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.

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.

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.

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.

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-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.

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.

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-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.

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.

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.

miR-15a-5p suppresses endometrial cancer cell growth via Wnt/ß-catenin signaling pathway by inhibiting WNT3A.

OBJECTIVE: Endometrial cancer is one of the three most common types of gynecologic cancer. The global incidence has increased in recent years. microRNAs (miRNAs) regulate numerous biological processes by binding to the 3'UTR of target mRNA to down-regulate protein synthesis. PATIENTS AND METHODS: Endometrial cancer patients received surgeries in our hospital were enrolled. MiR-15a-5p mimic or miR-15a-5p inhibitor was transfected into HEC-1-A cells by lentivirus. Colony formation assay was applied for detecting cell proliferation. Real-time PCR was performed to test miRNA and mRNA expression. Western blot was used to detect protein level. ChIP was adopted to test transcription activation. TOP/FOP was tested to determine Wnt signaling pathway activity. A dual-luciferase reporter assay was used to confirm miRNA target. RESULTS: miR-15a-5p was decreased in endometrial cancer cells and tissues. miR-15a-5p overexpression restrained HEC-1-A cell proliferation and stemness. miR-15a-5p mimic transfection reduced mRNA and protein levels of the proteins which are related to cell proliferation and Wnt signaling pathway. MiR-15a-5p targeted a putative binding site in the 3'-UTR of Wnt3a gene, thus regulating Wnt signaling pathway. miR-15a-5p overexpression decreased Wnt3a protein expression. Wnt3a presented significant negative correlation with the miR-15a-5p level in endometrial cancer patients. CONCLUSIONS: miR-15a-5p is a regulator of endometrial cancer cell proliferation by directly targeting Wnt3a to block Wnt signaling pathway.

MicroRNA­214 targets Wnt3a to suppress liver cancer cell proliferation.

MicroRNAs (miRNAs/miRs) are crucial molecules that act as tumor suppressor genes or oncogenes in human cancer progression. The dysregulation of miRNA expression has been detected in liver cancer. The present study aimed to explore the molecular mechanisms by which miR­214 affects liver cancer cell proliferation. Reverse transcription­quantitative polymerase chain reaction was used to determine the expression of miR­214 in liver cancer cell lines and hepatocellular carcinoma (HCC) tissues. A luciferase reporter assay was performed to determine whether Wnt3a is a target gene of miR­214. Cell Counting kit­8 and cell cycle analysis were used to explore the effects of miR­214 on liver cancer cell proliferation. Immunohistochemistry was used to detect protein expression levels. Wnt3a knockdown was used to determine the function of Wnt3a in liver cancer cell proliferation. The results demonstrated that the expression levels of human miR­214 were reduced in HCC tissues and liver cancer cell lines compared with in control tissues and cells. Overexpression of miR­214 and Wnt3a silencing each inhibited liver cancer cell growth. Conversely, inhibition of miR­214 promoted liver cancer cell growth. The present study indicated that miR­214 acts as a tumor suppressor and may be considered a promising therapeutic target for the treatment of liver cancer.

The Wnt inhibitor LGK-974 enhances radiosensitivity of HepG2 cells by modulating Nrf2 signaling.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of antioxidant and detoxification activities that can eliminate reactive oxygen species (ROS) produced via irradiation. However, Nrf2 overexpression in liver cancer cells may cause both radioresistance and chemoresistance. Reducing Nrf2 levels can enhance the radiosensitivity of HepG2 cells. Wingless/int-3A (Wnt3A) is a Wnt family protein that mainly activates the canonical Wnt signaling pathway. Recent studies showed that the Axin1-GSK-3ß protein complex, a component of the canonical Wnt signaling pathway, can capture Nrf2 and facilitate its ubiquitination and proteasomal degradation in the cytoplasm. This protein complex is degraded upon activation of the Wnt signaling pathway. In the present study, we treated HepG2 cells with the Wnt3A inhibitor LGK-974, an effective and specific PORCN inhibitor that can prevent the formation of a proper folding of the Wnt protein in the endoplasmic reticulum. We found that HepG2 cells became more sensitive to radiation with increasing LGK-974 concentrations. Upon 2 Gy or 4 Gy irradiation, the cells treated with LGK-974 more frequently underwent apoptosis and grew less rapidly. PCR and western blot results showed that inhibiting the secretion of Wnt3A blocked the Wnt signaling pathway and prevented Nrf2 signaling. Notably, the Wnt inhibitor may serve as a radiosensitizing drug.

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.

Bone Metastasis of Prostate Cancer Can Be Therapeutically Targeted at the TBX2-WNT Signaling Axis.

Identification of factors that mediate visceral and bone metastatic spread and subsequent bone remodeling events is highly relevant to successful therapeutic intervention in advanced human prostate cancer. TBX2, a T-box family transcription factor that negatively regulates cell-cycle inhibitor p21, plays critical roles during embryonic development, and recent studies have highlighted its role in cancer. Here, we report that TBX2 is overexpressed in human prostate cancer specimens and bone metastases from xenograft mouse models of human prostate cancer. Blocking endogenous TBX2 expression in PC3 and ARCaPM prostate cancer cell models using a dominant-negative construct resulted in decreased tumor cell proliferation, colony formation, and invasion in vitro Blocking endogenous TBX2 in human prostate cancer mouse xenografts decreased invasion and abrogation of bone and soft tissue metastasis. Furthermore, blocking endogenous TBX2 in prostate cancer cells dramatically reduced bone-colonizing capability through reduced tumor cell growth and bone remodeling in an intratibial mouse model. TBX2 acted in trans by promoting transcription of the canonical WNT (WNT3A) promoter. Genetically rescuing WNT3A levels in prostate cancer cells with endogenously blocked TBX2 partially restored the TBX2-induced prostate cancer metastatic capability in mice. Conversely, WNT3A-neutralizing antibodies or WNT antagonist SFRP-2 blocked TBX2-induced invasion. Our findings highlight TBX2 as a novel therapeutic target upstream of WNT3A, where WNT3A antagonists could be novel agents for the treatment of metastasis and for skeletal complications in prostate cancer patients. Cancer Res; 77(6); 1331-44. ©2017 AACR.

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.

Inhibition of canonical WNT/ß-catenin signaling is involved in leflunomide (LEF)-mediated cytotoxic effects on renal carcinoma cells.

Leflunomide (LEF), an inhibitor of dihydroorotate dehydrogenase (DHODH) in pyrimidine biosynthetic pathway, is an immunomodulatory agent approved for the treatment of rheumatoid arthritis. In this study, we show that LEF significantly reduced cell proliferation of renal carcinoma cells in a concentration-dependent manner. LEF at 50 µM induced S-phase arrest and autophagy. Higher doses of LEF (>50 µM) effectively induced cell apoptosis. Modulating the concentration of LEF resulted in distinct effects on the expression of regulatory proteins associated with cell cycle, apoptosis, and autophagy. In particular, high concentrations of LEF inhibited canonical WNT signaling by promoting nucleo-cytoplasmic shuttling and proteasome-dependent degradation of ß-catenin. Mechanistic studies showed that the repression of AKT activation partly accounted for LEF-mediated WNT inhibition. Gene expression microarray revealed that LEF treatment greatly inhibited the expression of FZD10 gene, a receptor mediating WNT/ß-catenin activation. In vivo xenograft study in NOD/SCID mice further validated the inhibitory effects of LEF on tumor growth and Wnt/ß-catenin signaling. However, LEF treatment also triggered cell autophagy and elevated the expression of WNT3a, which ameliorated its cytotoxic effects. The combination of LEF with a WNT inhibitor IWP-2 or autophagy inhibitor HCQ could yield an enhanced anti-tumor outcome. Taken together, these results identify the potential utility and pharmacological feature of LEF in the chemotherapy of renal cell carcinoma (RCC).

Cucurbitacin B inhibits the stemness and metastatic abilities of NSCLC via downregulation of canonical Wnt/ß-catenin signaling axis.

Lack of effective anti-metastatic drugs creates a major hurdle for metastatic lung cancer therapy. For successful lung cancer treatment, there is a strong need of newer therapeutics with metastasis-inhibitory potential. In the present study, we determined the anti-metastatic and anti-angiogenic potential of a natural plant triterpenoid, Cucurbitacin B (CuB) against non-small cell lung cancer (NSCLC) both in vitro and in vivo. CuB demonstrated a strong anti-migratory and anti-invasive ability against metastatic NSCLC at nanomolar concentrations. CuB also showed significant tumor angiogenesis-inhibitory effects as evidenced by the inhibition of migratory, invasive and tube-forming capacities of human umbilical vein endothelial cells. CuB-mediated inhibition of angiogenesis was validated by the inhibition of pre-existing vasculature in chick embryo chorio-allantoic membrane and matrigel plugs. Similarly, CuB inhibited the migratory behavior of TGF-ß1-induced experimental EMT model. The CuB-mediated inhibition of metastasis and angiogenesis was attributable to the downregulation of Wnt/ß-catenin signaling axis, validated by siRNA-knockdown of Wnt3 and Wnt3a. The CuB-mediated downregulation of Wnt/ß-catenin signaling was also validated using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis model in vivo. Collectively, our findings suggest that CuB inhibited the metastatic abilities of NSCLC through the inhibition of Wnt/ß-catenin signaling axis.