TOPflash Activity Research Articles

A small molecule compound 759 inhibits the wnt/beta-catenin signaling pathway via increasing the Axin protein stability.

Wnt/β-catenin signaling plays important role in cancers. Compound 759 is one of the compounds previously screened to identify inhibitors of the Wnt/β-catenin pathway in A549 cells [Lee et al. in Bioorg Med Chem Lett 20:5900-5904, 2010]. However, the mechanism by which Compound 759 induces the inhibition of the Wnt/β-catenin pathway remains unknown. In our study, we employed various assays to comprehensively evaluate the effects of Compound 759 on lung cancer cells. Our results demonstrated that Compound 759 significantly suppressed cell proliferation and Wnt3a-induced Topflash activity and arrested the cell cycle at the G1 stage. Changes in Wnt/β-catenin signaling-related protein expression, gene activity, and protein stability including Axin, and p21, were achieved through western blot and qRT-PCR analysis. Compound 759 treatment upregulated the mRNA level of p21 and increased Axin protein levels without altering the mRNA expression in A549 cells. Co-treatment of Wnt3a and varying doses of Compound 759 dose-dependently increased the amounts of Axin1 in the cytosol and inhibited β-catenin translocation into the nucleus. Moreover, Compound 759 reduced tumor size and weight in the A549 cell-induced tumor growth in the in vivo tumor xenograft mouse model. Our findings indicate that Compound 759 exhibits potential anti-cancer activity by inhibiting the Wnt/β-catenin signaling pathway through the increase of Axin1 protein stability.

The anti-HIV drug abacavir stimulates β-catenin activity in osteoblast lineage cells.

Bone mineral density (BMD) loss in people living with HIV occurs with the initiation of combined antiretroviral therapy (cART), particularly with tenofovir disoproxil fumarate (TDF) containing cART. Switching from TDF to abacavir (ABC) or dolutegravir (DTG) leads to increased BMD. Whether BMD gains are due to cessation of TDF or anabolic effects of ABC or DTG is unclear. We investigated the effects of ABC and DTG on osteoblast lineage cells in vitro and in vivo. Primary human osteoblasts and male C57BL/6 mice were treated with individual antiretrovirals (ARVs) or a combination of ABC/DTG/lamivudine (3TC). Nearly all ARVs and cART inhibited osteogenic activity in vitro. Due to the importance of Wnt/β-catenin in bone formation, we further investigated ARV effects on the Wnt/β-catenin pathway. ABC, alone and as part of ABC/DTG/3TC, increased osteoblastic β-catenin activity as indicated by increased TOPFlash activity, hypo-phosphorylated (active) β-catenin staining, and β-catenin targeted gene expression. Mice treated with TDF had decreased lumbar spine BMD and trabecular connectivity density in the vertebrae, while those treated with ABC/DTG/3TC reduced cortical area and thickness in the femur. Mice treated with ABC alone had no bone structural changes, increased circulating levels of the bone formation marker, P1NP, and elevated expression of the Wnt/β-catenin target gene, Lef1, in osteocyte enriched samples. Further, bones from ARV-treated mice were isolated to evaluate ARV distribution. All ARVs were detected in the bone tissue, which was inclusive of bone marrow, but when bone marrow was removed, only TDF, ABC, and DTG were detected at ~0.1% of the circulating levels. Overall, our findings demonstrate that ABC activates Wnt/β-catenin signaling, but whether this leads to increased bone formation requires further study. Assessing the impact of ARVs on bone is critical to informing ARV selection and/or discovery of regimens that do not negatively impact the skeleton.

Effects of Hyperbaric Oxygen Intervention on the Degenerated Intervertebral Disc: From Molecular Mechanisms to Animal Models.

MicroRNA (miRNA) 107 expression is downregulated but Wnt3a protein and β-catenin are upregulated in degenerated intervertebral disc (IVD). We investigated mir-107/Wnt3a-β-catenin signaling in vitro and in vivo following hyperbaric oxygen (HBO) intervention. Our results showed 96 miRNAs were upregulated and 66 downregulated in degenerated nucleus pulposus cells (NPCs) following HBO treatment. The 3' untranslated region (UTR) of the Wnt3a mRNA contained the "seed-matched-sequence" for miR-107. MiR-107 was upregulated and a marked suppression of Wnt3a was observed simultaneously in degenerated NPCs following HBO intervention. Knockdown of miR-107 upregulated Wnt3a expression in hyperoxic cells. HBO downregulated the protein expression of Wnt3a, phosphorylated LRP6, and cyclin D1. There was decreased TOP flash activity following HBO intervention, whereas the FOP flash activity was not affected. HBO decreased the nuclear translocation of β-catenin and decreased the secretion of MMP-3 and -9 in degenerated NPCs. Moreover, rabbit serum KS levels and the stained area for Wnt3a and β-catenin in repaired cartilage tended to be lower in the HBO group. We observed that HBO inhibits Wnt3a/β-catenin signaling-related pathways by upregulating miR-107 expression in degenerated NPCs. HBO may play a protective role against IVD degeneration and could be used as a future therapeutic treatment.

Marinobazzanan, a Bazzanane-Type Sesquiterpenoid, Suppresses the Cell Motility and Tumorigenesis in Cancer Cells.

Marinobazzanan (1), a new bazzanane-type sesquiterpenoid, was isolated from a marine-derived fungus belonging to the genus Acremonium. The chemical structure of 1 was elucidated using NMR and mass spectroscopic data, while the relative configurations were established through the analysis of NOESY data. The absolute configurations of 1 were determined by the modified Mosher's method as well as vibrational circular dichroism (VCD) spectra calculation and it was determined as 6R, 7R, 9R, and 10R. It was found that compound 1 was not cytotoxic to human cancer cells, including A549 (lung cancer), AGS (gastric cancer), and Caco-2 (colorectal cancer) below the concentration of 25 μM. However, compound 1 was shown to significantly decrease cancer-cell migration and invasion and soft-agar colony-formation ability at concentrations ranging from 1 to 5 μM by downregulating the expression level of KITENIN and upregulating the expression level of KAI1. Compound 1 suppressed β-catenin-mediated TOPFLASH activity and its downstream targets in AGS, A549, and Caco-2 and slightly suppressed the Notch signal pathway in three cancer cells. Furthermore, 1 also reduced the number of metastatic nodules in an intraperitoneal xenograft mouse model.

Revisiting Lightning Activity and Parameterization Using Geostationary Satellite Observations

The Geostationary Lightning Mapper (GLM) on the Geostationary Operational Environmental Satellite 16 (GOES-16) detects total lightning continuously, with a high spatial resolution and detection efficiency. Coincident data from the GLM and the Advanced Baseline Imager (ABI) are used to explore the correlation between the cloud top properties and flash activity across the continental United States (CONUS) sector from May to September 2020. A large number of collocated infrared (IR) brightness temperature (TBB), cloud top height (CTH) and lightning data provides robust statistics. Overall, the likelihood of lightning occurrence and high flash density is higher if the TBB is colder than 225 K. The higher CTH is observed to be correlated with a larger flash rate, a smaller flash size, stronger updraft, and larger optical energy. Furthermore, the cloud top updraft velocity (w) is estimated based on the decreasing rate of TBB, but it is smaller than the updraft velocity of the convective core. As a result, the relationship between CTH and lightning flash rate is investigated independently of w over the continental, oceanic and coastal regimes in the tropics and mid-latitudes. When the CTH is higher than 12 km, the flash rates of oceanic lightning are 38% smaller than those of both coastal and continental lightning. In addition, it should be noted that more studies are necessary to examine why the oceanic lightning with low clouds (CTH < 8 km) has higher flash rates than lightning over land and coast. Finally, the exponents of derived power relationship between CTH and lightning flash rate are smaller than four, which is underestimated due to the GLM detection efficiency and the difference between IR CTH and 20 dBZ CTH. The results from combining the ABI and GLM products suggest that merging multiple satellite datasets could benefit both lightning activity and parameterization studies, although the parallax corrections should be considered.

Residue 6.43 defines receptor function in class F GPCRs

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P6.43. Functional comparison of FZD and FZD P6.43F mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO.

USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling.

The tumour suppressors RNF43 and ZNRF3 play a central role in development and tissue homeostasis by promoting the turnover of the Wnt receptors LRP6 and Frizzled (FZD). The stem cell growth factor R‐spondin induces auto‐ubiquitination and membrane clearance of ZNRF3/RNF43 to promote Wnt signalling. However, the deubiquitinase stabilising ZNRF3/RNF43 at the plasma membrane remains unknown. Here, we show that the USP42 antagonises R‐spondin by protecting ZNRF3/RNF43 from ubiquitin‐dependent clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R‐spondin‐LGR4‐ZNRF3 ternary complex by deubiquitinating ZNRF3. Accordingly, USP42 increases the turnover of LRP6 and Frizzled (FZD) receptors and inhibits Wnt signalling. Furthermore, we show that USP42 functions as a roadblock for paracrine Wnt signalling in colon cancer cells and mouse small intestinal organoids. We provide new mechanistic insights into the regulation R‐spondin and conclude that USP42 is crucial for ZNRF3/RNF43 stabilisation at the cell surface.

Identification of dinactin, a macrolide antibiotic, as a natural product-based small molecule targeting Wnt/β-catenin signaling pathway in cancer cells

Despite the fact that hyper-activation of Wnt/β-catenin signaling pathway has been seen in many cancers, including liver, colorectal and lung carcinoma, no small molecule inhibitors are available that specifically target this pathway. In this study, we analyzed the impact of dinactin (DA), an antibiotic ionophore produced by Streptomycesspecies, as an effective small molecule targeting Wnt/β-catenin signaling pathway in cancer cells. We performed MTT assays to investigate cell viability and proliferation after exposure to small molecules. Protein expression analysis was carried out by western blotting. Top-Flash reporter assays were used to score for β-catenin signaling and cell cycle analysis was carried out by flow cytometry. In the first set of experiments, DA was seen to selectively inhibit the proliferation of HCT-116 and HepG2 cancer cells, unlike HEK-293 cells (a lowtumorigenic cell line), in apoptosis-independent manner. Further, DA was seen to block the G1/S progression and decrease the expression of cyclin D1 in cancer cells. Since cyclin D1 is the downstream target gene of Wnt/β-catenin signaling, we examined the impact of DA on TCF-dependent β-catenin activity using Top-Flash reporter assay. Interestingly, DA significantly decreased Top-Flash activity at lower nano-molar concentrations when compared with salinomycin in HCT-116 and HepG2 cells. We report the identification of dinactin as a natural product-based small molecule that effectively blocks the Wnt/β-catenin signaling pathway in cancer cells at nano-molar concentration. We anticipate that DA could be developed as a novel drug for anti-cancer therapy and for the management of neuropathic pain.

Long non-coding RNA LINC01503 predicts worse prognosis in glioma and promotes tumorigenesis and progression through activation of Wnt/β-catenin signaling.

Long-noncoding RNAs (lncRNAs) have been recently shown to be involved in the regulation of numerous biological processes including tumor progression. In this study, we aimed to explore the role of lncRNA LINC01503 (LINC01503) in the development and progression of glioma. Relative levels of LINC01503 were evaluated in tumor tissues from 133 patients with glioma as well as from cultured glioma cell lines. The correlation among LINC01503 levels, pathological types, and survivals of glioma patients were also determined using the Kaplan-Meier method and multivariate analysis. Next, we investigated the effect of LINC01503 on the proliferation, colony formation, apoptosis, migration and invasion in the U251 and LN299 cells. Relative protein expression was analyzed by Western blot assays. We found that LINC01503 expression level was significantly up-regulated in glioma tissue and cells, and that its overexpression was significantly correlated with KPS, tumor size and WHO grade in glioma patients. Kaplan-Meier analysis showed that patients with higher levels of LINC01503 had significantly poorer overall survival and disease-free survival than those with lower expression of this lncRNA in glioma patients. Multivariate analysis further confirmed that LINC01503 is an independent prognostic factor in patients with glioma. Functional assays with in vitro showed that knockdown of LINC01503 in the U251 and LN299 cell lines suppressed cells growth, colony formation, invasion and migration, and promoted apoptosis. Mechanistic investigation showed that LINC01503 can modulate Wnt/β-catenin signaling, as determined by that knockdown of LINC01503 decreased the TOP-FLASH activity and β-catenin, cyclin D1 and c-myc. Our findings suggested that LINC01503 conferred oncogenic function in glioma and may be a new prognostic biomarker and novel therapeutic strategy for this malignancy.

SRC-like adaptor protein negatively regulates Wnt signaling in intrahepatic cholangiocarcinoma.

Currently, the molecular mechanisms underlying intrahepatic cholangiocarcinoma (IHCC) are poorly understood. In the present study, the focus was primarily on SRC-like adaptor protein (SLAP), an adaptor protein, which is aberrantly expressed in various cancer types. To the best of our knowledge, the present study was the first to demonstrate that SLAP was decreased in IHCC tissues and cells, compared with controls. Further study indicated that SLAP overexpression suppressed IHCC cell proliferation and induced cell cycle arrest, indicating the tumor suppressor role of SLAP in IHCC progression. To demonstrate the effects of SLAP on Wnt signaling, the β-catenin/T cell factor transcription reporter assay was conducted. Compared with the negative adenovirus vector control (Ad-NC), overexpression of SLAP reduced TOPflash activity, and no changes in FOPflash activity were identified. Furthermore, the expression levels of Wnt target genes, including β-catenin, c-Myc, cluster of differentiation 44, Slug, Vimentin and matrix metallopeptidase-9, were reduced in RBE and Huh28 cells overexpressing SLAP. Additionally, the effects of SLAP on IHCC cell invasion and migration were determined. Compared with the Ad-NC control, the migration and invasion capacity was reduced following overexpression of SLAP in RBE and Huh28 cells. In summary, reduced SLAP expression may enhance IHCC malignant progression by activating Wnt signaling.

Abstract 3864: Development of high-specific and sensitive reporter assays for the detection of Wnt/β-catenin signaling activity

Abstract The Wnt/β-catenin signaling pathway is involved in development, tissue homeostasis, and regeneration. Since aberrant activation of this pathway plays a crucial role in human carcinogenesis, inhibition of this pathway has been shown to be a promising therapeutic strategy for cancers with de-regulated Wnt signaling. Although cell-based assays using synthetic TCF/LEF reporters (also known as TOPFLASH) have contributed to the discovery of small molecules that modulate Wnt signaling pathway, these assays often raise a problem of the specificity in the screening. In this study, we aimed to develop a specific reporter for the detection of Wnt signaling activity. We performed integrated transcriptome analysis and found that expression of histidine ammonia-lyase gene (HAL) is negatively regulated by β-catenin/TCF. In addition to TOPFLASH, we leveraged a promoter region of HAL as another transcriptional readout of Wnt signaling. Subsequently, HepG2 cells stably expressing both optimized HAL promoter reporter and TOPFLASH reporter were established. Since inhibition of the β-catenin/TCF transcriptional activity decreased TOPFLASH activity and simultaneously increased HAL reporter activity, we termed this assay as a bidirectional reporter assay. To verify the effectiveness of this assay, we compared the number of hits in the screening of a test library using the bidirectional assay with that using the conventional TOPFLASH/FOPFLASH assay. Among the 361 compounds in the library, the bidirectional reporter assay identified 9 hits, while the TOPFLASH/FOPFLASH assay did 79 hits. Four compounds were overlapped between these assays, suggesting that the bidirectional assay decreased false positives compared with the conventional assay. The concept of bidirectional assay might be useful for the screening of compounds that regulate other transcriptional factors and/or disease-associated signaling pathways. Citation Format: Kiyoshi Yamaguchi, Chi Zhu, Tomoyuki Ohsugi, Akari Saku, Tsuneo Ikenoue, Yoichi Furukawa. Development of high-specific and sensitive reporter assays for the detection of Wnt/β-catenin signaling activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3864.

Mechanism of MicroRNA-708 Targeting BAMBI in Cell Proliferation, Migration, and Apoptosis in Mice With Melanoma via the Wnt and TGF-β Signaling Pathways.

Objective:The aim of this study was to evaluate the mechanisms involved with miRNA-708 and its targeting of bone morphogenetic protein and activin membrane-bound inhibitor in cell proliferation, migration, and apoptosis in mice with melanoma via the Wnt and transforming growth factor β signaling pathways.Methods:Sixty mice were recruited of which 40 were subsequently assigned into the experimental group (22 mice were successfully established as melanoma model and 18 mice used in tumor xenograft), and the normal control group consisted of 20 mice. B16 cells were assigned to the normal, blank, and negative control, miR-708 mimics, miR-708 inhibitors, si-BAMBI, and miR-708 inhibitors + si-bone morphogenetic protein and activin membrane-bound inhibitor groups. Western blotting and reverse transcription quantitative polymerase chain reaction were employed to detect the expression levels within the tissues and cell lines. TCF luciferase reporter (TOP-FLASH) or a control vector (FOP-FLASH) was applied to detect the activity of the Wnt signaling pathway. MTT3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay, flow cytometry, scratch test, and Transwell assay were conducted, respectively, for cell proliferation, apoptosis, migration, and invasion, while tumor xenograft procedures were performed on the nude mice recruited for the study.Results:Compared to the normal control group, the model group displayed increased expressions of bone morphogenetic protein and activin membrane-bound inhibitor, Wnt10B, P53, and Bcl-2; TOPflash activity; β-catenin expression; cell proliferation; migration; and invasion capabilities while decreased expressions of miR-708, vascular endothelial growth factor, Fas, Bax, Caspase-3, and cleaved Caspase-3 and apoptosis rate. Compared to the blank and negative control groups, the miR-708 mimics and small-interfering RNA-bone morphogenetic protein and activin membrane-bound inhibitor groups exhibited decreases expressions of bone morphogenetic protein and activin membrane-bound inhibitor, Wnt10B, P53, and Bcl-2 and decreased proliferation, migration, and invasion capabilities, while increases in the apoptosis rate, expressions of vascular endothelial growth factor, Fas, Bax, Caspase-3, and cleaved Caspase-3; however, downregulated levels of TOPflash activity and β-catenin expression were recorded. The miR-708 inhibitors group displayed an opposite trend.Conclusion:Downregulation of miR-708-targeted bone morphogenetic protein and activin membrane-bound inhibitor inhibits the proliferation and migration of melanoma cells through the activation of the transforming growth factor β pathway and the suppression of Wnt pathway.

The lichen secondary metabolite atranorin suppresses lung cancer cell motility and tumorigenesis

Lichens are symbiotic organisms that produce various secondary metabolites. Here, different lichen extracts were examined to identify secondary metabolites with anti-migratory activity against human lung cancer cells. Everniastrum vexans had the most potent inhibitory activity, and atranorin was identified as an active subcomponent of this extract. Atranorin suppressed β-catenin-mediated TOPFLASH activity by inhibiting the nuclear import of β-catenin and downregulating β-catenin/LEF and c-jun/AP-1 downstream target genes such as CD44, cyclin-D1 and c-myc. Atranorin decreased KAI1 C-terminal interacting tetraspanin (KITENIN)-mediated AP-1 activity and the activity of the KITENIN 3′-untranslated region. The nuclear distribution of the AP-1 transcriptional factor, including c-jun and c-fos, was suppressed in atranorin-treated cells, and atranorin inhibited the activity of Rho GTPases including Rac1, Cdc42, and RhoA, whereas it had no effect on epithelial-mesenchymal transition markers. STAT-luciferase activity and nuclear STAT levels were decreased, whereas total STAT levels were moderately reduced. The human cell motility and lung cancer RT² Profiler PCR Arrays identified additional atranorin target genes. Atranorin significantly inhibited tumorigenesis in vitro and in vivo. Taken together, our results indicated that E. vexans and its subcomponent atranorin may inhibit lung cancer cell motility and tumorigenesis by affecting AP-1, Wnt, and STAT signaling and suppressing RhoGTPase activity.

Cytotoxic activities of Telectadium dongnaiense and its constituents by inhibition of the Wnt/β-catenin signaling pathway

BackgroundWnt/β-catenin signaling pathway is a potential target for the treatment of human colon cancer. Thus, the inhibitory effects of various plant extracts on cell proliferation and Wnt signal transduction were evaluated to discover a Wnt signaling inhibitor. PurposeThe present study aimed to investigate the cytotoxicity involved in Wnt pathway of the MeOH extract from Telectadium dongnaiense bark (TDB) and to identify its bioactive constituents by bioassay-guided fractionation. MethodsThe sulforhodamine B-based proliferation assay and the β-catenin/TCF-responsive reporter gene assay were employed as screening systems. The isolation and identification of compounds were elucidated on the basis of spectroscopic methods. Inhibitory effects on the expression levels of Wnt target genes were determined by real-time PCR and western blotting. ResultsThe extract of TDB most strongly inhibited cell proliferation and TOPflash activity (IC50 = 1.5 and 2.0 µg/ml), which was correlated with its inhibitory effects on the expression of Wnt target genes. Three major compounds were isolated from bioactive fractions and were identified as 1,4-dicaffeoylquinic acid (1), quercetin 3-rutinoside (2), and periplocin (3). Only compound 3 showed anti-proliferative activity (IC50 = 0.06 µM) and exhibited Wnt signaling inhibitory effects in HCT116 colon cancer cells. ConclusionsThis study contributes to understanding the cytotoxic properties of TDB extract and its constituents and provides a potent strategy for its further application.

Exposure to febrile-range hyperthermia potentiates Wnt signalling and epithelial–mesenchymal transition gene expression in lung epithelium

Background: As environmental and body temperatures vary, lung epithelial cells experience temperatures significantly different from normal core temperature. Our previous studies in human lung epithelium showed that: (i) heat shock accelerates wound healing and activates profibrotic gene expression through heat shock factor-1 (HSF1); (ii) HSF1 is activated at febrile temperatures (38–41 °C) and (iii) hypothermia (32 °C) activates and hyperthermia (39.5 °C) reduces expression of a subset of miRNAs that target protein kinase-Cα (PKCα) and enhance proliferation.Methods: We analysed the effect of hypo- and hyperthermia exposure on Wnt signalling by exposing human small airway epithelial cells (SAECs) and HEK293T cells to 32, 37 or 39.5 °C for 24 h, then analysing Wnt-3a-induced epithelial–mesenchymal transition (EMT) gene expression by qRT-PCR and TOPFlash reporter plasmid activity. Effects of miRNA mimics and inhibitors and the HSF1 inhibitor, KNK437, were evaluated.Results: Exposure to 39.5 °C for 24 h increased subsequent Wnt-3a-induced EMT gene expression in SAECs and Wnt-3a-induced TOPFlash activity in HEK293T cells. Increased Wnt responsiveness was associated with HSF1 activation and blocked by KNK437. Overexpressing temperature-responsive miRNA mimics reduced Wnt responsiveness in 39.5 °C-exposed HEK293T cells, but inhibitors of the same miRNAs failed to restore Wnt responsiveness in 32 °C-exposed HEK293T cells.Conclusions: Wnt responsiveness, including expression of genes associated with EMT, increases after exposure to febrile-range temperature through an HSF1-dependent mechanism that is independent of previously identified temperature-dependent miRNAs. This process may be relevant to febrile fibrosing lung diseases, including the fibroproliferative phase of acute respiratory distress syndrome (ARDS) and exacerbations of idiopathic pulmonary fibrosis (IPF).

Reversal effect of ouabain on multidrug resistance in esophageal carcinoma EC109/CDDP cells by inhibiting the translocation of Wnt/β-catenin into the nucleus.

The incidence of esophageal carcinoma is increasing throughout the world. A major obstacle to its treatment is acquired multidrug resistance (MDR) which results in the failure of chemotherapy and patient relapse. Here, we identified that ouabain is capable of reversing MDR to cisplatin (CDDP) in EC109/CDDP cells and explore the possible mechanisms of action. The parental and the MDR cell lines were both sensitive to ouabain with 50% inhibitory concentration (IC50) values of 258.11 and 710.63nM, respectively. Cisplatin cytotoxicity increased in the EC109/CDDP cells by the addition of ouabain which helps promote CDDP-induced apoptosis. Ouabain at 20nM effectively reduced the IC50 of CDDP in EC109/CDDP cells from 36.54 to 3.39μM. This represents a 10.78-fold increase in sensitization to CDDP. We also found that ouabain was capable of down regulating the expression of P-glycoprotein (P-gp) and Bcl-2 in a dose- and time-dependent manner. Finally, the results indicated that ouabain suppressed Wnt luciferase report (TOPFlash) activity obviously in EC109/CDDP and depressed the translocation of β-catenin into the nucleus. Moreover, ouabain showed reversal effect of MDR to CDDP in nude mouse xenograft model, and reduced the protein level of β-catenin (Y333) in tumor tissue of CDDP plus ouabain group. All data proved that ouabain has a potent β-catenin-dependent anti-MDR effect.

Gastrin promotes the metastasis of gastric carcinoma through the β-catenin/TCF-4 pathway.

Gastric cancer is the most common epithelial malignancy and the second leading cause of cancer-related death worldwide; metastasis is a crucial factor in the progression of gastric cancer. The present study applied gastrin-17 amide (G-17) in SGC7901 cells. The results showed that G-17 promoted the cell cycle by accelerating the G0/G1phase and by increasing the cell proliferation rate by binding to the gastrin receptor. The migratory and invasive abilities of the SGC7901 cells were increased by G-17. The expression levels of matrix metalloproteinase (MMP)-7, MMP-9 and vascular endothelial growth factor (VEGF) were enhanced by G-17 as well. Moreover, G-17 caused the overexpression of β-catenin and TCF-4. G-17 also caused a preferential cytoplasmic and nuclear localization of β-catenin with a high TOP-FLASH activity. Finally, axin reduced the migratory and invasive abilities of the SGC7901 cells, and inhibited the expression of β-catenin, TCF-4, MMP-7, MMP-9 and VEGF; these effects were counteracted by adding G-17. In summary, the present study confirmed the proliferation and metastasis-promoting role of G-17 via binding to the gastrin receptor, and the β-catenin/TCF-4 pathway was found to be essential for mediating G-17-induced metastasis in gastric cancer. These results may provide a novel gene target for the treatment of gastric cancer.

Dysregulation of WNT5A/ROR2 Signaling Characterizes the Progression of Barrett-Associated Esophageal Adenocarcinoma.

The mechanism underlying the progression of normal esophageal mucosa to esophageal adenocarcinoma remains elusive. WNT5A is a noncanonical WNT, which mainly functions via the receptor tyrosine kinase-like orphan receptor 2 (ROR2), and has an unclear role in carcinogenesis. In this study, we aimed to determine the role of WNT5A/ROR2 signaling in esophageal adenocarcinoma. Analysis of WNT5A and ROR2 expression patterns in healthy controls, Barrett and esophageal adenocarcinoma patients' esophageal clinical specimens as well as in various esophageal cell lines demonstrated a ROR2 overexpression in esophageal adenocarcinoma tissues compared with Barrett and healthy mucosa, whereas WNT5A expression was found significantly downregulated toward esophageal adenocarcinoma formation. Treatment of esophageal adenocarcinoma OE33 cells with human recombinant WNT5A (rhWNT5A) significantly suppressed proliferation, survival, and migration in a dose-dependent fashion. rhWNT5A was found to inhibit TOPflash activity in ROR2 wild-type cells, whereas increased TOPflash activity in ROR2-knockdown OE33 cells. In addition, ROR2 knockdown alone abolished cell proliferation and weakened the migration properties of OE33 cells. These findings support an early dysregulation of the noncanonical WNT5A/ROR2 pathway in the pathogenesis of esophageal adenocarcinoma, with the loss of WNT5A expression together with the ROR2 overexpression to be consistent with tumor promotion. The dysregulation of WNT5A/ROR2 noncanonical WNT signaling in Barrett-associated esophageal adenocarcinoma introduces possible prognostic markers and novel targets for tailored therapy of this malignancy. Mol Cancer Res; 14(7); 647-59. ©2016 AACR.

WNT7A Regulation by miR-15b in Ovarian Cancer.

WNT signaling is well known to play an important role in the regulation of development, cell proliferation and cell differentiation in a wide variety of normal and cancerous tissues. Despite the wealth of knowledge concerning when and where various WNT genes are expressed and downstream events under their control, there is surprisingly little published evidence of how they are regulated. We have recently reported that aberrant WNT7A is observed in serous ovarian carcinomas, and WNT7A is the sole ligand accelerating ovarian tumor progression through CTNNB1 (β-catenin)/TCF signaling in the absence of CTNNB1 mutations. In the present study, we report that WNT7A is a direct target of miR-15b in ovarian cancer. We showed that a luciferase reporter containing the putative binding site of miR-15b in the WNT7A 3’-UTR was significantly repressed by miR-15b. Mutation of the putative binding site of miR-15b in the WNT7A 3’-UTR restored luciferase activity. Furthermore, miR-15b was able to repress increased levels of TOPFLASH activity by WNT7A, but not those induced by S33Y. Additionally, miR-15b dose-dependently decreased WNT7A expression. When we evaluated the prognostic impact of WNT7A and miR-15b expression using TCGA datasets, a significant inverse correlation in which high-expression of WNT7A and low-expression of miR-15b was associated with reduced survival rates of ovarian cancer patients. Treatment with decitabine dose-dependently increased miR-15b expression, and silencing of DNMT1 significantly increased miR-15b expression. These results suggest that WNT7A is post-transcriptionally regulated by miR-15b, which could be down-regulated by promoter hypermethylation, potentially via DNMT1, in ovarian cancer.

Pin1-mediated Modification Prolongs the Nuclear Retention of β-Catenin in Wnt3a-induced Osteoblast Differentiation

The canonical Wnt signaling pathway, in which β-catenin nuclear localization is a crucial step, plays an important role in osteoblast differentiation. Pin1, a prolyl isomerase, is also known as a key enzyme in osteogenesis. However, the role of Pin1 in canonical Wnt signal-induced osteoblast differentiation is poorly understood. We found that Pin1 deficiency caused osteopenia and reduction of β-catenin in bone lining cells. Similarly, Pin1 knockdown or treatment with Pin1 inhibitors strongly decreased the nuclear β-catenin level, TOP flash activity, and expression of bone marker genes induced by canonical Wnt activation and vice versa in Pin1 overexpression. Pin1 interacts directly with and isomerizes β-catenin in the nucleus. The isomerized β-catenin could not bind to nuclear adenomatous polyposis coli, which drives β-catenin out of the nucleus for proteasomal degradation, which consequently increases the retention of β-catenin in the nucleus and might explain the decrease of β-catenin ubiquitination. These results indicate that Pin1 could be a critical target to modulate β-catenin-mediated osteogenesis.