Wnt family member 4 (WNT4) and WNT3A activate cell-autonomous Wnt signaling independent of porcupine O-acyltransferase or Wnt secretion.

Porcupine O-acyltransferase (PORCN) is considered essential for Wnt secretion and signaling. However, we observed that PORCN inhibition does not phenocopy the effects of WNT4 knockdown in WNT4-dependent breast cancer cells. This suggests a unique relationship between PORCN and WNT4 signaling. To examine the role of PORCN in WNT4 signaling, here we overexpressed WNT4 or WNT3A in breast cancer, ovarian cancer, and fibrosarcoma cell lines. Conditioned media from these lines and co-culture systems were used to assess the dependence of Wnt secretion and activity on the critical Wnt secretion proteins PORCN and Wnt ligand secretion (WLS) mediator. We observed that WLS is universally required for Wnt secretion and paracrine signaling. In contrast, the dependence of WNT3A secretion and activity on PORCN varied across the cell lines, and WNT4 secretion was PORCN-independent in all models. Surprisingly, WNT4 did not exhibit paracrine activity in any tested context. Absent the expected paracrine activity of secreted WNT4, we identified cell-autonomous Wnt signaling activation by WNT4 and WNT3A, independent of PORCN or Wnt secretion. The PORCN-independent, cell-autonomous Wnt signaling demonstrated here may be critical in WNT4-driven cellular contexts or in those that are considered to have dysfunctional Wnt signaling.

Treatment of cancer stem cells from human colon adenocarcinoma cell line HT-29 with resveratrol and sulindac induced mesenchymal-endothelial transition rate.

In the current experiment, the combined regime of resveratrol and a Wnt-3a inhibitor, sulindac, were examined on the angiogenic potential of cancer stem cells from human colon adenocarcinoma cell line HT-29 during 7 days. Cancer stem cells were enriched via a magnetic-activated cell sorter technique and cultured in endothelial induction medium containing sulindac and resveratrol. Expression of endothelial markers such as the von Willebrand factor (vWF) and vascular endothelial cadherin (VE-cadherin) and genes participating in mesenchymal-to-epithelial transition was studied by real-time PCR assay. Protein levels of Wnt-3a and angiogenic factor YKL-40 were examined by western blotting. ELISA was used to determine the level of N-acetylgalactosaminyltransferase 11 (GALNT11) during mesenchymal-endothelial transition. Autophagy status was monitored by PCR array under treatment with the resveratrol plus sulindac. Results showed that resveratrol and sulindac had the potential to decrease the cell survival of HT-29 cancer cells and the clonogenic capacity of cancer stem cells compared with the control (p < 0.05). The expression of VE-cadherin and vWF was induced in cancer stem cells incubated with endothelial differentiation medium enriched with resveratrol (p < 0.05). Interestingly, the Wnt-3a level was increased in the presence of resveratrol and sulindac (p < 0.05). YKL-40 was reduced after cell exposure to sulindac and resveratrol. The intracellular content of resistance factor GALNT11 was diminished after treatment with resveratrol (p < 0.05). Resveratrol had the potential to induce the transcription of autophagy signaling genes in cancer stem cells during endothelial differentiation (p < 0.05). These data show that resveratrol could increase cancer stem cell trans-differentiation toward endothelial lineage while decrease cell resistance by modulation of autophagy signaling and GALNT11 synthesis.

miR-103/107 promote ER stress-mediated apoptosis via targeting the Wnt3a/ß-catenin/ATF6 pathway in preadipocytes.

Both miR-103 and miR-107 have been demonstrated to restrain cell proliferation and regulate lipid metabolism and inflammation. However, the effects of miR-103/107 on preadipocyte apoptosis remain unknown. In the present research, we have investigated how miR-103/107 regulated preadipocyte apoptosis. We found that miR-103/107 aggravated endoplasmic reticulum (ER) stress-mediated apoptosis in preadipocytes. We confirmed that miR-103/107 targeted WNT family member 3a (Wnt3a) in preadipocytes. It was found that overexpressing Wnt3a resulted in suppression of ER stress-mediated apoptosis, while restoration of miR-103/107 counteracted the effects of Wnt3a in preadipocytes. Moreover, bioinformatics and luciferase assays indicated that activating transcription factor (ATF)6 is a key player linking miR-103/107-induced ER stress to apoptosis. ATF6 is regulated by lymphoid enhancer-binding factor 1, a transcription factor downstream of the Wnt3a/ß-catenin signaling pathway, and ATF6 binds to the B-cell lymphoma 2 (Bcl2) promoter to regulate apoptosis further. In conclusion, miR-103/107 promoted ER stress-mediated apoptosis by targeting the Wnt3a/ß-catenin/ATF6 signaling pathway in preadipocytes. This study revealed that the miR-103/107-Wnt3a/ß-catenin-ATF6 pathway is critical to the progression of apoptosis in preadipocytes, which suggested that approaches to activate miR-103/107 could potentially be useful as new therapies for treating obesity and metabolic syndrome-related disorders.

Iron chelator-induced up-regulation of Ndrg1 inhibits proliferation and EMT process by targeting Wnt/ß-catenin pathway in colon cancer cells.

Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), as the novel iron chelator, has been reported to inhibit the tumorigenesis and progression of various cancer cells. However, whether Dp44mT has anticancer effects in colon cancer cells is still unknown. Here, we investigated the antitumor action of Dp44mT in colon cancer and its underlying mechanisms, and the connections between Dp44mT and N-myc downstream-regulated genes 1(Ndrg1). We used cell viability, migration and invasion assay, flow cytometry, western blot and qRT-PCR to examine the anticancer effects of Dp44mT and Ndrg1. We found that Dp44mT suppressed cell viability, migration, invasion and induced apoptosis of colon cancer cells and over-expression of Ndrg1 also suppressed cell viability, migration, invasion and induced apoptosis of colon cancer cells. Dp44mT attenuated the TGF-ß1-induced EMT in colon cancer cells, and Dp44mT could up-regulate Ndrg1 expression level. Overexpression of Ndrg1 attenuates the TGF-ß1-induced EMT, Dp44mT and Ndrg1 suppressed EMT through activation of Wnt/ß catenin signaling pathway. In conclusion, our data demonstrated that Dp44mT/Ndrg1 have effective anticancer capability in colon cancer cells and that may represent a promising treatment strategy for human colon cancer.

MiR-195 suppresses colon cancer proliferation and metastasis by targeting WNT3A.

MicroRNAs (miRNAs) are a novel class of diagnostic and therapeutic target in cancer. Here, we aimed to explore the effects and mechanism of miR-195 regulation in colon cancer. The expressions of several putative miRNAs in colon tumors, compared to those in normal tissues, were investigated by bioinformatical analysis of a Gene Expression Omnibus database. Quantitative real-time PCR analysis (qRT-PCR) was used to validate the identified changes in normal tissues, primary tumors, and metastatic tumors. MTT, soft agar colony formation, and transwell assays were used to evaluate the effects of miR-195 overexpression or inhibition on cell viability, proliferation, migration, and invasion. Targets of miR-195 were identified by TargetScan, and subsequently verified by qRT-PCR and Western blot. The role of miR-195 in the ß-catenin pathway was also studied using RT-PCR and Western blot. MiR-195 expression was downregulated in colon carcinoma tissues and negatively correlated with the metastatic potential. While transfecting miR-195 mimics decreased the proliferation, migration, and invasion of colon cancer cells, miR-195 inhibition exerted opposing effects. WNT3A was identified as a direct target of miR-195. ß-catenin was also downregulated by miR-195 in colon cancers. MiR-195 downregulation is associated with the enhanced proliferation, migration, and invasion of colon cancer. MiR-195 directly downregulates WNT3A. Our results indicate that miR-195 is a potential diagnostic marker and therapeutic target for improving the clinical management of colon cancer.

Dickkopf-1 inhibits Wnt3a-induced migration and epithelial-mesenchymal transition of human lens epithelial cells.

Posterior capsular opacification (PCO) is a major post-operative complication of cataract surgery. Epithelial-mesenchymal transition (EMT) contributes to PCO. We previously indicated that Wnt3a induces the EMT of human lens epithelial cells (LECs) and plays an important role in the development of PCO. The present study aimed to test the potential effect of Dickkopf-1 (Dkk1) on Wnt3a-induced cell migration and the EMT of LECs and to explore possible cellular mechanisms. The secretion of Dkk1 was reduced in the rabbit PCO model, and Dkk1 injected into the eyes post-surgical manipulation prevented PCO formation. Cultured HLE-B3 cells were then transfected with Wnt3a in the presence or absence of Dkk1. Dkk1 treatment restored the epithelial phenotype and reversed the expression of EMT-associated proteins induced by Wnt3a. Dkk1 suppressed LEC migration and the expression of matrix metalloproteinase-1 (MMP-1), and the activity of MMP-2 and MMP-9. Dkk1 inhibited the nuclear accumulation of ß-catenin, which is the key regulator of the canonical Wnt signaling. Our results indicate that Dkk1 inhibits Wnt3a-induced migration and the EMT of human LECs.The results contribute to the prevention of PCO formation and development.

Wnt-mediated protein stabilization ensures proper mitotic microtubule assembly and chromosome segregation.

Wnt signaling stimulates cell proliferation by promoting the G1/S transition of the cell cycle through ß-catenin/TCF4-mediated gene transcription. However, Wnt signaling peaks in mitosis and contributes to the stabilization of proteins other than ß-catenin, a pathway recently introduced as Wnt-dependent stabilization of proteins (Wnt/STOP). Here, we show that Wnt/STOP regulated by basal Wnt signaling during a normal cell cycle is required for proper spindle microtubule assembly and for faithful chromosome segregation during mitosis. Consequently, inhibition of basal Wnt signaling results in increased microtubule assembly rates, abnormal mitotic spindle formation and the induction of aneuploidy in human somatic cells.

Wnt4 antagonises Wnt3a mediated increases in growth and glucose stimulated insulin secretion in the pancreatic beta-cell line, INS-1.

The Wnt signalling pathway in beta-cells has been linked to the development of type 2 diabetes. Investigating the impact of a non-canonical Wnt ligand, Wnt4, on beta-cell function we found that in INS-1 cells, Wnt4 was able to completely block Wnt3a stimulated cell growth and insulin secretion. However, despite high levels of Wnt4 protein being detected in INS-1 cells, reducing the expression of Wnt4 had no impact on cell growth or Wnt3a signalling. As such, the role of the endogenously expressed Wnt4 in beta-cells is unclear, but the data showing that Wnt4 can act as a negative regulator of canonical Wnt signalling in beta-cells suggests that this pathway could be a potential target for modulating beta-cell function.

EGF Inhibits Wnt/ß-Catenin-Induced Osteoblast Differentiation by Promoting ß-Catenin Degradation.

Bone morphogenetic protein (BMP) and canonical Wnts are representative developmental signals that enhance osteoblast differentiation and bone formation. Previously, we demonstrated that epidermal growth factor (EGF) inhibits BMP2-induced osteoblast differentiation by inducing Smurf1 expression. However, the regulatory role of EGF in Wnt/ß-catenin-induced osteoblast differentiation has not been elucidated. In this study, we investigated the effect of EGF on Wnt/ß-catenin signaling-induced osteoblast differentiation using the C2C12 cell line. EGF significantly suppressed the expression of osteoblast marker genes, which were induced by Wnt3a and a GSK-3ß inhibitor. EGF increased the expression levels of Smurf1 mRNA and protein. Smurf1 knockdown rescued Wnt/ß-catenin-induced osteogenic marker gene expression in the presence of EGF. EGF treatment or Smurf1 overexpression did not affect ß-catenin mRNA expression levels, but reduced ß-catenin protein levels and TOP-Flash activity. EGF and Smurf1 promoted ß-catenin ubiquitination. Co-immunoprecipitation and GST pull-down assays showed that Smurf1 associates with ß-catenin. These results suggest that EGF/Smurf1 inhibits Wnt/ß-catenin-induced osteogenic differentiation and that Smurf1 downregulates Wnt/ß-catenin signaling by enhancing proteasomal degradation of ß-catenin.

Protein kinase PKN1 represses Wnt/ß-catenin signaling in human melanoma cells.

Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of ß-catenin-responsive transcription (ß-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of ß-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/ß-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A.

Dickkopf-1 is regulated by the mevalonate pathway in breast cancer.

INTRODUCTION: Amino-bisphosphonates and statins inhibit the mevalonate pathway, and may exert anti-tumor effects. The Wnt inhibitor dickkopf-1 (DKK-1) promotes osteolytic bone lesions by inhibiting osteoblast functions and has been implicated as an adverse marker in multiple cancers. We assessed the effects of mevalonate pathway inhibition on DKK-1 expression in osteotropic breast cancer. METHODS: Regulation of DKK-1 by bisphosphonates and statins was assessed in human breast cancer cell lines, and the role of the mevalonate pathway and downstream targets was analyzed. Moreover, the potential of breast cancer cells to modulate osteoblastogenesis via DKK-1 was studied in mC2C12 cells. Clinical relevance was validated by analyzing DKK-1 expression in the tissue and serum of women with breast cancer exposed to bisphosphonates. RESULTS: DKK-1 was highly expressed in receptor-negative breast cancer cell lines. Patients with receptor-negative tumors displayed elevated levels of DKK-1 at the tissue and serum level compared to healthy controls. Zoledronic acid and atorvastatin potently suppressed DKK-1 in vitro by inhibiting geranylgeranylation of CDC42 and Rho. Regulation of DKK-1 was strongest in osteolytic breast cancer cell lines with abundant DKK-1 expression. Suppression of DKK-1 inhibited the ability of breast cancer cells to block WNT3A-induced production of alkaline phosphates and bone-protective osteoprotegerin in preosteoblastic C2C12 cells. In line with the in vitro data, treatment of breast cancer patients with zoledronic acid decreased DKK-1 levels by a mean of 60% after 12 months of treatment. CONCLUSION: DKK-1 is a novel target of the mevalonate pathway that is suppressed by zoledronic acid and atorvastatin in breast cancer.

Blocking of frizzled signaling with a homologous peptide fragment of wnt3a/wnt5a reduces infarct expansion and prevents the development of heart failure after myocardial infarction.

BACKGROUND: The molecular pathways that control the wound healing after myocardial infarction (MI) are not completely elucidated. One of these pathways is the Wnt/Frizzled pathway. In this study, we evaluated Frizzled as a novel therapeutic target for MI. These Frizzled proteins act as receptors for Wnt proteins and were previously shown to be expressed in the healing infarct. METHODS AND RESULTS: Wnt/Frizzled signaling has been studied for decades, but synthetic ligands that interfere with the interaction between Wnts and Frizzled have not been described to date. Here we report the selection of 3 peptides derived from regions of high homology between Wnt3a and Wnt5a that act as antagonists for Frizzled proteins. UM206, the peptide with the highest affinity, antagonized the effect of Wnt3a and Wnt5a in different in vitro assays. Administration of UM206 to mice for 5 weeks, starting immediately after the induction of MI, reduced infarct expansion and increased the numbers of capillaries and myofibroblasts in the infarct area. Moreover, heart failure development was inhibited by this therapy. CONCLUSIONS: Blocking of Frizzled signaling reduces infarct expansion and preserves cardiac function after MI. Our findings underscore the potential of Frizzled receptors as a target for pharmacotherapy of cardiac remodeling after MI.

Blockers of Wnt3a, Wnt10a, or ß-Catenin Prevent Chemotherapy-Induced Neuropathic Pain In Vivo.

Although chemotherapy is a key cancer treatment, many chemotherapeutic drugs produce chronic neuropathic pain, called chemotherapy-induced neuropathic pain (CINP), which is a dose-limiting adverse effect. To date, there is no medicine that prevents CINP in cancer patients and survivors. We determined whether blockers of the canonical Wnt signaling pathway prevent CINP. Neuropathic pain was induced by intraperitoneal injection of paclitaxel (PAC) on four alternate days in male Sprague-Dawley rats or male Axin2-LacZ knock-in mice. XAV-939, LGK-974, and iCRT14, Wnt/ß-catenin blockers, were administered intraperitoneally as a single or multiple doses before or after injury. Mechanical allodynia, phosphoproteome profiling, Wnt ligands, and inflammatory mediators were measured by von Frey filament, phosphoproteomics, reverse transcription-polymerase chain reaction, and Western blot analysis. Localization of ß-catenin was determined by immunohistochemical analysis in the dorsal root ganglia (DRGs) in rats and human. Our phosphoproteome profiling of CINP rats revealed significant phosphorylation changes in Wnt signaling components. Importantly, repeated systemic injections of XAV-939 or LGK-974 prevented the development of CINP in rats. In addition, XAV-939, LGK-974, and iCRT14 ameliorated CINP. PAC increased Wnt3a and Wnt10a, activated ß-catenin in DRG, and increased monocyte chemoattractant protein-1 and interleukin-1ß in DRG. PAC also upregulated rAxin2 in mice. Furthermore, ß-catenin was expressed in neurons, including calcitonin gene-related protein-expressing neurons and satellite cells in rat and human DRG. In conclusion, chemotherapy increases Wnt3a, Wnt10a, and ß-catenin in DRG and their pharmacological blockers prevent and ameliorate CINP, suggesting a target for the prevention and treatment of CINP.

Piperine suppresses the Wnt/ß-catenin pathway and has anti-cancer effects on colorectal cancer cells.

More than 94% of colorectal cancer cases have mutations in one or more Wnt/ß-catenin signaling pathway components. Inactivating mutations in APC or activating mutations in ß-catenin (CTNNB1) lead to signaling overactivation and subsequent intestinal hyperplasia. Numerous classes of medicines derived from synthetic or natural small molecules, including alkaloids, have benefited the treatment of different diseases, including cancer, Piperine is a true alkaloid, derived from lysine, responsible for the spicy taste of black pepper (Piper nigrum) and long pepper (Piper longum). Studies have shown that piperine has a wide range of pharmacological properties; however, piperine molecular mechanisms of action are still not fully understood. By using Wnt/ß-catenin pathway epistasis experiment we show that piperine inhibits the canonical Wnt pathway induced by overexpression of ß-catenin, ß-catenin S33A or dnTCF4 VP16, while also suppressing ß-catenin nuclear localization in HCT116 cell line. Additionally, piperine impairs cell proliferation and migration in HCT116, SW480 and DLD-1 colorectal tumor cell lines, while not affecting the non-tumoral cell line IEC-6. In summary, piperine inhibits the canonical Wnt signaling pathway and displays anti-cancer effects on colorectal cancer cell lines.

Directed differentiation of telencephalic precursors from embryonic stem cells.

We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells ( approximately 90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation ( approximately 35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6(+) cells yield up to 75% of Bf1(+) cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1(+) and/or Islet1/2(+) cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.

Deoxynivalenol induces inhibition of cell proliferation via the Wnt/ß-catenin signaling pathway.

The type B trichothecene mycotoxin deoxynivalenol (DON), colloquially known as "vomitoxin", is the most commonly detected trichothecene in cereal-based foods, exerting acute and chronic toxic effects on animals and causing serious safety-related concerns. At the cellular and molecular levels, DON can inhibit macromolecule synthesis by binding to ribosomes and may disrupt cell proliferation, differentiation and apoptosis. However, the molecular mechanisms underlying the cytotoxicity of DON remain to be determined. Here, we identified ß-catenin, the key signal transducer of the Wnt cascade, as a novel target of DON by quantitative real-time PCR and Western blot analysis in human embryonic kidney 293T and colorectal SW480 cells treated with DON at half IC50 (50 ng/mL for HEK293T and 1 µg/mL for SW480). Further analysis showed that neither posttranslationalmodification nor nuclear accumulation of ß-catenin was affected post DON treatment. Moreover, we found that the ß-catenin-dependent canonical Wnt signaling pathway, which regulates many biological processes during embryonic development and adult tissue homeostasis, was involved in DON-induced inhibition of cell proliferation. Then, we determined that the ß-catenin/c-Myc axis was essential for this process, as the DON-induced inhibition of cell proliferation was efficiently rescued by restoration of ß-catenin or c-Myc levels. Our results advance the current understanding of the molecular toxicological mechanism of DON and provide a new perspective on strategies for the prevention and control of mycotoxins.

Apigenin inhibits fibroblast proliferation and reduces epidural fibrosis by regulating Wnt3a/ß-catenin signaling pathway.

BACKGROUND: Failed back surgery syndrome (FBSS) is a common complication after the laminectomy. Epidural fibrosis is the major cause of lower back pain and other complications. Numerous studies have shown that apigenin (API) could treat various fibrotic diseases by regulating various signaling pathways, whereas no study has discussed whether API can inhibit fibroblast proliferation and reduce epidural fibrosis after the laminectomy by regulating Wnt3a/ß-catenin signaling pathway. METHODS: Human fibroblasts were cultured and treated with API in different concentrations for 24 h. CCK-8 detection and EdU incorporation assay were performed to detect cell viability and cell proliferation. Western blotting analysis was applied to detect expressions of proliferative proteins, Wnt3a, and its downstream proteins. Moreover, the Wnt3a gene was overexpressed in fibroblasts to define the relationship between Wnt3a/ß-catenin signaling pathway and fibroblast proliferation. Wnt3a overexpressed fibroblasts were treated with API to verify if it could reverse the effects of API treatment. Twenty-four Sprague-Dawley rats were randomly divided into four groups. Laminectomy was performed and the rats were gavaged with different doses of API or 5% sodium carboxyl methyl cellulose (CMC-Na) solution for 1 month. The abilities of API to inhibit fibroblast proliferation and to reduce epidural fibrosis were evaluated using histological and immunohistochemical analysis. RESULTS: CCK-8 detection and EdU incorporation assay demonstrated that API could inhibit the viability and proliferation rate of fibroblasts in a concentration-dependent manner. The Western blotting analysis revealed that API could inhibit the expressions of PCNA, cyclinD1, Wnt3a, and its downstream proteins. The overexpression of Wnt3a in fibroblasts could upregulate the expressions of proliferative proteins such as PCNA and cyclinD1. The inhibitory effect of API on PCNA, Wnt3a, and its downstream proteins was partially reversed by overexpression of Wnt3a. Moreover, the results of the histological and immunohistochemical analysis revealed that API could reduce the epidural fibrosis in rats by inhibiting fibroblast proliferation in a dose-dependent manner. CONCLUSIONS: API can inhibit fibroblast proliferation and reduce epidural fibrosis by suppressing Wnt3a/ß-catenin signaling pathway, which can be adopted as a new option to prevent epidural fibrosis after the laminectomy.

Wnt3a Neutralization Enhances T-cell Responses through Indirect Mechanisms and Restrains Tumor Growth.

The Wnt/ß-catenin pathway regulates T-cell functions, including the repression of effector functions to the advantage of memory development via Tcf1. In a companion study, we demonstrate that, in human cancers, Wnt3a/ß-catenin signaling maintains tumor-infiltrating T cells in a partially exhausted status. Here, we have investigated the effects of Wnt3a neutralization in vivo in a mouse tumor model. Abundant Wnt3a was released, mostly by stromal cells, in the tumor microenvironment. We tested whether Wnt3a neutralization in vivo could rescue the effector capacity of tumor-infiltrating T cells, by administering an antibody to Wnt3a to tumor-bearing mice. This therapy restrained tumor growth and favored the expansion of tumor antigen-specific CD8+ effector memory T cells with increased expression of Tbet and IFNγ and reduced expression of Tcf1. However, the effect was not attributable to the interruption of T-cell-intrinsic ß-catenin signaling, because Wnt3a/ß-catenin activation correlated with enhanced, not reduced, T-cell effector functions both ex vivo and in vitro Adoptively transferred CD8+ T cells, not directly exposed to the anti-Wnt3a antibody but infiltrating previously Wnt3a-neutralized tumors, also showed improved functions. The rescue of T-cell response was thus secondary to T-cell-extrinsic changes that likely involved dendritic cells. Indeed, tumor-derived Wnt3a strongly suppressed dendritic cell maturation in vitro, and anti-Wnt3a treatment rescued dendritic cell activities in vivo Our results clarify the function of the Wnt3a/ß-catenin pathway in antitumor effector T cells and suggest that Wnt3a neutralization might be a promising immunotherapy for rescuing dendritic cell activities. Cancer Immunol Res; 6(8); 953-64. ©2018 AACR.

miR-708-5p promotes fibroblast-like synoviocytes' cell apoptosis and ameliorates rheumatoid arthritis by the inhibition of Wnt3a/ß-catenin pathway.

AIM: MicroRNAs (miRNA) are a class of small, highly conserved noncoding RNA molecules, which contain 18-28 nucleotides and are involved in the regulation of gene expression. It has been proved that microRNAs play a very important role in several key cellular processes, such as cell differentiation, cell cycle progression, and apoptosis, as well as in autoimmune disease. One recently identified miRNA, miR-708-5p, has been demonstrated to have profound roles in suppressing oncogenesis in different types of tumors. However, the role of miR-708-5p in rheumatoid arthritis (RA) remains to be fully elucidated. Therefore, in this study, we are aiming to identify the role of miR-708-5p in RA. METHODS: The expression level of miR-708-5p in synovial tissues of patients with RA is much lower than in non-RA controls. The effects of miR-708-5p on cell apoptosis, colony formation, and migration in fibroblast-like synoviocytes were assessed in MH7A cells. RESULTS: Results showed that delivery of miR-708-5p mimics into synovial fibroblasts MH7A could induce cell apoptosis and inhibit colony formation and migration. In addition, miR-708-5p mimics significantly inhibit Wnt3a/ß-catenin pathway activity both in transcription and protein level, which could be reversed by the addition of R-spondin 1, an activator of Wnt pathway. R-spondin 1 could also reverse the inhibition of cell survival and proliferation, which was induced by miR-708-5p mimics in MH7A. Moreover, injection of miR-708-5p mimics into collagen-induced rat RA model could ameliorate the RA index and decrease Wnt3a/ß-catenin expression in rat joint tissues. CONCLUSION: Therefore, we concluded that miR-708-5p is likely to be involved in RA pathogenesis via inhibition of Wnt3a/ß-catenin pathway.

Neuroprotective and regenerative roles of intranasal Wnt-3a administration after focal ischemic stroke in mice.

Wnt signaling is a conserved pathway involved in expansion of neural progenitors and lineage specification during development. However, the role of Wnt signaling in the post-stroke brain has not been well-elucidated. We hypothesized that Wnt-3a would play an important role for neurogenesis and brain repair. Adult male mice were subjected to a focal ischemic stroke targeting the sensorimotor cortex. Mice that received Wnt-3a (2 µg/kg/day, 1 h after stroke and once a day for the next 2 days, intranasal delivery) had reduced infarct volume compared to stroke controls. Wnt-3a intranasal treatment of seven days upregulated the expression of brain-derived growth factor (BDNF), increased the proliferation and migration of neuroblasts from the subventricular zone (SVZ), resulting in increased numbers of newly formed neurons and endothelial cells in the peri-infarct zone. Both the molecular and cellular effects of Wnt-3a were blocked by the Wnt specific inhibitors XAV-939 or Dkk-1. In functional assays, Wnt-3a treatment enhanced the local cerebral blood flow (LCBF) in the peri-infarct, as well as improved sensorimotor functions in a battery of behavioral tests. Together, our data demonstrates that the Wnt-3a signaling can act as a dual neuroprotective and regenerative factor for the treatment of ischemic stroke.