Wnt Inhibitor Research Articles

Wif1 Mediates Coordination of Bone Morphogenetic Protein and Wnt Signaling in Neural and Glioma Stem Cells

Wnts, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) are paracrine signaling pathways implicated in the niche control of stem cell fate decisions. BMP-on and Wnt-off are the dominant quiescent niche signaling pathways in many cell types, including neural stem cells (NSCs). However, among the multiple inhibitory family members of the Wnt pathway, those with direct action after BMP4 stimulation in NSCs remain unclear. We examined 11 Wnt inhibitors in NSCs after BMP4 treatment. Wnt inhibitory factor 1 (Wif1) has been identified as the main factor reacting to BMP4 stimuli. RNA sequencing confirmed that Wif1 was markedly upregulated after BMP4 treatment in different gene expression analyses. Similar to the functional role of BMP4, Wif1 significantly decreased the cell cycle of NSCs and significantly inhibited cell proliferation (P < 0.05). Combined treatment with BMP4 and Wif1 significantly enhanced the inhibition of cell growth compared with the single treatment (P < 0.05). Wif1 expression was clearly lower in glioblastoma and low-grade glioma samples than in normal samples (P < 0.05). A functional analysis revealed that both BMP4 and Wif1 could decrease glioma cell growth. These effects were abrogated by the BMP inhibitor Noggin. The collective findings demonstrate that Wif1 plays a key role in quiescent NSC homeostasis and glioma cell growth downstream of BMP-on signaling. The functional roles of Wif1/BMP4 in glioma cells may provide a technical basis for regenerative medicine, drug discovery, and personal molecular therapy in future clinical treatments.

Effects of inhibition of Wnt/beta‐catenin pathway on Amacrine and Muller cells in retinas of zebrafish embryos at different development stages

AbstractPurposeThe aim of this study was to investigate the changes of Wnt pathway inhibition on Amacrine and Muller cells in the embryonic period of zebrafish.MethodsZebrafishes divided into two subgroups: control and Wnt signaling pathway inhibited groups. The effect of Wnt inhibition (IWR1) on Amacrine and Müller cells were examined at 24th, 38th, 60th and 72nd hours of Zebrafish embryos. Immunofluorescence imaging and in situ hybridization methods were used to compare the ocular structures of the groups treated with glutamine synthetase, calretinin, Neuro D1 (Amacrine and Muller cell markers), Pax6 and Rx3 (genes effective in eye formation).ResultsThe most striking results of our study are; the diminished overall cellular structures of neuroretina at the 24th, 60th hour with Neuro D1, decreased Phospho‐Beta‐catenin staining at the 24th hour, apoptosis of whole ocular tissues at the 24th hour with Cleaved Caspase 3, no staining with glutamine synthase at the 38th hour, reduced β‐Catenin staining in the 60th hour and increased Caspase 3 staining at 72nd hour in IWR group. The related figures (Fig 1–7) are shown below.ConclusionsThe results of our study revealed that the inhibition of the Wnt/beta‐catenin pathway could be responsible for the maldevelopment of ocular tissues especially on the neuroretinal tissues including Amacrine and Muller cells at the early stages. Our results could shed a light on the pathophysiology of developmental anomalies of ocular tissues like colobomas and be a new model for other experimental researches like retinopathies in the future.

LIM Homeobox-2 Suppresses Hallmarks of Adult and Pediatric Liver Cancers by Inactivating MAPK/ERK and Wnt/Beta-Catenin Pathways

Introduction: Hepatocellular carcinoma and hepatoblastoma are two liver cancers characterized by gene deregulations, chromosomal rearrangements, and mutations in Wnt/beta-catenin (Wnt) pathway-related genes. LHX2, a transcriptional factor member of the LIM homeobox gene family, has important functions in embryogenesis and liver development. LHX2 is oncogenic in many solid tumors and leukemia, but its role in liver cancer is unknown. Methods: We analyzed the expression of LHX2 in hepatocellular carcinoma and hepatoblastoma samples using various transcriptomic datasets and biological samples. The role of LHX2 was studied using lentiviral transduction, in vitro cell-based assays (growth, migration, senescence, and apoptosis), molecular approaches (phosphokinase arrays and RNA-seq), bioinformatics, and two in vivo models in chicken and Xenopus embryos. Results: We found a strong connection between LHX2 downregulation and Wnt activation in these two liver cancers. In hepatoblastoma, LHX2 downregulation correlated with multiple poor outcome parameters including higher patient age, intermediate- and high-risk tumors, and low patient survival. Forced expression of LHX2 reduced the proliferation, migration, and survival of liver cancer cells in vitro through the inactivation of MAPK/ERK and Wnt signals. In vivo, LHX2 impeded the development of tumors in chick embryos and repressed the Wnt pathway in Xenopus embryos. RNA-sequencing data and bioinformatic analyses confirmed the deregulation of many biological functions and molecular processes associated with cell migration, cell survival, and liver carcinogenesis in LHX2-expressing hepatoma cells. At a mechanistic level, LHX2 mediated the disassembling of beta-catenin/T-cell factor 4 complex and induced expression of multiple inhibitors of Wnt (e.g., TLE/Groucho) and MAPK/ERK (e.g., DUSPs) pathways. Conclusion: Collectively, our findings demonstrate a tumor suppressive function of LHX2 in adult and pediatric liver cancers.

The Small-Molecule Wnt Inhibitor ICG-001 Efficiently Inhibits Colorectal Cancer Stemness and Metastasis by Suppressing MEIS1 Expression.

Recurrence and metastasis remain major obstacles in colorectal cancer (CRC) treatment. Recent studies suggest that a small subpopulation of cells with a self-renewal ability, called cancer stem-like cells (CSCs), promotes recurrence and metastasis in CRC. Unfortunately, no CSC inhibitor has been demonstrated to be more effective than existing chemotherapeutic drugs, resulting in a significant unmet need for effective CRC therapies. In this study, transcriptomic profiling of metastatic tumors from CRC patients revealed significant upregulation in the Wnt pathway and stemness genes. Thus, we examined the therapeutic effect of the small-molecule Wnt inhibitor ICG-001 on cancer stemness and metastasis. The ICG-001 treatment efficiently attenuated self-renewal activity and metastatic potential. Mechanistically, myeloid ecotropic viral insertion site 1 (MEIS1) was identified as a target gene of ICG-001 that is transcriptionally regulated by Wnt signaling. A series of functional analyses revealed that MEIS1 enhanced the CSC behavior and metastatic potential of the CRC cells. Collectively, our findings suggest that ICG-001 efficiently inhibits CRC stemness and metastasis by suppressing MEIS1 expression. These results provide a basis for the further clinical investigation of ICG-001 as a targeted therapy for CSCs, opening a new avenue for the development of novel Wnt inhibitors for the treatment of CRC metastasis.

Wnt Inhibitory Factor 1 Binds to and Inhibits the Activity of Sonic Hedgehog.

The hedgehog (Hh) and Wnt pathways, crucial for the embryonic development and stem cell proliferation of Metazoa, have long been known to have similarities that argue for their common evolutionary origin. A surprising additional similarity of the two pathways came with the discovery that WIF1 proteins are involved in the regulation of both the Wnt and Hh pathways. Originally, WIF1 (Wnt Inhibitory Factor 1) was identified as a Wnt antagonist of vertebrates, but subsequent studies have shown that in Drosophila, the WIF1 ortholog serves primarily to control the distribution of Hh. In the present, work we have characterized the interaction of the human WIF1 protein with human sonic hedgehog (Shh) using Surface Plasmon Resonance spectroscopy and reporter assays monitoring the signaling activity of human Shh. Our studies have shown that human WIF1 protein binds human Shh with high affinity and inhibits its signaling activity efficiently. Our observation that the human WIF1 protein is a potent antagonist of human Shh suggests that the known tumor suppressor activity of WIF1 may not be ascribed only to its role as a Wnt inhibitor.

Hyperbaric Oxygen Therapy Does Not Have a Negative Impact on Bone Signaling Pathways in Humans.

Introduction: Oxygen is emerging as an important factor in the local regulation of bone remodeling. Some preclinical data suggest that hyperoxia may have deleterious effects on bone cells. However, its clinical relevance is unclear. Hence, we studied the effect of hyperbaric oxygen therapy (HBOT) on serum biomarkers reflecting the status of the Wnt and receptor activator of NF-κB ligand (RANKL) pathways, two core pathways for bone homeostasis. Materials and methods: This was a prospective study of 20 patients undergoing HBOT (mean age 58 yrs., range 35–82 yrs.) because of complications of radiotherapy or chronic anal fissure. Patients were subjected to HBOT (100% oxygen; 2.4 atmospheres absolute for 90 min). The average number of HBOT sessions was 20 ± 5 (range 8–31). Serum hypoxia-inducible factor 1- (HIF1-, osteoprotegerin (OPG), RANKL, and the Wnt inhibitors sclerostin and dickkopf-1 (DKK1) were measured at baseline and after HBOT by using specific immunoassays. Results: HIF-1α in eight patients with measurable serum levels increased from 0.084 (0.098) ng/mL at baseline to 0.146 (0.130) ng/mL after HBOT (p = 0.028). However, HBOT did not induce any significant changes in the serum levels of OPG, RANKL, sclerostin or DKK1. This was independent of the patients’ diagnosis, either neoplasia or benign. Conclusion: Despite the potential concerns about hyperoxia, we found no evidence that HBOT has any detrimental effect on bone homeostasis.

CircEAF2 counteracts Epstein-Barr virus-positive diffuse large B-cell lymphoma progression via miR-BART19-3p/APC/\u03b2-catenin axis

BackgroundEpstein-Barr virus (EBV) represents an important pathogenic factor of lymphoma and is significantly associated with poor clinical outcome of diffuse large B-cell lymphoma (DLBCL). Circular RNAs (circRNAs) play an essential role in lymphoma progression. However, the underlying mechanism of circRNA on DLBCL progression related to EBV remains largely unknown.MethodsCircRNA was screened by high-throughput sequencing in tumor samples of 12 patients with DLBCL according to EBV infection status. Expression of circEAF2, as well as the relationship with clinical characteristics and prognosis, were further analyzed in tumor samples of 100 DLBCL patients using quantitative real-time PCR. Gain- and loss-of-function experiments were conducted to investigate the biological functions of circEAF2 both in vitro and in vivo. The underlying mechanism of circRNA on DLBCL progression were further determined by RNA sequencing, RNA pull down assay, dual-luciferase reporter assay, rescue experiments and western blotting.ResultsWe identified a novel circRNA circEAF2, which was downregulated in EBV + DLBCL and negatively correlated with EBV infection and DLBCL progression. In EBV-positive B lymphoma cells, circEAF2 overexpression induced lymphoma cell apoptosis and sensitized lymphoma cells to epirubicin. As mechanism of action, circEAF2 specifically targeted EBV-encoded miR-BART19-3p, upregulated APC, and suppressed downstream β-catenin expression, resulting in inactivation of Wnt signaling pathway and inhibition of EBV + DLBCL cell proliferation. In EBV-positive B-lymphoma murine models, xenografted tumors with circEAF2 overexpression presented decreased Ki-67 positivity, increased cell apoptosis and retarded tumor growth.ConclusionsCircEAF2 counteracted EBV + DLBCL progression via miR-BART19-3p/APC/β-catenin axis, referring circEAF2 as a potential prognostic biomarker. Therapeutic targeting EBV-encoded miRNA may be a promising strategy in treating EBV-associated lymphoid malignancies.

Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species.

ABSTRACTDespite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated ‘The people behind the papers’ interview.

A novel mutation of SATB2 inhibits odontogenesis of human dental pulp stem cells through Wnt/\u03b2-catenin signaling pathway

BackgroundSATB2-associated syndrome (SAS) is a multisystem disorder caused by mutation of human SATB2 gene. Tooth agenesis is one of the most common phenotypes observed in SAS. Our study aimed at identifying novel variant of SATB2 in a patient with SAS, and to investigate the cellular and molecular mechanism of tooth agenesis caused by SATB2 mutation.MethodsWe applied whole exome sequencing (WES) to identify the novel mutation of SATB2 in a Chinese patient with SAS. Construction and overexpression of wild-type and the mutant vector was performed, followed by functional analysis including flow cytometry assay, fluorescent immunocytochemistry, western blot, quantitative real-time PCR and Alizarin Red S staining to investigate its impact on hDPSCs and the underlying mechanisms.ResultsAs a result, we identified a novel frameshift mutation of SATB2 (c. 376_378delinsTT) in a patient with SAS exhibiting tooth agenesis. Human DPSCs transfected with mutant SATB2 showed decreased cell proliferation and odontogenic differentiation capacity compared with hDPSCs transfected with wild-type SATB2 plasmid. Mechanistically, mutant SATB2 failed to translocate into nucleus and distributed in the cytoplasm, failing to activate Wnt/β-catenin signaling pathway, whereas the wild-type SATB2 translocated into the nucleus and upregulated the expression of active β-catenin. When we used Wnt inhibitor XAV939 to treat hDPSCs transfected with wild-type SATB2 plasmid, the increased odontogenic differentiation capacity was attenuated. Furthermore, we found that SATB2 mutation resulted in the upregulation of DKK1 and histone demethylase JHDM1D to inhibit Wnt/β-catenin signaling pathway.ConclusionWe identified a novel frameshift mutation of SATB2 (c.376_378delinsTT, p.Leu126SerfsX6) in a Chinese patient with SATB2-associated syndrome (SAS) exhibiting tooth agenesis. Mechanistically, SATB2 regulated osteo/odontogenesis of human dental pulp stem cells through Wnt/β-catenin signaling pathway by regulating DKK1 and histone demethylase JHDM1D.

Stage-specific regulation of DNA methylation by TET enzymes during human cardiac differentiation.

Changes in DNA methylation are associated with normal cardiogenesis, whereas altered methylation patterns can occur in congenital heart disease. Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) and promote locus-specific DNA demethylation. Here, we characterize stage-specific methylation dynamics and the function of TETs during human cardiomyocyte differentiation. Human embryonic stem cells (hESCs) in which all three TET genes are inactivated fail to generate cardiomyocytes (CMs), with altered mesoderm patterning and defective cardiac progenitor specification. Genome-wide methylation analysis shows TET knockout causes promoter hypermethylation of genes encoding WNT inhibitors, leading to hyperactivated WNT signaling and defects in cardiac mesoderm patterning. TET activity is also needed to maintain hypomethylated status and expression of NKX2-5 for subsequent cardiac progenitor specification. Finally, loss of TETs causes a set of cardiac structural genes to fail to be demethylated at the cardiac progenitor stage. Our data demonstrate key roles for TET proteins in controlling methylation dynamics at sequential steps during human cardiac development.

Computational and in vitro validation of cardiogenic induction of quercetin on adipose-derived mesenchymal stromal cells through the inhibition of Wnt and non-Smad-dependent TGF-β pathways.

Exploiting human mesenchymal stem cells (hMSCs) was proposed as a promising therapeutic approach for cardiovascular disease due to their capacity to differentiate into cardiac cells. Though modulation of the intracellular signaling pathways dominantly WNT/β catenin and transforming growth factor-β (TGF-β) have been reported to promote differentiation of hMSCs into cardiomyocytes in the prevailing literature, a safe and reproducible system for their clinical application has not yet turned into reality. In the present study, the molecular docking-based strategy was first applied for evaluating the potency of some natural phenolic compounds in the modulation of Wnt and TGF-β signaling pathways using a vital class of crystallographic protein structures of WNT signaling regulators such as Frizzled, Disheveled, GSK3-β, β-catenin, LRP 5/6 extracellular domain, Tankyrase and their variety of active pockets. Then, the impacts of plant-derived chemical compounds on the regulation of the relevant signals for the differentiation of hMSCs into the definitive mesoderm lineage and cardiac progenitors were assessed in vitro. Data obtained revealed the synergistic activity of Wnt and TGF-β superfamily to direct cardiac differentiation in human cardiogenesis by comparing cardiac gene expression in the presence and absence of the TGF-β inhibitors. We found that the inhibitory effect of canonical Wnt/β-catenin is sufficient to cause proper cardiomyocyte differentiation, but the TGF-β pathway plays a vital role in enhancing the expression of the cardiomyocyte-specific marker (cTnT). It was found that quercetin, a p38MAPK inhibitor with the high energy dock to the active pocket of Wnt receptors, promotes cardiac differentiation via the inhibition of both Wnt and non-Smad TGF-β pathways. Altogether, data presented here can contribute to the development of a feasible and efficient cardiac differentiation protocol as an "off-the-shelf" therapeutic source using novel natural agents for cardiac repair or regeneration.

CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids.

Human pluripotent stem cells (PSCs) represent a powerful tool to investigate human eye development and disease. When grown in 3D, they can self-assemble into laminar organized retinas; however, variation in the size, shape and composition of individual organoids exists. Neither the microenvironment nor the timing of critical growth factors driving retinogenesis are fully understood. To explore early retinal development, we developed a SIX6-GFP reporter that enabled the systematic optimization of conditions that promote optic vesicle formation. We demonstrated that early hypoxic growth conditions enhanced SIX6 expression and promoted eye formation. SIX6 expression was further enhanced by sequential inhibition of Wnt and activation of sonic hedgehog signaling. SIX6 + optic vesicles showed RNA expression profiles that were consistent with a retinal identity; however, ventral diencephalic markers were also present. To demonstrate that optic vesicles lead to bona fide “retina-like” structures we generated a SIX6-GFP/POU4F2-tdTomato dual reporter line that labeled the entire developing retina and retinal ganglion cells, respectively. Additional brain regions, including the hypothalamus and midbrain-hindbrain (MBHB) territories were identified by harvesting SIX6 + /POU4F2- and SIX6- organoids, respectively. Using RNAseq to study transcriptional profiles we demonstrated that SIX6-GFP and POU4F2-tdTomato reporters provided a reliable readout for developing human retina, hypothalamus, and midbrain/hindbrain organoids.

Abstract 11611: The Wnt Inhibitor Secreted Frizzled-Related Protein 3 Protects Mitral Valve Endothelium from Mi-Induced Endothelial to Mesenchymal Transition

Introduction: There are changes in the mitral valve (MV) leaflets seen within 2-6 months post-myocardial infarction (MI) that include endothelial to mesenchymal transition (EndMT) and fibrosis. Early modulation of EndMT and fibrosis in MV leaflets may limit ischemic mitral regurgitation and heart failure. The onset of EndMT and fibrosis in MV leaflets and the mechanism of their instigation are not known. Hypothesis: We hypothesized that circulating molecules present in plasma within days after MI incite EndMT and fibrotic processes in MV leaflets. Methods: Using flow cytometric analysis and immunofluorescence staining, we examined the onset of EndMT in 20 adult sheep: 8 sheep with IMI, 8 with sham surgery, and 4 naïve controls with immediate excision of hearts under general anesthesia. The effect of plasma on EndMT induction in primary MV endothelial cells (VECs) was assessed by qPCR, migration assays, and immunofluorescence staining. Plasma cytokine levels were assessed using a cytokine array. Immunoblotting, immunofluorescence staining, RNA sequencing and bioinformatic analysis were used to identify biochemical changes by post-MI plasma. Results: Ovine MVs harvested 8-10 days after inferior MI revealed an onset of EndMT, shown by increased CD31/α-SMA-positive cells in post-MI MV leaflets. In contract to sham plasma, post-MI plasma induced EndMT and pro-fibrotic markers and enhanced migration of primary mitral VECs. Analysis of sham versus post-MI plasma revealed a significant drop in the Wnt antagonist sFRP3 in post-MI plasma. Addition of recombinant sFRP3 to post-MI plasma reversed its EndMT-inducing effect on mitral VECs, measured by restored VE-cadherin, reduced α-SMA, and reduced TGFβ1-3 expression. RNA-seq analysis of mitral VECs exposed to post-MI showed upregulated FOXM1. Blocking FOXM1 with Siomycin A reduced EndMT and pro-fibrotic transcripts in mitral VECs treated with post-MI plasma. Finally, FOXM1 induced by post-MI plasma was downregulated by sFRP3. Conclusions: Reduced sFRP3 in post-MI plasma facilitates EndMT and fibrosis in mitral VECs by increasing the transcription factor FOXM1. Restoring sFRP3 levels and/or inhibiting FOXM1 may provide new strategies to minimize MI-induced maladaptive changes that occur in the MV.

Abstract 13459: Human iPS-Derived Pre-Epicardial Cells Direct Cardiomyocyte Aggregation Expansion and Organization in vitro

During heart maturation, epicardial-derived cells emanating from the pro-epicardial organ envelope the developing heart, then integrate within the myocardium and undergo epithelial-mesenchymal transition (EMT) to become fibroblasts, smooth muscle cells, and endothelial cells that enable several processes of healthy heart morphogenesis (i.e., ventricular thickening, compaction, and angiogenesis). Directed cardiac differentiation of human induced-pluripotent stem cells (hiPSCs) holds great potential for generating several cardiovascular cell types, which can theoretically be combined to model cardiac development more completely and engineer sophisticated myocardial grafts. Here, we show that differentiating hiPSC-derived lateral plate mesoderm (LPM) with BMP4, RA and VEGF (BVR) for 96 hours can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) with high efficiency (86.8 ± 4.1% WT1 + by FACS), that also show significant increases in WT1, TBX18, SEMA3D , TBX5, BNC, and SCX transcript expression within 7 days. This protocol was successfully reproduced in three commercial lines of hiPSCs. BVR stimulation after Wnt inhibition of LPM demonstrated efficient co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidated CMs into dense aggregates, which then formed a connected beating syncytium with significantly enhanced contractility (0.029 ± 0.002 mN/mm 2 ; p&lt;0.05 vs. CMs-alone and co-culture controls), improved calcium handling (i.e. amplitude, upstroke, and decay), sarcomere length (1.82 ± 0.02 μm vs. 1.72 ± 0.03 μm, p=0.00264) and mitochondrial density (79.4 ± 3% vs. 54.9 ± 6%, p=0.0029). Co-cultured PECs demonstrated enhanced maturity to epicardial cells with significant upregulation of UPK1B, ITGA4, and ALDH1A2 transcript expressions. Our study also demonstrated that PECs secrete IGF2 and stimulate CM proliferation in co-culture (37.2 ± 2.1% vs. 28.1 ± 1%, p=0.016). These characteristics suggest PECs could play a key role in enhancing cell-cell signaling and tissue organization, which may help to recapitulate important aspects of cardiac morphogenesis in vitro and advance the maturation of bioengineered cardiac constructs.

DDRE-15. Sema3C SIGNALING CONFERS RESISTANCE TO Wnt INHIBITION IN GLIOBLASTOMA

Abstract BACKGROUND Wnt signaling is widely dysregulated in cancer. The therapeutic potential of Wnt inhibitors appears promising in preclinical studies. However, they have uniformly failed clinical trials. How cancer cells develop Wnt inhibitor resistance is poorly understood. Current Wnt inhibitors are designed targeting either ligand or receptor. We hypothesized cancer cells will bypass ligand-receptor interaction through an unknown mechanism. We focused on the neurodevelopmental signaling program of Semaphorin 3C (Sema3C) that is upregulated in 85% of GBM and regulates glioma stem-cell-driven tumor progression. RESULTS Porcupine inhibitor LGK974 reduced TCF1 expression in the GBM tumor mouse models, suggesting successful target engagement in vivo. However, it failed to prolong the overall survival. Sema3C expression strongly correlated with TCF1 expression in human GBM samples by immunohistochemical analysis. Genetic inhibition of Sema3C and TCF1 together prolonged animal survival more than either alone, indicating better control of Wnt pathway signaling with dual pathway blockade. Immunofluorescence and cell fractionation studies revealed that Sema3C signaling drove β-catenin nuclear accumulation. Sema3C regulates transactivation of Wnt target genes including TCF1, c-Myc and c-Met. Sema3C pathway activates Rac1. It is reported that Rac1 activates β-catenin and promotes β-catenin nuclear accumulation. In GSCs, constitutively active Rac1 restored β-catenin nuclear localization and rescued TCF1 and c-Myc down-regulation in the setting of Sema3C silencing. Sema3C can drive canonical Wnt signaling even when Wnt ligand secretion is blocked. Together, the data support that GSCs can escape Wnt inhibition through Sema3C and Rac1. CONCLUSIONS Sema3C signaling drives canonical Wnt signaling, providing an escape mechanism for cancer cells despite Wnt ligand-receptor interruption. Sema3C-β-catenin signaling promotes GSC self-renewal and tumor progression. Upstream Wnt pathway inhibition alone is insufficient to control tumors. Our data provide a therapeutic strategy of dual blockade of Wnt and Sema3C pathways to provide clinically significant tumor control.

Activation of the WNT-BMP-FGF Regulatory Network Induces the Onset of Cell Death in Anterior Mesodermal Cells to Establish the ANZ

The spatiotemporal control of programmed cell death (PCD) plays a significant role in sculpting the limb. In the early avian limb bud, the anterior necrotic zone (ANZ) and the posterior necrotic zone are two cell death regions associated with digit number reduction. In this study, we evaluated the first events triggered by the FGF, BMP, and WNT signaling interactions to initiate cell death in the anterior margin of the limb to establish the ANZ. This study demonstrates that in a period of two to 8 h after the inhibition of WNT or FGF signaling or the activation of BMP signaling, cell death was induced in the anterior margin of the limb concomitantly with the regulation of Dkk, Fgf8, and Bmp4 expression. Comparing the gene expression profile between the ANZ and the undifferentiated zone at 22HH and 25HH and between the ANZ of 22HH and 25HH stages correlates with functional programs controlled by the regulatory network FGF, BMP, and WNT signaling in the anterior margin of the limb. This work provides novel insights to recognize a negative feedback loop between FGF8, BMP4, and DKK to control the onset of cell death in the anterior margin of the limb to the establishment of the ANZ.

A Tumor-Targeting Metal-Organic Nanoparticle Constructed by Dynamic Combinatorial Chemistry toward Accurately Redressing Carcinogenic Wnt Cascade.

Targeted and immunological therapy have revolutionized the malignancy treatment, but is suffering from the dose-limiting side effects and inadequate responsiveness. The emerging nanoscale infinite coordination polymers provide a feasible strategy for tumor targeting and immune sensitization. Herein, a "one-pot" self-assembled strategy based on dynamic combinatorial chemistry (DCC) principle is designed to construct a tumor-targeting metal-organic nanoparticle (MOICP) through a spontaneous co-assembling among three metal-organic coordination polymers tuned by a Wnt-inhibitor carnosic acid (CA). Responding to the tumor microenvironment, MOICP presents an optimized tumor-preferential accumulation and the satisfactory biosafety. MOICP is more active in vitro and in vivo than CA in suppressing of Wnt signaling pathway, and potently inhibits tumor growth in a patient-derived xenograft model of Wnt-activated pancreatic carcinoma. Moreover, MOICP reverses the lack of intratumoral infiltration of T lymphocytes, and hence augments the action of Anti-PD1 (programmed cell death protein 1) immunotherapy in B16F10 melanoma allograft mice model. This clinically viable MOICP can not only be applied to Wnt inhibition for cancer targeted therapy and immunotherapeutic sensitization, but also provides a de novo pattern for nanomedicine architecture with cargo-initiated co-self-assembly guided by DCC, thereby bringing new inspiration in general for disease intervention.

Wnt/β-Catenin Signaling Stabilizes Hemidesmosomes in Keratinocytes

Hemidesmosomes (HDs) are adhesion complexes that promote epithelial-stromal attachment in stratified and complex epithelia, including the epidermis. In various biological processes, such as differentiation and migration of epidermal keratinocytes during wound healing or carcinoma invasion, quick assembly and disassembly of HDs are prerequisites. In this study, we show that inhibition of Wnt/β-catenin signaling disturbs HD organization in keratinocytes. Screening with inhibitors identified the depletion of HD components and HD-like structures through Wnt inhibition, but keratinocyte differentiation was not affected. Wnt inhibition significantly diminished plectin and type XVII collagen expression in the basal side of Wnt-inhibited cells and the dermo-epidermal junction of the Wnt-inactive murine basal epidermis. Similar to Wnt inhibition, PLEC-knockout cells or cells with plectin-type XVII collagen binding defects showed type XVII collagen reduction in the basal side of the cells, implying the possible involvement of Wnt/β-catenin signaling in HD assembly. Atypical protein kinase C inhibition ameliorated the phenotypes of Wnt-inhibited cells. These findings show that Wnt/β-catenin signaling regulates the localization of HD components in keratinocytes and that the atypical protein kinase C pathway is involved in Wnt inhibition‒induced HD disarrangement. Our study suggests that the Wnt signaling pathway could be a potential therapeutic target for treating HD-defective diseases, such as epidermolysis bullosa.

Auto/paracrine factors and early Wnt inhibition promote cardiomyocyte differentiation from human induced pluripotent stem cells at initial low cell density

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) have received increasing attention for their clinical use. Many protocols induce cardiomyocytes at an initial high cell density (confluence) to utilize cell density effects as hidden factors for cardiomyocyte differentiation. Previously, we established a protocol to induce hiPSC differentiation into cardiomyocytes using a defined culture medium and an initial low cell density (1% confluence) to minimize the hidden factors. Here, we investigated the key factors promoting cardiomyocyte differentiation at an initial low cell density to clarify the effects of cell density. Co-culture of hiPSCs at an initial low cell density with those at an initial high cell density showed that signals secreted from cells (auto/paracrine factors) and not cell–cell contact signals, played an important role in cardiomyocyte differentiation. Moreover, although cultures with initial low cell density showed higher expression of anti-cardiac mesoderm genes, earlier treatment with a Wnt production inhibitor efficiently suppressed the anti-cardiac mesoderm gene expression and promoted cardiomyocyte differentiation by up to 80% at an initial low cell density. These results suggest that the main effect of cell density on cardiomyocyte differentiation is inhibition of Wnt signaling at the early stage of induction, through auto/paracrine factors.

Targeting Wnt Signaling in the Tumor Immune Microenvironment to Enhancing EpCAM CAR T-Cell therapy.

Colorectal cancer (CRC) patients are still lacking viable treatments. Chimeric antigen receptor (CAR) T cells have shown promise in hematologic malignancies, but their efficacy in solid tumors has been limited due to the immunosuppressive tumor microenvironment. We found that cancer antigen- EpCAM expression increased in the metastatic stage compared with the primary stage in cancers and the activation of Wnt and TGFβ pathways was positively correlated with EpCAM expression in multiple cancers, including colorectal cancer. We constructed CAR T cells targeting EpCAM that successfully showed selective cytotoxicity in highly EpCAM-expressing cancer cell lines. The combination of EpCAM CAR-T with the Wnt inhibitor-hsBCL9CT-24 displayed synergetic effect against EpCAM-positive colon cells in vitro and also in vivo. A mechanistic study showed that hsBCL9CT-24 treatment could modulate the tumor environment and improve infiltration of T cells, while possibly promoting the effector T cells at the early stages and postponing the exhaustion of CAR T cells at advanced stages. Overall, these results demonstrated that the combination of EpCAM CAR T-cell therapy with the Wnt inhibitor can overcome the limitations of CAR T cells in treating solid tumors.