1.Modeling hepatoblastoma development with human fetal liver organoids reveals YAP1 activation is sufficient for tumorigenesis.
Li YANG ; Jin CHEN ; Jianqing LIANG ; Yufeng ZHANG ; Qingzhe WANG ; Xiaojun REN ; Jinsong WEI ; Qianchun GONG ; Jiting ZHANG ; Ning JIANG ; Xinhua LIN ; Jin LI ; Bing ZHAO
Protein & Cell 2022;13(9):683-688
2.Activation of Yes-associated protein (YAP) improves mouse acute liver failure by alleviating ferroptosis.
Wen ZHAO ; Miao LEI ; Hailin ZHANG ; Jiawei WANG ; Dongdong LI ; Chuanmiao LIU
Chinese Journal of Cellular and Molecular Immunology 2023;39(10):910-916
Objective To investigate the effects of YAP on the occurrence and progression of acute liver failure by regulating the ferroptosis pathway and its underlying mechanism. Methods A total of 20 8-week-old C57BL/6 mice were randomly divided into four groups: a control group, an acute liver failure model group, a YAP agonist XMU-MP-1 treatment group and a YAP inhibitor verteporfin treatment group, five mice for each group. HE staining was used to observe the pathological changes of hepatic inflammation and necrosis. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected by liver biochemistry. Iron (Fe), malondialdehyde (MDA), glutathione (GSH) determination kits were used to measure their levels in liver tissues of each group. The changes of hepatocyte mitochondrial in each group were observed by electron microscopy. Real time PCR and Western blot analysis were used to detect the mRNA and protein expressions of YAP, glutathione peroxidase 4 (GPX4) and 5-lipoxygenase (5-LOX). Results Compared with the control group, mice in the acute liver failure model group and the YAP inhibitor verteporfin treatment group showed severe liver tissue congestion with inflammatory cell infiltration and structural damage to hepatic lobules. Liver injury was alleviated in the XMU-MP-1 treatment group. With the occurrence of liver failure, plasma ALT and AST levels significantly increased, and liver function was improved in XMU-MP-1 treatment group. Electron microscopy showed that mitochondria in hepatocytes of mice with liver failure became smaller and bilayer membrane density increased, while mitochondria changes in the XMU-MP-1 group were alleviated. In addition, the acute liver failure model group showed an increase in Fe and MDA contents, decreased protein expressions of GPX4, and enhanced expression of 5-LOX, suggesting that ferroptosis was involved in acute liver failure in C57BL/6 mice. Ferroptosis was inhibited by activation of YAP. Conclusion Activation of YAP may ameliorate liver injury by inhibiting ferroptosis.
Animals
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Mice
;
Ferroptosis
;
Glutathione
;
Liver Failure
;
Liver Failure, Acute/drug therapy*
;
Mice, Inbred C57BL
;
Verteporfin
;
YAP-Signaling Proteins/metabolism*
3.A YAP/TAZ-CD54 axis is required for CXCR2-CD44- tumor-specific neutrophils to suppress gastric cancer.
Pingping NIE ; Weihong ZHANG ; Yan MENG ; Moubin LIN ; Fenghua GUO ; Hui ZHANG ; Zhenzhu TONG ; Meng WANG ; Fan CHEN ; Liwei AN ; Yang TANG ; Yi HAN ; Ruixian YU ; Wenjia WANG ; Yuanzhi XU ; Linxin WEI ; Zhaocai ZHOU ; Shi JIAO
Protein & Cell 2023;14(7):513-531
As an important part of tumor microenvironment, neutrophils are poorly understood due to their spatiotemporal heterogeneity in tumorigenesis. Here we defined, at single-cell resolution, CD44-CXCR2- neutrophils as tumor-specific neutrophils (tsNeus) in both mouse and human gastric cancer (GC). We uncovered a Hippo regulon in neutrophils with unique YAP signature genes (e.g., ICAM1, CD14, EGR1) distinct from those identified in epithelial and/or cancer cells. Importantly, knockout of YAP/TAZ in neutrophils impaired their differentiation into CD54+ tsNeus and reduced their antitumor activity, leading to accelerated GC progression. Moreover, the relative amounts of CD54+ tsNeus were found to be negatively associated with GC progression and positively associated with patient survival. Interestingly, GC patients receiving neoadjuvant chemotherapy had increased numbers of CD54+ tsNeus. Furthermore, pharmacologically enhancing YAP activity selectively activated neutrophils to suppress refractory GC, with no significant inflammation-related side effects. Thus, our work characterized tumor-specific neutrophils in GC and revealed an essential role of YAP/TAZ-CD54 axis in tsNeus, opening a new possibility to develop neutrophil-based antitumor therapeutics.
Humans
;
Animals
;
Mice
;
Adaptor Proteins, Signal Transducing/metabolism*
;
Transcription Factors/metabolism*
;
Stomach Neoplasms/pathology*
;
Neutrophils/pathology*
;
Signal Transduction/genetics*
;
YAP-Signaling Proteins
;
Tumor Microenvironment
;
Hyaluronan Receptors/genetics*
4.Doublecortin-like kinase 1 activates Hippo pathway to promote migration, invasion and proliferation of pancreatic cancer cells.
Rui YAN ; Zi Wei LIANG ; He Shu LIU ; Yang GE ; Guang Yu AN
Chinese Journal of Oncology 2023;45(7):594-604
Objective: To explore the mechanism of Doublecortin-like kinase 1 (DCLK1) in promoting cell migration, invasion and proliferation in pancreatic cancer. Methods: The correlation between DCLK1 and Hippo pathway was analyzed using TCGA and GTEx databases and confirmed by fluorescence staining of pancreatic cancer tissue microarrays. At the cellular level, immunofluorescence staining of cell crawls and western blot assays were performed to clarify whether DCLK1 regulates yes associated protein1 (YAP1), a downstream effector of the Hippo pathway. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyze the expressions of YAP1 binding transcription factor TEA-DNA binding proteins (TEAD) and downstream malignant behavior-promoting molecules CYR61, EDN1, AREG, and CTGF. Transwell test of the DCLK1-overexpressing cells treated with the Hippo pathway inhibitor Verteporfin was used to examine whether the malignant behavior-promoting ability was blocked. Analysis of changes in the proliferation index of experimental cells used real-time label-free cells. Results: TCGA combined with GTEx data analysis showed that the expressions of DCLK1 and YAP1 molecules in pancreatic cancer tissues were significantly higher than those in adjacent tissues (P<0.05). Moreover, DCLK1was positively correlated with the expressions of many effectors in the Hippo pathway, including LATS1 (r=0.53, P<0.001), LATS2 (r=0.34, P<0.001), MOB1B (r=0.40, P<0.001). In addition, the tissue microarray of pancreatic cancer patients was stained with multicolor fluorescence, indicated that the high expression of DCLK1 in pancreatic cancer patients was accompanied by the up-regulated expression of YAP1. The expression of DCLK1 in pancreatic cancer cell lines was analyzed by the CCLE database. The results showed that the expression of DCLK1 in AsPC-1 and PANC-1 cells was low. Thus, we overexpressed DCLK1 in AsPC-1 and PANC-1 cell lines and found that DCLK1 overexpression in pancreatic cancer cell lines promoted YAP1 expression and accessible to the nucleus. In addition, DCLK1 up-regulated the expression of YAP1 binding transcription factor TEAD and increased the mRNA expression levels of downstream malignant behavior-promoting molecules. Finally, Verteporfin, an inhibitor of the Hippo pathway, could antagonize the cell's malignant behavior-promoting ability mediated by high expression of DCLK1. We found that the number of migrated cells with DCLK1 overexpressing AsPC-1 group was 68.33±7.09, which was significantly higher than 22.00±4.58 of DCLK1 overexpressing cells treated with Verteporfin (P<0.05). Similarly, the migration number of PANC-1 cells overexpressing DCLK1 was 65.66±8.73, which was significantly higher than 37.00±6.00 of the control group and 32.33±9.61 of Hippo pathway inhibitor-treated group (P<0.05). Meanwhile, the number of invasive cells in the DCLK1-overexpressed group was significantly higher than that in the DCLK1 wild-type group cells, while the Verteporfin-treated DCLK1-overexpressed cells showed a significant decrease. In addition, we monitored the cell proliferation index using the real-time cellular analysis (RTCA) assay, and the proliferation index of DCLK1-overexpressed AsPC-1 cells was 0.66±0.04, which was significantly higher than 0.38±0.01 of DCLK1 wild-type AsPC-1 cells (P<0.05) as well as 0.05±0.03 of DCLK1-overexpressed AsPC1 cells treated with Verteporfin (P<0.05). PANC-1 cells showed the same pattern, with a proliferation index of 0.77±0.04 for DCLK1-overexpressed PANC-1 cells, significantly higher than DCLK1-overexpressed PANC1 cells after Verteporfin treatment (0.14±0.05, P<0.05). Conclusion: The expression of DCLK1 is remarkably associated with the Hippo pathway, it promotes the migration, invasion, and proliferation of pancreatic cancer cells by activating the Hippo pathway.
Humans
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Doublecortin-Like Kinases
;
Hippo Signaling Pathway
;
Verteporfin/pharmacology*
;
Cell Line, Tumor
;
Protein Serine-Threonine Kinases/metabolism*
;
Pancreatic Neoplasms/pathology*
;
YAP-Signaling Proteins
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Transcription Factors/metabolism*
;
Cell Proliferation/genetics*
;
Gene Expression Regulation, Neoplastic
;
Tumor Suppressor Proteins/genetics*
5.YAP regulates intestinal epithelial cell proliferation through activation of STAT3 in DSS-induced colitis and associated cancer.
Journal of Central South University(Medical Sciences) 2022;47(12):1637-1645
OBJECTIVES:
Ulcerative colitis (UC) is a chronic, relapsing inflammation of the colon. Impaired epithelial repair is an important biological features of UC. Accelerating intestinal epithelial repair to achieve endoscopic mucosal healing has become a key goal in UC. Yes-associated protein (YAP) is a key transcriptional coactivator that regulates organ size, tissue growth and tumorigenesis. Growing studies have focused on the role of YAP in intestinal epithelial regeneration. This study explore the molecular mechanism for the role YAP in modulating colonic epithelial proliferation, repair, and the development of colitis associated cancer.
METHODS:
We constructed the acute colitis mouse model through successive 5 days of 3% dextran sulfate sodium salt (DSS) induction. Then YAP-overexpressed mouse model was constructed by intraperitoneal injection the YAP overexpressed and negative control lentivirus into DSS mice. On the 5th day of DSS induction and the 5th day of normal drinking water after removing DSS (5+5 d), the mice were killed by spinal dislocation. The colon was taken to measure the length, and the bowel 1-2 cm near the anal canal was selected for immunohistochemical and Western blotting. We used YAP over-expressed colonic epithelial cells and small interfering signal transducer and activator of transcription 3 (STAT3) RNA to probe the regulation of YAP on STAT3, using cell counting kit-8 and scratch assays to explore the role of YAP on colonic epithelial cell proliferation. Finally, we conducted co-immunoprecipitation to test the relationship between YAP and STAT3.
RESULTS:
After DSS treatment, the expression of YAP was dramatically diminished in crypts. Compared with the empty control mice, overexpression of YAP drastically accelerated epithelial regeneration after DSS induced colitis, presenting with more intact of structural integrity in intestinal epithelium and a reduction in the number of inflammatory cells in the mucosa. Further Western blotting, functional experiment and co-immunoprecipitation analyses showed that the expression of YAP in nucleus was significantly increased by 2 h post DSS cessation, accompanied with up-regulated total protein levels of STAT3 and phosphorylated-STAT3 (p-STAT3). Overexpression of YAP enhanced the expression of STAT3, p-STAT3, and their transcriptional targets including c-Myc and Cyclin D1. In addition, it promoted the proliferation and the "wound healing" of colonic cells. However, these effects were reversed when silencing STAT3 on YAP-overexpressed FHC cells. Moreover, protein immunoprecipitation indicated that YAP could directly interact with STAT3 in the nucleus, up-regulatvng the expressvon of STAT3. Finally, during the process of CAC, overexpression of YAP mutant caused the down-regulated expression of STAT3 and inhibited the development and progress of CAC.
CONCLUSIONS
YAP activates STAT3 signaling in regulation of epithelial cell proliferation and promotes mucosal regeneration after DSS induced colitis, which may serve as a potential therapeutic target in UC. However, persistent and excessive YAP activation may promote CAC development.
Animals
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Mice
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Cell Proliferation
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Colitis/drug therapy*
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Colon/metabolism*
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Dextran Sulfate/adverse effects*
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Disease Models, Animal
;
Intestinal Mucosa
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Mice, Inbred C57BL
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Neoplasm Recurrence, Local/metabolism*
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STAT3 Transcription Factor/metabolism*
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YAP-Signaling Proteins/metabolism*
6.Changes of YAP activity at the early stage of nonalcoholic steatohepatitis and its spatiotemporal relationship with ductular reaction in mice.
Ya Xue LIU ; Jia En LIANG ; Wei Lan ZENG ; Yan WANG
Journal of Southern Medical University 2022;42(9):1324-1334
OBJECTIVE:
To explore the changes in Yes-associated protein (YAP) activity at the early stage of nonalcoholic steatohepatitis (NASH) and the spatiotemporal relationship between YAP and ductular reaction (DR).
METHODS:
Male C57BL/6J mouse models of NASH were established by feeding with a methionine- and choline-deficient (MCD) diet or a thioacetamide (TAA) diet for 12 weeks. At different time points during the feeding, liver histology of the mice was observed with HE and Masson trichrome staining. The mRNA expressions of YAP and its target genes (Ctgf, Cyr61, Acta2) were determined by qPCR, and the total protein expression level of YAP was measured with immunoblotting. The expression and distribution of YAP and the markers of DR (K19 and Sox9) were observed with immunohistochemical staining.
RESULTS:
At the early stage of NASH induced by MCD diet (1 to 4 weeks), the mRNA expression of YAP and its target genes and the total protein expression of YAP increased significantly (P < 0.01). The number of YAP-positive hepatocytes reached the peak level of 90.8 (cells per ×400 field of view) at week 2 and then decreased to 30.8 at week 4 (P < 0.001); YAP-positive ductular cells appeared near the portal area, where DR began to occur. From 8 to 12 weeks, numerous K19/Sox9-positive DR cells were observed in the hepatic lobules around the central vein (P < 0.01), while only a few YAP-positive hepatocytes were present in the liver tissue (P > 0.05), and the number of YAP-positive ductular cells gradually increased with time (P < 0.001). At the early stage of NASH induced by TAA diet (3 days to 2 weeks), the mRNA expression of YAP and its target genes and the total protein expression of YAP increased significantly (P < 0.05), and the number of YAP-positive hepatocytes reached the peak of 69.2 at week 2 and then decreased to 55.2 at week 4 (P < 0.001); YAP-positive ductular cells first appeared at the initial location of DR near the central vein. From 6 to 12 weeks, numerous K19/Sox9-positive DR cells occurred in the hepatic lobules around the central vein (P < 0.01). While the number of YAP-positive hepatocytes decreased (P < 0.001), the number of YAP-positive ductular cells continued to increase (P < 0.001).
CONCLUSION
During the development of NASH, YAP activation occurs earlier than DR but they are spatiotemporally correlated. YAP activation in hepatocytes may participate in DR by promoting hepatocyte dedifferentiation.
Animals
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Choline
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Disease Models, Animal
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Hepatocytes
;
Liver/metabolism*
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Male
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Methionine/metabolism*
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Mice
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Mice, Inbred C57BL
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Non-alcoholic Fatty Liver Disease/metabolism*
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RNA, Messenger/metabolism*
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Thioacetamide/metabolism*
;
YAP-Signaling Proteins
7.RGFP966 inactivation of the YAP pathway attenuates cardiac dysfunction induced by prolonged hypothermic preservation.
Xiao-He ZHENG ; Lin-Lin WANG ; Ming-Zhi ZHENG ; Jin-Jie ZHONG ; Ying-Ying CHEN ; Yue-Liang SHEN
Journal of Zhejiang University. Science. B 2020;21(9):703-715
Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4-6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP-transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway.
Acrylamides/pharmacology*
;
Animals
;
Apoptosis/drug effects*
;
Cryopreservation
;
Disaccharides/pharmacology*
;
Electrolytes/pharmacology*
;
Glutamates/pharmacology*
;
Glutathione/pharmacology*
;
Heart/physiology*
;
Heart Transplantation/methods*
;
Hepatocyte Growth Factor/antagonists & inhibitors*
;
Histidine/pharmacology*
;
Histone Deacetylase Inhibitors/pharmacology*
;
Intracellular Signaling Peptides and Proteins/antagonists & inhibitors*
;
Male
;
Mannitol/pharmacology*
;
Oxidative Stress/drug effects*
;
Phenylenediamines/pharmacology*
;
Proto-Oncogene Proteins/antagonists & inhibitors*
;
Rats
;
Rats, Sprague-Dawley
;
Signal Transduction/drug effects*
;
YAP-Signaling Proteins
8.Kindlin-2 regulates endometrium development via mTOR and Hippo signaling pathways in mice.
Jing ZHANG ; Jia Gui SONG ; Zhen Bin WANG ; Yu Qing GONG ; Tian Zhuo WANG ; Jin Yu ZHOU ; Jun ZHAN ; Hong Quan ZHANG
Journal of Peking University(Health Sciences) 2022;54(5):846-852
OBJECTIVE:
To investigate the effects and mechanisms of Kindlin-2 on uterus development and reproductive capacity in female mice.
METHODS:
Cdh16-Cre tool mice and Kindlin-2flox/flox mice were used to construct the mouse model of uterus specific knockout of Kindlin-2, and the effects of Kindlin-2 deletion on uterine development and reproduction capacity of female mice were observed. High expression and knockdown of Kindlin-2 in endometrial cancer cell lines HEC-1 and Ish were used to detect the regulation of mammalian target of rapamycin (mTOR) signaling pathway. In addition, uterine proteins of the female mice with specific knockout of Kindlin-2 and female mice in the control group were extracted to detect the protein levels of key molecules of mTOR signaling pathway and Hippo signaling pathway.
RESULTS:
The mouse model of uterine specific knockout of Kindlin-2 was successfully constructed. The knockout efficiency of Kindlin-2 in mouse uterus was identified and verified by mouse tail polymerase chain reaction (PCR), Western blot protein identification, immunohistochemical staining (IHC) and other methods. Compared with the control group, the female mice with uterus specific deletion of Kindlin-2 lost weight, seriously impaired reproductive ability, and the number of newborn mice decreased, but the proportion of the female mice and male mice in the newborn mice did not change. Hematoxylin eosin staining (HE) experiment showed that the endometrium of Kindlin-2 knockout group was incomplete and the thickness of uterine wall became thinner. In terms of mechanism, the deletion of Kindlin-2 in endo-metrial cancer cell lines HEC-1 and Ish could downregulate the protein levels of mTOR, phosphorylated mTOR, adenosine monophosphate-activated protein kinase (AMPK), phosphorylated AMPK and phosphorylated ribosomal protein S6 (S6), and the mTOR signal pathway was inhibited. It was found that the specific deletion of Kindlin-2 could upregulate the protein levels of Mps one binding 1 (MOB1) and phosphorylated Yes-associated protein (YAP) in the uterus of the female mice, and the Hippo signal pathway was activated.
CONCLUSION
Kindlin-2 inhibits the development of uterus by inhibiting mTOR signal pathway and activating Hippo signal pathway, thereby inhibiting the fertility of female mice.
AMP-Activated Protein Kinases/metabolism*
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Adenosine Monophosphate/metabolism*
;
Animals
;
Cadherins/metabolism*
;
Cytoskeletal Proteins/metabolism*
;
Endometrium/metabolism*
;
Eosine Yellowish-(YS)/metabolism*
;
Female
;
Hematoxylin/metabolism*
;
Hippo Signaling Pathway
;
Male
;
Mammals/metabolism*
;
Mice
;
Muscle Proteins
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Ribosomal Protein S6/metabolism*
;
Sirolimus/metabolism*
;
TOR Serine-Threonine Kinases/metabolism*
;
YAP-Signaling Proteins