult form. In the Xenopus laevis intestine, some of the larval epithelial cells dedifferentiate into adult stem cells, which newly form the adult epithelium similar to the mammalian one. We have previously shown that TH-activated Shh, Wnt and Notch signaling pathways play important roles in adult epithelial development. Here we focus on the Hippo signaling pathway, which is known to interact with these pathways in the mammalian intestine. Our quantitative RT-PCR analysis indicates that the expression of genes involved in this pathway including YAP1, TAZ, TEAD1 and core kinases is differently regulated by TH in the metamorphosing intestine. Additionally, we show by in situ hybridization and immunohistochemistry that the transcriptional co-activator YAP1, a major effector of the Hippo signaling, is expressed in the adult stem cells and connective tissue cells surrounding them and that YAP1 protein is localized in either nucleus or cytoplasm of the stem cells. We further show that YAP1 binds its binding partner TEAD1 (transcription factor) in vivo and that their interaction is inhibited by verteporfin (VP). More importantly, by using VP in organ cultureof the tadpole intestine, we experimentally demonstrate that the inhibition of YAP1-TEAD1 interaction decreases both TH-induced stem cells expressing LGR5 and nearby connective tissue cells in number and proliferation, leading to the failure of adult epithelial development. Our results indicate that YAP-TEAD complex is required for stem cell development during intestinal remodeling.

HTLV-1 activates YAP via NF-κB/p65 to promote oncogenesis

Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) infection. HTLV-1 exerts its oncogenic functions by interacting with signaling pathways involved in cell proliferation and transformation. Dysregulation of the Hippo/YAP pathway is associated with multiple cancers, including virus-induced malignancies. In the present study, we observe that expression of YAP, which is the key effector of Hippo signaling, is elevated in ATL cells by the action of the HTLV-1 Tax protein. YAP transcriptional activity is remarkably enhanced in HTLV-1-infected cells and ATL patients. In addition, Tax activates the YAP protein via a mechanism involving the NF-κB/p65 pathway. As a mechanism for this cross talk between the Hippo and NF-κB pathways, we found that p65 abrogates the interaction between YAP and LATS1, leading to suppression of YAP phosphorylation, inhibition of ubiquitination-dependent degradation of YAP, and YAP nuclear accumulation. Finally, knockdown of YAP suppresses the proliferation of ATL cells invitro and tumor formation in ATL-engrafted mice. Taken together, our results suggest that p65-induced YAP activation is essential for ATL pathogenesis and implicate YAP as a potential therapeutic target for ATL treatment.

RNA-binding protein complex LIN28/MSI2 enhances cancer stem cell-like properties by modulating Hippo-YAP1 signaling and independently of Let-7

The RNA binding protein LIN28 directly modulates the stability and translation of target mRNAs independently of Let-7; however, the key downstream targets of LIN28 in this process are largely unknown. Here, we revealed that Hippo signaling effector YAP1 functioned as a key downstream regulator of LIN28 to modulate the cancer stem cell (CSC)-like properties and tumor progressions in triple negative breast cancer (TNBC). LIN28 was overexpressed in BC tissues and cell lines, and significantly correlated with poorer overall survivals in patients. Ectopic LIN28 expression enhanced, while knockdown of LIN28A inhibited the CSC-like properties, cell growth and invasive phenotypes of TNBC cells in vitro and in vivo. Transcriptome analysis demonstrated LIN28 overexpression significantly induced the expressions of YAP1 downstream genes, while reduced the transcripts of YAP1 upstream kinases, such as MST1/2 and LATS1/2, and knockdown of LIN28A exhibited the opposite effects. Furthermore, constitutive activation of YAP1 in LIN28 knockdown TNBC cells could rescue the cell growth and invasive phenotypes in vitro and in vivo. Mechanistically, instead of the dependence of Let-7, LIN28 recruited RNA binding protein MSI2 in a manner dependent on the LIN28 CSD domain and MSI2 RRM domain, to directly induce the mRNA decay of YAP1 upstream kinases, leading to the inhibition of Hippo pathway and activation of YAP1, which eventually gave rise to increased CSC populations, enhanced tumor cell growth and invasive phenotypes. Accordingly, co-upregulations of LIN28 and MSI2 in TNBC tissues were strongly associated with YAP1 protein level and tumor malignance. Taken together, our findings unravel a novel LIN28/MSI2-YAP1 regulatory axis to induce the CSC-like properties, tumor growth and metastasis, independently of Let-7, which may serve as a potential therapeutic strategy for the treatment of a subset of TNBC with LIN28 overexpression.

The Galapagos Chip Platform for High-Throughput Screening of Cell Adhesive Chemical Micropatterns.

In vivo cells reside in a complex extracellular matrix (ECM) that presents spatially distributed biochemical and -physical cues at the nano- to micrometer scales. Chemical micropatterning is successfully used to generate adhesive islands to control where and how cells attach and restore cues of the ECM in vitro. Although chemical micropatterning has become a powerful tool to study cell-material interactions, only a fraction of the possible micropattern designs was covered so far, leaving many other possible designs still unexplored. Here, a high-throughput screening platform called "Galapagos chip" is developed. It contains a library of 2176 distinct subcellular chemical patterns created using mathematical algorithms and a straightforward UV-induced two-step surface modification. This approach enables the immobilization of ligands in geometrically defined regions onto cell culture substrates. To validate the system, binary RGD/polyethylene glycol patterns are prepared on which human mesenchymal stem cells are cultured, and the authors observe how different patterns affect cell and organelle morphology. As proof of concept, the cells are stained for the mechanosensitive YAP protein, and, using a machine-learning algorithm, it is demonstrated that cell shape and YAP nuclear translocation correlate. It is concluded that the Galapagos chip is a versatile platform to screen geometrical aspects of cell-ECM interaction.

Live imaging YAP signalling in mouse embryo development.

YAP protein is a critical regulator of mammalian embryonic development. By generating a near-infrared fusion YAP reporter mouse line, we have achieved high-resolution live imaging of YAP localization during mouse embryonic development. We have validated the reporter by demonstrating its predicted responses to blocking LATS kinase activity or blocking cell polarity. By time lapse imaging preimplantation embryos, we revealed a mitotic reset behaviour of YAP nuclear localization. We also demonstrated deep tissue live imaging in post-implantation embryos and revealed an intriguing nuclear YAP pattern in migrating cells. The YAP fusion reporter mice and imaging methods will open new opportunities for understanding dynamic YAP signalling in vivo in many different situations.

Neuropeptide Calcitonin Gene-related Peptide Signaling via RAMP1 Stimulates Liver Fibrosis and Regulates Yes-associated Protein Activity during Chronic Liver Damage

Background & aims: Chronic liver damage can ultimately lead to liver cirrhosis. Sensory nerves innervating the liver secrete the neuropeptide calcitonin gene-related peptide (CGRP), which binds to receptor activity-modifying protein (RAMP)1. We investigated whether the loss of RAMP1 affects liver fibrosis in chronic liver inflammation. Methods: Wild-type and RAMP1-deficient mice were injected with carbon tetrachloride (CCl4) to induce liver injury. CGRP/RAMP1 levels in CCl4-treated livers were determined by RT-PCR. Fibrosis was assessed by Sirius red staining and Western blot (α-SMA and collagen). The influence of CGRP/RAMP1 on the Hippo signaling pathway was assessed by Western blot. In addition, we stimulated the human hepatic stellate cell line (LX-2) in vitro with CGRP. The expression of α-SMA, collagen and YAP proteins was quantified by Western blot. Results: Chronic liver injury resulted in upregulation of hepatic CGRP/RAMP1 mRNA expression. In the absence of RAMP1, murine livers exhibited less fibrosis. In addition, expression of markers for hepatocyte proliferation was decreased. Also, the expression of YAP was diminished in RAMP1-deficient mice. At the same time, phosphorylation of YAP on Ser127, which promotes inactivation of YAP, was increased in RAMP1-deficient livers. In vitro, stimulation of LX-2 cells with CGRP promoted hepatic stellate cell activation. Moreover, treatment with CGRP resulted in inactivation of YAP, confirming our in vivo results. Conclusion: RAMP1/CGRP signaling promotes liver fibrosis and controls YAP activity, as well as the activation of LX-2 cells in vitro. The CGRP receptor RAMP1 may serve as a potential target of antifibrotic therapy in liver disease.

Circ_0014235 confers Gefitinib resistance and malignant behaviors in non-small cell lung cancer resistant to Gefitinib by governing the miR-146b-5p/YAP/PD-L1 pathway

ABSTRACT Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as Gefitinib, have been recommended as the first-line treatment reagent for advanced EGFR-mutant non-small cell lung cancer (NSCLC). However, the mechanisms of drug resistance development are not fully determined. This study aimed to explore the role of circular RNA (circ_0014235) in Gefitinib-resistant NSCLC. The expression of circ_0014235, microRNA-146b-5p (miR-146b-5p) and Yes1 associated transcriptional regulator (YAP) mRNA was detected by quantitative real-time PCR (qPCR). Cell viability was detected by CCK-8 assay. Cell proliferation was assessed by colony formation assay and EdU assay. Cell cycle and cell apoptosis were determined by flow cytometry assay. The expression of marker proteins, YAP protein and programmed death ligand 1 (PD-L1) protein was detected by Western blot. The putative relationship between miR-146b-5p and circ_0014235 or YAP was ensured by dual-luciferase reporter assay and RIP assay. Animal models were established to explore the role of circ_0014235 in vivo. Circ_0014235 was highly expressed in Gefitinib-resistant NSCLC cells. Circ_0014235 downregulation reduced Gefitinib IC50, inhibited cell proliferation and induced cell apoptosis and cell cycle arrest in Gefitinib-resistant NSCLC cells, while these effects were reversed by the inhibition of miR-146b-5p, a target of circ_0014235. In addition, YAP was a target gene of miR-146b-5p, and circ_0014235 relieved miR-146b-5p-mediated inhibition on YAP by targeting miR-146b-5p. MiR-146b-5p restoration-blocked Gefitinib IC50 and cell malignant behaviors were recovered by YAP overexpression. YAP positively regulated PD-L1 expression, and YAP overexpression contributes to Gefitinib IC50 and cell malignant behaviors by upregulating PD-L1. Circ_0014235 confers Gefitinib resistance and malignant behaviors in Gefitinib-resistant NSCLC by governing the miR-146b-5p/YAP/PD-L1 pathway.

Activation of the Hippo Pathway in Rana sylvatica: Yapping Stops in Response to Anoxia.

Wood frogs (Rana sylvatica) display well-developed anoxia tolerance as one component of their capacity to endure prolonged whole-body freezing during the winter months. Under anoxic conditions, multiple cellular responses are triggered to efficiently cope with stress by suppressing gene transcription and promoting activation of mechanisms that support cell survival. Activation of the Hippo signaling pathway initiates a cascade of protein kinase reactions that end with phosphorylation of YAP protein. Multiple pathway components of the Hippo pathway were analyzed via immunoblotting, qPCR or DNA-binding ELISAs to assess the effects of 24 h anoxia and 4 h aerobic recovery, compared with controls, on liver and heart metabolism of wood frogs. Immunoblot results showed significant increases in the relative levels of multiple proteins of the Hippo pathway representing an overall activation of the pathway in both organs under anoxia stress. Upregulation of transcript levels further confirmed this. A decrease in YAP and TEAD protein levels in the nuclear fraction also indicated reduced translocation of these proteins. Decreased DNA-binding activity of TEAD at the promoter region also suggested repression of gene transcription of its downstream targets such as SOX2 and OCT4. Furthermore, changes in the protein levels of two downstream targets of TEAD, OCT4 and SOX2, established regulated transcriptional activity and could possibly be associated with the activation of the Hippo pathway. Increased levels of TAZ in anoxic hearts also suggested its involvement in the repair mechanism for damage caused to cardiac muscles during anoxia. In summary, this study provides the first insights into the role of the Hippo pathway in maintaining cellular homeostasis in response to anoxia in amphibians.