Urolithin A attenuates hexavalent chromium-induced small intestinal injury by modulating PP2A/Hippo/YAP1 pathway.

Hexavalent chromium (Cr(VI)) exposure has been linked with gastrointestinal toxicity, whereas the molecular pathways and key targets remain elusive. Computational toxicology analysis predicted the correlation between protein phosphatase 2A (PP2A) and genes regarding Cr(VI)-induced intestinal injury. Here, we generated a mouse model with intestinal epithelium-specific knock-out of Ppp2r1a (encoding PP2A Aα subunit) to investigate the mechanisms underlying Cr(VI)-induced small intestinal toxicity. Heterozygous mice (HE) and matched wild-type (WT) littermates were administrated with Cr(VI) at 0, 5, 20, 80 mg/L for 28 successive days. Cr(VI) treatment led to crypt hyperplasia, epithelial cell apoptosis, and intestinal barrier dysfunction, accompanied by the decline of goblet cell counts and Occludin expression in WT mice. Notably, these effects were aggravated in HE mice, indicating that PP2A Aα deficiency conferred mice with susceptibility to Cr(VI)-induced intestinal injury. Integrated data analysis and biological experiments revealed Cr(VI) exposure could decrease YAP1 phosphorylation at Ser127 but increase protein expression and activity, together with elevated TAZ protein driving epithelial crypt cells proliferation following damage, suggesting the involvement of Hippo/YAP1 signaling pathway in Cr(VI)-induced intestinal toxicity. Nevertheless, the enhanced phosphorylation of YAP1 in HE mice resulted in proliferation/repair defects in intestinal epithelium, thereby exacerbating Cr(VI)-induced gut barrier dysfunction. Notably, by molecular docking and further studies, we identified Urolithin A, a microbial metabolite, attenuated Cr(VI)-induced disruption of intestinal barrier function, partly by modulating YAP1 expression and activity. Our findings reveal the novel molecular pathways participated in Cr(VI)-caused small intestinal injury and urolithin A could potentially protect against environmental hazards-induced intestinal diseases.

Interference with ENO2 promotes ferroptosis and inhibits glycolysis in clear cell renal cell carcinoma by regulating Hippo­YAP1 signaling.

Glycolytic enzyme enolase 2 (ENO2) is dysregulated in various cancer types. Nevertheless, the role and underlying mechanism of ENO2 in clear cell renal cell carcinoma (ccRCC) remain unclear. Therefore, the current study investigated the effect and mechanism of ENO2 in ccRCC. ENO2 expression in a ccRCC cell line was assessed using reverse transcription-quantitative PCR and western blotting. Analysis of glycolysis was performed by estimating the extracellular acidification rate, lactic acid concentration, glucose uptake and the expression of glucose transporter 1, pyruvate kinase muscle isozyme M2 and hexokinase 2. Moreover, ferroptosis was assessed by detecting the level of total iron, lipid peroxide, reactive oxygen species and the expression of ferroptosis-related protein. In addition, mitochondrial function was assessed using JC-1 staining and detection kits. The results indicated that ENO2 is expressed at high levels in ccRCC cell lines, and interference with ENO2 expression inhibits glycolysis, promotes ferroptosis and affects mitochondrial function in ccRCC cells. Further investigation demonstrated that interference with ENO2 expression affected ferroptosis levels in ccRCC cells by inhibiting the glycolysis process. Mechanistically, the present results indicated that ENO2 may affect ferroptosis, glycolysis and mitochondrial functions by regulating Hippo-yes-associated protein 1 (YAP1) signaling in ccRCC cells. In conclusion, the present study showed that ENO2 affects ferroptosis, glycolysis and mitochondrial functions in ccRCC cells by regulating Hippo-YAP1 signaling, hence demonstrating its potential as a therapeutic target in ccRCC.

FTO attenuates the cytotoxicity of cisplatin in KGN granulosa cell-like tumour cells by regulating the Hippo/YAP1 signalling pathway.

Premature ovarian failure (POF) is a devastating condition for women under 40 years old. Chemotherapy, especially the use of cisplatin, has been demonstrated to promote the apoptosis of granulosa cells in primary and secondary follicles, leading to POF. Our previous studies demonstrated that fat mass- and obesity-associated (FTO) plays an essential role in protecting granulosa cells from cisplatin-induced cytotoxicity. Various studies have suggested that the Hippo/YAP signalling pathway plays a significant role in regulating cell apoptosis and proliferation. Additionally, YAP1 is the main downstream target of the Hippo signalling pathway and is negatively regulated by the Hippo signalling pathway. However, whether the Hippo/YAP signalling pathway is involved in the protective effect of FTO on granulosa cells has not been determined. In this study, we found that after cisplatin treatment, the apoptosis of granulosa cells increased in a concentration-dependent manner, accompanied by the downregulation of FTO and YAP1. Furthermore, overexpression of FTO decreased cisplatin-induced granulosa cell apoptosis, inhibited the Hippo/YAP kinase cascade-induced phosphorylation of YAP1, and promoted the entry of YAP1 into the nucleus. The downstream targets of YAP1 (CTGF, CYR61, and ANKRD1) were also increased. Si-RNA-mediated downregulation of FTO promoted cisplatin-induced granulosa cell apoptosis, activated the Hippo/YAP kinase cascade, and inhibited the YAP1 entry into the nucleus. These effects were completely reversed by the small molecule inhibitor of YAP1-verteporfin (VP). Taken together, these data suggested that FTO-YAP1 plays a positive role in regulating the proliferation of injured granulosa cells induced by cisplatin.

Hippo/YAP1 inhibition by verteporfin attenuates osteophyte formation in a mouse surgical osteoarthritis model.

Osteophyte is an outgrowth of cartilage formed at the margins of the affected joint through endochondral ossification-like processes, and is one of the most common radiographic features of osteoarthritis (OA) that has been used to define the stage of disease. Osteophyte has been regarded to adapt the joint to the altered biomechanics of OA patients, limits joint movement and represent a source of joint pain, however, the mechanism of osteophyte formation, the morphology characteristics and biomechanical properties of osteophyte cells are remained unclear. In the present study, we isolated osteophyte cells and chondrocytes from late-stage OA patients who underwent total knee replacement surgeries, by applying Atomic Force Microscopy (AFM), we identified osteophyte cells were in irregular shape with dendrites, shrunk cell body, smooth surface and high elastic modulus (23.3 ± 5.4 kPa) when compared with chondrocytes (6.5 ± 1.8 kPa). In addition, osteophyte cells showed higher proliferation ability and colony formation capacity than chondrocytes. Mechanistically, we identified YAP1, the core transcriptional factor of Hippo signaling pathway, was highly expressed in osteophyte cell both at protein and RNA levels. Inactivation of Hippo/YAP1 signaling pathway by Verteporfin is sufficient to inhibit osteophyte cell proliferation in vitro and attenuate osteophyte formation in vivo. In conclusion, the morphology characteristic and biomechanical property of osteophyte cells at single cell level are quite different from chondrocytes, although we could not exclude other regulatory mechanisms, our findings suggested that Hippo/YAP1 is of great importance for osteophyte formation.

Helicobacter pylori-induced aberrant demethylation and expression of GNB4 promotes gastric carcinogenesis via the Hippo-YAP1 pathway.

BACKGROUND: Helicobacter pylori (H. pylori) infection causes aberrant DNA methylation and contributes to the risk of gastric cancer (GC). Guanine nucleotide-binding protein subunit beta-4 (GNB4) is involved in various tumorigenic processes. We found an aberrant methylation level of GNB4 in H. pylori-induced GC in our previous bioinformatic analysis; however, its expression and underlying molecular mechanisms are poorly understood. METHODS: The expression, underlying signaling pathways, and clinical significance of GNB4 were analyzed in a local cohort of 107 patients with GC and several public databases. H. pylori infection was induced in in vitro and in vivo models. Methylation-specific PCR, pyrosequencing, and mass spectrometry analysis were used to detect changes in methylation levels. GNB4, TET1, and YAP1 were overexpressed or knocked down in GC cell lines. We performed gain- and loss-of-function experiments, including CCK-8, EdU, colony formation, transwell migration, and invasion assays. Nude mice were injected with genetically manipulated GC cells, and the growth of xenograft tumors and metastases was measured. Real-time quantitative PCR, western blotting, immunofluorescence, immunohistochemistry, chromatin immunoprecipitation, and co-immunoprecipitation experiments were performed to elucidate the underlying molecular mechanisms. RESULTS: GNB4 expression was significantly upregulated in GC and correlated with aggressive clinical characteristics and poor prognosis. Increased levels of GNB4 were associated with shorter survival times. Infection with H. pylori strains 26695 and SS1 induced GNB4 mRNA and protein expression in GC cell lines and mice. Additionally, silencing of GNB4 blocked the pro-proliferative, metastatic, and invasive ability of H. pylori in GC cells. H. pylori infection remarkably decreased the methylation level of the GNB4 promoter region, particularly at the CpG#5 site (chr3:179451746-179451745). H. pylori infection upregulated TET1 expression via activation of the NF-κB. TET binds to the GNB4 promoter region which undergoes demethylation modification. Functionally, we identified that GNB4 induced oncogenic behaviors of tumors via the Hippo-YAP1 pathway in both in vitro and in vivo models. CONCLUSIONS: Our findings demonstrate that H. pylori infection activates the NF-κB-TET1-GNB4 demethylation-YAP1 axis, which may be a potential therapeutic target for GC.

FTO promotes cervical cancer cell proliferation, colony formation, migration and invasion via the regulation of the BMP4/Hippo/YAP1/TAZ pathway.

Cervical cancer is the fourth most common malignancy tumor worldwide with high incidence and mortality. Accumulating evidence indicated that through an m6A-dependent or m6A-independent mechanism, fat mass and obesity associated gene (FTO) exhibits the tumor-promoting and suppressive roles of FTO involved in various cancers, including cervical cancer. This study aims to verify the biological function and potential mechanisms of FTO in cervical cancer cell proliferation, colony formation, migration, and invasion in vitro as well as tumor growth in vivo. Herein, we confirmed that knockdown of FTO inhibits cell proliferation, colony formation, migration, and invasion of cervical cancer cells in vitro via cell counting kit-8 (CCK8) assay, colony formation assay, and transwell migration and invasion assay. The demethylase activity of FTO is required for cell proliferation, colony formation, migration, and invasion of cervical cancer cells in vitro. RNA sequencing, online database analysis, and western blotting revealed that FTO regulated the BMP4/Hippo/YAP1/TAZ pathway. In addition, FTO upregulates the expression of BMP4 in an m6A-dependent manner and binds to the N-terminal of BMP4 to form a dimer at the C-terminal in cervical cancer cells through protein-protein interaction. We further discovered that BMP4 treatment promoted cell proliferation, colony formation, migration, and invasion of cervical cancer cells, and rescue experiments validated that BMP4 treatment reversed the inhibition of FTO knockdown on the Hippo/YAP1/TAZ pathway and the progression of cervical cancer cells in vitro. Notably, the knockdown of FTO significantly suppressed xenograft tumor growth and the protein level of BMP4 in vivo. Collectively, our results demonstrate that the FTO promotes cervical cancer progression in vitro and in vivo via the regulation of the BMP4/Hippo/YAP1/TAZ pathway, suggesting that FTO acts as an oncogenic molecule and the FTO/BMP4 Hippo/YAP1/TAZ axis may serve as valuable targets for cervical cancer treatment.

TPM2 attenuates progression of prostate cancer by blocking PDLIM7-mediated nuclear translocation of YAP1.

BACKGROUND: Prostate cancer (PCa) is a common malignant tumor of the genitourinary system. Clinical intervention in advanced PCa remains challenging. Tropomyosins 2 (TPM2) are actin-binding proteins and have been found as a biomarker candidate for certain cancers. However, no studies have explored the role of TPM2 in PCa and its regulatory mechanism. METHODS: TPM2 expression was assessed in Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) PCa patient dataset. The effect of TPM2 on PCa progression was assessed in vitro and in vivo by quantifying proliferation, migration, invasion and tumor growth assays, and the mechanism of TPM2 in PCa progression was gradually revealed by Western blotting, immunoprecipitation, and immunofluorescence staining arrays. RESULTS: TPM2 was found to be severely downregulated in tumor tissues of PCa patients compared with tumor-adjacent normal tissues. In vitro experiments revealed that TPM2 overexpression inhibited PCa cell proliferation, invasion and androgen-independent proliferation. Moreover, TPM2 overexpression inhibited the growth of subcutaneous xenograft tumors in vivo. Mechanistically, this effect was noted to be dependent on PDZ-binding motif of TPM2. TPM2 competed with YAP1 for binding to PDLIM7 through the PDZ-binding motif. The binding of TPM2 to PDLIM7 subsequently inhibited the nuclear transport function of PDLIM7 for YAP1. YAP1 sequestered in the cytoplasm phosphorylated at S127, resulting in its inactivation or degradation which in turn inhibited the expression of YAP1 downstream target genes. CONCLUSIONS: This study investigated the role of TPM2, PDLIM7, and YAP1 in PCa progression and castration resistance. TPM2 attenuates progression of PCa by blocking PDLIM7-mediated nuclear translocation of YAP1. Accordingly, targeting the expression or functional modulation of TPM2, PDLIM7, or YAP1 has the potential to be an effective therapeutic approach to reduce PCa proliferation and prevent the progression of castration-resistant prostate cancer (CRPC).

Tumor-secreted exosomal miR-141 activates tumor-stroma interactions and controls premetastatic niche formation in ovarian cancer metastasis.

BACKGROUND: Metastatic colonization is one of the critical steps in tumor metastasis. A pre-metastatic niche is required for metastatic colonization and is determined by tumor-stroma interactions, yet the mechanistic underpinnings remain incompletely understood. METHODS: PCR-based miRNome profiling, qPCR, immunofluorescent analyses evaluated the expression of exosomal miR-141 and cell-to-cell communication. LC-MS/MS proteomic profiling and Dual-Luciferase analyses identified YAP1 as the direct target of miR-141. Human cytokine profiling, ChIP, luciferase reporter assays, and subcellular fractionation analyses confirmed YAP1 in modulating GROα production. A series of in vitro tumorigenic assays, an ex vivo model and Yap1 stromal conditional knockout (cKO) mouse model demonstrated the roles of miR-141/YAP1/GROα/CXCR1/2 signaling cascade. RNAi, CRISPR/Cas9 and CRISPRi systems were used for gene silencing. Blood sera, OvCa tumor tissue samples, and tissue array were included for clinical correlations. RESULTS: Hsa-miR-141-3p (miR-141), an exosomal miRNA, is highly secreted by ovarian cancer cells and reprograms stromal fibroblasts into proinflammatory cancer-associated fibroblasts (CAFs), facilitating metastatic colonization. A mechanistic study showed that miR-141 targeted YAP1, a critical effector of the Hippo pathway, reducing the nuclear YAP1/TAZ ratio and enhancing GROα production from stromal fibroblasts. Stromal-specific knockout (cKO) of Yap1 in murine models shaped the GROα-enriched microenvironment, facilitating in vivo tumor colonization, but this effect was reversed after Cxcr1/2 depletion in OvCa cells. The YAP1/GROα correlation was demonstrated in clinical samples, highlighting the clinical relevance of this research and providing a potential therapeutic intervention for impeding premetastatic niche formation and metastatic progression of ovarian cancers. CONCLUSIONS: This study uncovers miR-141 as an OvCa-derived exosomal microRNA mediating the tumor-stroma interactions and the formation of tumor-promoting stromal niche through activating YAP1/GROα/CXCRs signaling cascade, providing new insight into therapy for OvCa patients with peritoneal metastases.

Cancer-associated fibroblasts potentiate colorectal cancer progression by crosstalk of the IGF2-IGF1R and Hippo-YAP1 signaling pathways.

Colorectal cancer (CRC) is one of the most common cancers worldwide. The tumor microenvironment exerts crucial effects in driving CRC progression. Cancer-associated fibroblasts (CAFs) serve as one of the most important tumor microenvironment components promoting CRC progression. This study aimed to elucidate the novel molecular mechanisms of CAF-secreted insulin-like growth factor (IGF) 2 in colorectal carcinogenesis. Our results indicated that IGF2 was a prominent factor upregulated in CAFs compared with normal fibroblasts. CAF-derived conditioned media (CM) promoted tumor growth, migration, and invasion of HCT 116 and DLD-1 cells. IGF1R expression is significantly increased in CRC, serving as a potent receptor in response to IGF2 stimulation and predicting unfavorable outcomes for CRC patients. Apart from the PI3K-AKT pathway, RNA-seq analysis revealed that the YAP1-target signature serves as a prominent downstream effector to mediate the oncogenic signaling of IGF2-IGF1R. By single-cell RNA sequencing (scRNA-seq) and immunohistochemical validation, IGF2 was found to be predominantly secreted by CAFs, whereas IGF1R was expressed mainly by cancer cells. IGF2 triggers the nuclear accumulation of YAP1 and upregulates YAP1 target signatures; however, these effects were abolished by either IGF1R knockdown or inhibition with picropodophyllin (PPP), an IGF1R inhibitor. Using CRC organoid and in vivo studies, we found that cotargeting IGF1R and YAP1 with PPP and verteporfin (VP), a YAP1 inhibitor, enhanced antitumor effects compared with PPP treatment alone. In conclusion, this study revealed a novel molecular mechanism by which CAFs promote CRC progression. The findings highlight the translational potential of the IGF2-IGF1R-YAP1 axis as a prognostic biomarker and therapeutic target for CRC. © 2022 The Pathological Society of Great Britain and Ireland.

Virtual screening for small-molecule pathway regulators by image-profile matching.

Identifying the chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual, profile-based screening approach instead identifies compounds that match to pathways based on the phenotypic information in public cell image data, created using the Cell Painting assay. Our straightforward correlation-based computational strategy retrospectively uncovered the expected, known small-molecule regulators for 32% of positive-control gene queries. In prospective, discovery mode, we efficiently identified new compounds related to three query genes and validated them in subsequent gene-relevant assays, including compounds that phenocopy or pheno-oppose YAP1 overexpression and kill a Yap1-dependent sarcoma cell line. This image-profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.

GRK3 is a poor prognosticator and serves as a therapeutic target in advanced gastric adenocarcinoma.

BACKGROUND: G protein-coupled receptor (GPCR) is the most targeted protein family by the FDA-approved drugs. GPCR-kinase 3 (GRK3) is critical for GPCR signaling. Our genomic analysis showed that GRK3 expression correlated with poor prognosis of gastric adenocarcinoma (GAC) patients. However, GRK3's functions and clinical utility in GAC progression and metastases are unknown. METHODS: We studied GRK3 expression in normal, primary, and metastatic GAC tissues. We identified a novel GRK3 inhibitor, LD2, through a chemical-library screen. Through genetic and pharmacologic modulations of GRK3, a series of functional and molecular studies were performed in vitro and in vivo. Impact of GRK3 on YAP1 and its targets was determined. RESULTS: GRK3 was overexpressed in GAC tissues compared to normal and was even higher in peritoneal metastases. Overexpression (OE) of GRK3 was significantly associated with shorter survival. Upregulation of GRK3 in GAC cells increased cell invasion, colony formation, and proportion of ALDH1+ cells, while its downregulation reduced these attributes. Further, LD2 potently and specifically inhibited GRK3, but not GRK2, a very similar kinase to GRK3. LD2 highly suppressed GAC cells' malignant phenotypes in vitro. Mechanistically, GRK3 upregulated YAP1 in GAC tissues and its transcriptional downstream targets: SOX9, Birc5, Cyr61 and CTGF. Knockdown (KD) YAP1 rescued the phenotypes of GRK3 OE in GAC cells. GRK3 OE significantly increased tumor growth but LD2 inhibited tumor growth in the PDX model and dramatically suppressed peritoneal metastases induced by GRK3 OE. CONCLUSIONS: GRK3, a poor prognosticator for survival, conferred aggressive phenotype. Genetic silencing of GRK3 or its inhibitor LD2 blunted GRK3-conferred malignant attributes, suggesting GRK3 as a novel therapeutic target in advanced GAC.

N-Acetyl-l-tryptophan inhibits CCl4-induced hepatic fibrogenesis via regulating TGF-ß1/SMAD and Hippo/YAP1 signal.

BACKGROUND: Although 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. PURPOSE: The present study investigated the effect of l-NAT on liver fibrosis and explored it potential molecular mechanism. METHODS: To 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. RESULT: l-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-ß1/SMAD and matrix metalloproteinase 9 (MMP9) proteins, and reversed the expression of YAP1 protein in CCl4-induced liver fibrosis. CONCLUSION: These results clearly demonstrated that l-NAT attenuated CCl4-induced liver fibrosis in mice, and this protective mechanism might relate to TGF-ß1/SMAD and Hippo/YAP1 signaling pathway. Thus, this study provided data basis for the prevention and treatment of liver fibrosis.

Wnt5A and TGFß1 Converges through YAP1 Activity and Integrin Alpha v Up-Regulation Promoting Epithelial to Mesenchymal Transition in Ovarian Cancer Cells and Mesothelial Cell Activation.

In this paper, we investigate whether Wnt5A is associated with the TGF-ß1/Smad2/3 and Hippo-YAP1/TAZ-TEAD pathways, implicated in epithelial to mesenchymal transition (EMT) in epithelial ovarian cancer. We used 3D and 2D cultures of human epithelial ovarian cancer cell lines SKOV-3, OVCAR-3, CAOV-4, and different subtypes of human serous ovarian cancer compared to normal ovary specimens. Wnt5A showed a positive correlation with TAZ and TGFß1 in high- and low-grade serous ovarian cancer specimens compared to borderline serous and normal ovaries. Silencing Wnt5A by siRNAs significantly decreased Smad2/3 activation and YAP1 expression and nuclear shuttling in ovarian cancer (OvCa) cells. Furthermore, Wnt5A was required for TGFß1-induced cell migration and invasion. In addition, inhibition of YAP1 transcriptional activity by Verteporfin (VP) altered OvCa cell migration and invasion through decreased Wnt5A expression and inhibition of Smad2/3 activation, which was reverted in the presence of exogenous Wnt5A. We found that the activation of TGFß1 and YAP1 nuclear shuttling was promoted by Wnt5A-induced integrin alpha v. Lastly, Wnt5A was implicated in activating human primary omental mesothelial cells and subsequent invasion of ovarian cancer cells. Together, we propose that Wnt5A could be a critical mediator of EMT-associated pathways.

USP21 regulates Hippo signaling to promote radioresistance by deubiquitinating FOXM1 in cervical cancer.

The ectopic expression of ubiquitin-specific peptidase 21 (USP21) is common in different types of cancer. However, its relationship with radio-sensitivity in cervical cancer (CC) remains unclear. In this study, we aimed to uncover the effect of USP21 on CC radio-resistance and its underlying mechanism. Our results showed that the expression of USP21 was markedly increased in CC tissues of radio-resistant patients and CC cells treated with radiation. Besides, knockdown of USP21 restrained the survival fractions, and facilitated apoptosis of CC cells in the absence or presence of radiation. Additionally, USP21 in combination with FOXM1 regulated the stability and ubiquitination of FOXM1. However, FOXM1 reversed the effects of USP21 knockdown on the radio-resistance of CC cells. Furthermore, FOXM1 knockdown activated the Hippo pathway by inhibiting the nuclear translocation of Yes-associated protein 1 (YAP1), and FOXM1 knockdown attenuated the radio-resistance of CC cells via inhibiting the Hippo-YAP1 pathway. USP21 activated the Hippo pathway by mediating FOXM1. Knockdown of USP21 enhanced the radio-sensitivity of CC cells in vivo. In summary, USP21 contributed to the radio-resistance of CC cells via FOXM1/Hippo signaling, and may serve as a promising target for radio-sensitizers in the radiotherapy of CC.

GPNMB promotes the progression of diffuse large B cell lymphoma via YAP1-mediated activation of the Wnt/ß-catenin signaling pathway.

Glycoprotein non-metastatic melanoma protein B (GPNMB) has been confirmed to be related to the pathogenesis of tumors. However, the potential impact of GPNMB on the progression of diffuse large B-cell lymphoma (DLBCL) is unclear. In this study, the expression levels of GPNMB and Yes-associated protein (YAP) were analyzed using qRT-PCT and Western blot assay. Cell counting kit-8, EdU, and flow cytometry assays were used to detect the proliferation and apoptosis of DLBCL cells. A nude mice xenograft model was established for in vivo research. Results showed that GPNMB and YAP1 were upregulated in DLBCL cell lines. Knockdown of GPNMB inhibited cell proliferation and promoted apoptosis in DLBCL cells. Additionally, the expression levels of YAP1 and the downstream effector of Hippo pathway (c-myc) were markedly decreased when GPNMB was knocked down. Moreover, knockdown of GPNMB inhibited the nuclear translocation of ß-catenin protein, which could be abolished by YAP1 overexpression. Simultaneously, the anti-proliferative and pro-apoptotic effects of GPNMB knockdown could be reversed by YAP1 overexpression or LiCl (the activator of Wnt/ß-catenin pathway). Furthermore, the mice xenograft model confirmed that inhibition of GPNMB restrained the tumorigenesis of DLBCL in vivo. In conclusion, GPNMB could partly activate the Wnt/ß-catenin signaling pathway by targeting YAP1, so as to participate in tumorigenesis of DLBCL.

Upregulated microRNA let-7a accelerates apoptosis and inhibits proliferation in uterine junctional zone smooth muscle cells in adenomyosis under conditions of a normal activated hippo-YAP1 axis.

BACKGROUND: Let-7a is a small non-coding RNA that has been found to take part in cell proliferation and apoptosis. The hippo-YAP1 axis, known as a tumour suppressor pathway, also plays an important role in cell proliferation and apoptosis. YAP1, TAZ, and phospho-YAP1 play key roles in actions of the hippo-YAP1 axis. Adenomyosis (ADS) is a proliferative disease leading to a large uterus in patients with prolonged illness. Abnormal proliferation of smooth muscle cells (SMCs) in the uterine endometrial-myometrial junctional zone (JZ) is an important reason for developing ADS. This study aimed to explore the expression levels of let-7a and components of the hippo-YAP1 axis in SMCs in the uterine endometrial-myometrial JZ in ADS and to explore the roles of let-7a and the hippo-YAP1 axis of JZ SMC proliferation and apoptosis in ADS. METHODS: We collected JZ tissues for the primary culture of SMCs from 25 women diagnosed with ADS and 27 women without ADS. We used quantitative real-time polymerase chain reaction and western blotting to measure the mRNA and protein expression levels of let-7a, YAP1, TAZ, and phospho-YAP1 in ADS JZ SMCs. A CCK-8 assay and flow cytometry analysis of apoptosis were utilized to test the proliferation and apoptosis of JZ SMCs. The let-7a overexpression lentiviral vector GV280 was used to increase the expression level of let-7a. We added verteporfin to block the phosphorylation of components of the hippo-YAP1 axis. RESULTS: We found that the let-7a level was decreased, while the YAP1 and TAZ levels were increased in ADS JZ SMCs. Upregulated let-7a affected the expression levels of components of the hippo-YAP1 axis, accelerated apoptosis, and inhibited proliferation in JZ SMCs. Furthermore, accumulated YAP1 led to increasing proliferation of JZ SMCs after verteporfin treatment to block the phosphorylation of components of the hippo-YAP1 axis. If components of the hippo-YAP1 axis were unphosphorylated, upregulated let-7a could not inhibit the proliferation of ADS JZ SMCs. Upregulated let-7a could not activate the hippo-YAP1 axis in verteporfin treatment. CONCLUSIONS: Our findings suggest that the let-7a and hippo-YAP1 axis may act as important regulators of JZ SMCs proliferation, and upregulated let-7a may be an effective method to treat ADS.

Immortalization up-regulated protein promotes tumorigenesis and inhibits apoptosis of papillary thyroid cancer.

The incidence of thyroid cancer is increasing in recent years worldwide, but the underlying mechanisms await further exploration. We utilized the bioinformatic analysis to discover that Immortalization up-regulated protein (IMUP) could be a potential oncogene in the papillary thyroid cancer (PTC). We verified this finding in several databases and locally validated cohorts. Clinicopathological features analyses showed that high expression of IMUP is positively related to malignant clinicopathological features in PTC. Braf-like PTC patients with higher IMUP expression had shorter disease-free survival. The biological function of IMUP in PTC cell lines (KTC-1 and TPC-1) was investigated using small interfering RNA. Our results showed that silencing IMUP suppresses proliferation, migration and invasion while inducing apoptosis in PTC cell lines. Changes of the expression of apoptosis-related molecules were identified by real-time quantitative polymerase chain reaction and Western blotting. We also found that YAP1 and TAZ, the critical effectors in the Hippo pathway, were down-regulated when the IMUP is silenced. Rescue experiments showed that overexpression of YAP1 reverses the tumour inhibitory effect caused by IMUP knockdown. Our study demonstrated that IMUP has an oncogenic function in PTC and might be a new target gene in the treatment of PTC.

Up-regulated acylglycerol kinase (AGK) expression associates with gastric cancer progression through the formation of a novel YAP1-AGK-positive loop.

Acylglycerol kinase (AGK) uses adenosine triphosphate (ATP) and acylglycerol to generate adenosine diphosphate (ADP) and acyl-sn-glycerol 3-phosphate in cells. Recent evidence has demonstrated that dysregulated AGK expression is associated with the development of various human cancers. This study investigated the effects of AGK on gastric cancer cell proliferation and carcinogenesis and explored the underlying molecular events. AGK expression was up-regulated in gastric cancer and was associated with poor prognosis in gastric cancer patients. AGK overexpression increased gastric cancer proliferation, invasion capacity and the expression of the epithelial-mesenchymal transition markers in vitro. Conversely, the knockdown of AGK expression reduced gastric cancer cell proliferation in vitro and in nude mouse tumour cell xenografts. Importantly, AGK expression was associated with the YAP1 expression in gastric cancer cells and tissues. YAP1 expression also transcriptionally induced AGK expression through the binding of TEAD to the AGK gene promoter. However, AGK expression inhibited the activation of the Hippo pathway proteins and induced YAP1 nuclear localization to enhance the transcription activity of YAP1/TEADs. In conclusion, the study demonstrates that AGK is not only a novel target of the Hippo-YAP1 pathway, but that it also positively regulates YAP1 expression, thus forming a YAP1-AGK-positive feedback loop.

Endogenous YAP1 activation drives immediate onset of cervical carcinoma in situ in mice.

Cervical cancer (CC) is usually initiated by infection with high-risk types of human papillomavirus (HPV). The HPV E6 and E7 proteins target p53 and RB, respectively, but other cellular targets likely exist. We generated uterus-specific MOB1A/B double KO (uMob1DKO) mice, which immediately developed cervical squamous cell carcinoma in situ. Mutant cervical epithelial cells showed YAP1-dependent hyperproliferation, altered self-renewal, impaired contact inhibition, and chromosomal instability. p53 activation was increased in uMob1DKO cells, and additional p53 loss in uMob1DKO mice accelerated tumor invasion. In human CC, strong YAP1 activation was observed from the precancerous stage. Human cells overexpressing HPV16 E6/E7 showed inactivation of not only p53 and RB but also PTPN14, boosting YAP1 activation. Estrogen, cigarette smoke condensate, and PI3K hyperactivation all increased YAP1 activity in human cervical epithelial cells, and PTPN14 depletion along with PI3K activation or estrogen treatment further enhanced YAP1. Thus, immediate CC onset may initiate when YAP1 activity exceeds an oncogenic threshold, making Hippo-YAP1 signaling a major CC driver.

Targeting Hippo coactivator YAP1 through BET bromodomain inhibition in esophageal adenocarcinoma.

Hippo/YAP1 signaling is a major regulator of organ size, cancer stemness, and aggressive phenotype. Thus, targeting YAP1 may provide a novel therapeutic strategy for tumors with high YAP1 expression in esophageal cancer (EC). Chromatin immunoprecipitation (ChiP) and quantitative ChiP-PCR were used to determine the regulation of the chromatin remodeling protein bromodomain-containing protein 4 (BRD4) on YAP1. The role of the bromodomain and extraterminal motif (BET) inhibitor JQ1, an established BRD4 inhibitor, on inhibition of YAP1 in EC cells was dissected using western blot, immunofluorescence, qPCR, and transient transfection. The antitumor activities of BET inhibitor were further examined by variety of functional assays, cell proliferation (MTS), tumorsphere, and ALDH1+ labeling in vitro and in vivo. Here, we show that BRD4 regulates YAP1 expression and transcription. ChiP assays revealed that BRD4 directly occupies YAP1 promoter and that JQ1 robustly blocks BRD4 binding to the YAP1 promoter. Consequently, JQ1 strongly suppresses constitutive or induced YAP1 expression and transcription in EC cells and YAP1/Tead downstream transcriptional activity. Intriguingly, radiation-resistant cells that acquire strong cancer stem cell traits and an aggressive phenotype can be effectively suppressed by JQ1 as assessed by cell proliferation, tumorsphere formation, and reduction in the ALDH1+ cells. Moreover, effects of JQ1 are synergistically amplified by the addition of docetaxel in vitro and in vivo. Our results demonstrate that BRD4 is a critical regulator of Hippo/YAP1 signaling and that BRD4 inhibitor JQ1 represents a new class of inhibitor of Hippo/YAP1 signaling, primarily targeting YAP1 high and therapy-resistant cancer cells enriched with cancer stem cell properties.