Activation Of Non-canonical Wnt Signaling Research Articles

Microglia-induced activation of non-canonical Wnt signaling aggravates neurodegeneration in demyelinating disorders.

Oligodendrocytes are myelinating cells of the central nervous system. Multiple sclerosis (MS) is a demyelinating disease characterized by both myelin loss and neuronal degeneration. However, the molecular mechanisms underlying neuronal degeneration in demyelinating disorders are not fully understood. In the experimental autoimmune encephalomyelitis (EAE) demyelinating mouse model of MS, inflammatory microglia produce cytokines including interleukin-1β (IL-1β). Since microglia and non-canonical Wnt signaling components in neurons, such as the co-receptor Ror2, were observed in the spinal cord of EAE mice, we postulated that the interplay between activated microglia and spinal neurons under EAE conditions is mediated through non-canonical Wnt signaling. EAE treatment up-regulated in vivo expression of non-canonical Wnt signaling components in spinal neurons through microglial activation. In accordance with the neuronal degeneration detected in the EAE spinal cord in vivo, co-culture of spinal neurons with microglia or the application of recombinant IL-1β up-regulated non-canonical Wnt signaling, and induced neuronal cell death, which was suppressed by the inhibition of the Wnt-Ror2 pathway. Ectopic non-canonical Wnt signaling aggravated the demyelinating pathology in another MS mouse model due to Wnt5a-induced neurodegeneration. The linkage between activated microglia and neuronal Wnt-Ror2 signaling may provide a possible candidate target for therapeutic approaches to demyelinating disorders.

Abstract 2515: Pharmacological WNT-inhibition acts synergistically with chemo- and radiotherapy by overcoming treatment-resistance in glioma stem cells

Abstract Objective: Glioma Stem Cells (GSCs) are considered to be responsible for Glioblastoma's (GBM’s) dismal prognosis. WNT signaling is one of the major players in GSCs that promotes their radio- and chemoresistance. In this study we tested whether LGK974, an inhibitor of both canonical and non-canonical WNT signaling, might sensitize GBM cells to chemotherapeutics and irradiation and determined possible mechanisms causing treatment-resistance in GBM. Methods: At first we analyzed MGMT promoter methylation status of several GBM cell lines in order to define cells resistant to the standard chemotherapeutic drug Temozolomide (TMZ). For further investigations we chose two cell lines resistant or sensitive to TMZ, respectively. IC50 concentrations of TMZ and LGK974 as well as IC50 dose of γ-irradiation were tested via Titer Blue cell viability assay. Effectiveness of different concentration of each single therapy and its combination with LGK974 was determined to create a drug-response-curve due to the computerized simulation of synergism and antagonism in drug combination studies (Chou, 2006). A reporter assay for canonical WNT/β-catenin signaling was used to quantify the effect of TMZ and irradiation alone and in combination with LGK974 on the canonical and non-canonical WNT pathway. Western Blot and qPCR were applied to analyze the effects of combinatory therapy on protein and gene expression concerning WNT pathway activation. Results: We observed a significant synergy of combined irradiation/TMZ with LGK974 in all cell lines. There was no difference in effectiveness between cell lines resistant or sensitive to TMZ. Interestingly, following TMZ treatment or irradiation cells showed slightly decreased canonical WNT pathway activity. Furthermore, non-canonical target genes and receptors were upregulated under chemo- and radiotherapy, suggesting a reciprocal activation of the non-canonical branch of WNT signaling pathway. This phenomenon might possibly be responsible for the development of resistance against standard chemo- and radiotherapy. Conclusions: We propose that GSCs undergo a shift from canonical to non-canonical WNT signaling to become treatment-resistant under standard chemo- and radiotherapy. Treatment with LGK974 results in therapeutic synergy at least in part via suppressing the therapy-induced activation of non-canonical WNT signaling. Our results confirm the potential benefit of combining TMZ or irradiation with the WNT inhibitor LGK974 to overcome therapy-resistance in GBM. Citation Format: Abigail K. Suwala, Ulf D. Kahlert, Jaroslaw Maciaczyk. Pharmacological WNT-inhibition acts synergistically with chemo- and radiotherapy by overcoming treatment-resistance in glioma stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2515.

CAPE suppresses migration and invasion of prostate cancer cells via activation of non-canonical Wnt signaling.

Prostate cancer (PCa) was the fifth most common cancer overall in the world. More than 80% of patients died from PCa developed bone metastases. Caffeic acid phenethyl ester (CAPE) is a main bioactive component of honeybee hive propolis. Transwell and wound healing assays demonstrated that CAPE treatment suppressed the migration and invasion of PC-3 and DU-145 PCa cells. Gelatin zymography and Western blotting indicated that CAPE treatment reduced the abundance and activity of MMP-9 and MMP-2. Analysis using Micro-Western Array (MWA), a high-throughput antibody-based proteomics platform with 264 antibodies detecting signaling proteins involved in important pathways indicated that CAPE treatment induced receptor tyrosine kinase-like orphan receptor 2 (ROR2) in non-canonical Wnt signaling pathway but suppressed abundance of β-catenin, NF-κB activity, PI3K-Akt signaling, and epithelial-mesenchymal transition (EMT). Overexpression or knockdown of ROR2 suppressed or enhanced cell migration of PC-3 cells, respectively. TCF-LEF promoter binding assay revealed that CAPE treatment reduced canonical Wnt signaling. Intraperitoneal injection of CAPE reduced the metastasis of PC-3 xenografts in tail vein injection nude mice model. Immunohistochemical staining demonstrated that CAPE treatment increased abundance of ROR2 and Wnt5a but decreased protein expression of Ki67, Frizzle 4, NF-κB p65, MMP-9, Snail, β-catenin, and phosphorylation of IκBα. Clinical evidences suggested that genes affected by CAPE treatment (CTNNB1, RELA, FZD5, DVL3, MAPK9, SNAl1, ROR2, SMAD4, NFKBIA, DUSP6, and PLCB3) correlate with the aggressiveness of PCa. Our study suggested that CAPE may be a potential therapeutic agent for patients with advanced PCa.

Microglia-induced activation of non-canonical Wnt signaling aggravates neurodegeneration in demyelinating disorders

Microglia-induced activation of non-canonical Wnt signaling aggravates neurodegeneration in demyelinating disorders

RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance.

Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging; hence, sampling circulating tumor cells (CTCs) may reveal drug-resistance mechanisms. We established single-cell RNA-sequencing (RNA-Seq) profiles of 77 intact CTCs isolated from 13 patients (mean six CTCs per patient), by using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing under treatment with an AR inhibitor, compared with untreated cases, indicates activation of noncanonical Wnt signaling (P = 0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, whereas its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single-cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure.

GATA2 regulates Wnt signaling to promote primitive red blood cell fate

Primitive erythropoiesis is regulated in a non cell-autonomous fashion across evolution from frogs to mammals. In Xenopus laevis, signals from the overlying ectoderm are required to induce the mesoderm to adopt an erythroid fate. Previous studies in our lab identified the transcription factor GATA2 as a key regulator of this ectodermal signal. To identify GATA2 target genes in the ectoderm required for red blood cell formation in the mesoderm, we used microarray analysis to compare gene expression in ectoderm from GATA2 depleted and wild type embryos. Our analysis identified components of the non-canonical and canonical Wnt pathways as being reciprocally up- and down-regulated downstream of GATA2 in both mesoderm and ectoderm. We show that up-regulation of canonical Wnt signaling during gastrulation blocks commitment to a hematopoietic fate while down-regulation of non-canonical Wnt signaling impairs erythroid differentiation. Our results are consistent with a model in which GATA2 contributes to inhibition of canonical Wnt signaling, thereby permitting progenitors to exit the cell cycle and commit to a hematopoietic fate. Subsequently, activation of non-canonical Wnt signaling plays a later role in enabling these progenitors to differentiate as mature red blood cells.

Insight into the role of Wnt5a-induced signaling in normal and cancer cells.

Wnt5a is involved in the activation of noncanonical Wnt signaling, including planar cell polarity (PCP) and Wnt-Ca(2+) pathways. The Ror-family of receptor tyrosine kinases is composed of Ror1 and Ror2 in mammals. Ror2 acts as a receptor or coreceptor for Wnt5a and regulates Wnt5a-induced activation of PCP pathway, and Wnt5a-Ror2 axis indeed plays critical roles in the developmental morphogenesis by regulating cell polarity and migration. Furthermore, Wnt5a-Ror2 axis is constitutively activated in cancer cells and confers highly motile and invasive properties on cancer cells through the expression of matrix metalloproteinase genes and enhanced formation of invadopodia. Meanwhile, Wnt5a also exhibits a tumor-suppressive function in certain cancers, including breast and colorectal carcinomas. Thus, it is of great importance to understand the respective molecular mechanisms governing Wnt5a-mediated tumor-progressive and tumor-suppressive functions, in order to develop novel and proper diagnostic and therapeutic strategies targeting Wnt5a signaling for human cancers.

Dapper-1 is essential for Wnt5a induced cardiomyocyte hypertrophy by regulating the Wnt/PCP pathway.

The Wnt signaling pathway was identified as crucial mediator of cardiomyocyte hypertrophy. In this study we found that activation of non-canonical Wnt signaling by Wnt5a stimulates protein synthesis and enlargement of cardiomyocyte surface area. These hypertrophic features were inhibited in Dapper-1 (Dpr1) depleted cells. On the molecular level, we observed inhibition of the non-canonical Wnt/planar-cell-polarity (PCP) pathway denoted by reduction of c-jun-n-terminal-kinase (JNK) phosphorylation. Upstream of JNK, increased protein levels of the Wnt/PCP trans-membrane receptor van-Gogh-like-2 (Vangl2) were observed along with an enrichment of Vangl2 in perinuclear located vesicles. The findings suggest that Dpr1 is essential for execution of the Wnt/PCP pathway and regulation of the Vangl2/JNK axis. Depletion of Dpr1 inhibits non-canonical Wnt signaling induced cardiomyocyte hypertrophy by blocking Wnt/PCP signaling.

Activation of noncanonical Wnt signaling through WNT5A in visceral adipose tissue of obese subjects is related to inflammation.

Wingless-type mouse mammary tumor virus integration site family (WNT)-5A is a glycoprotein involved in the regulation of the inflammatory response by activating the noncanonical Wnt signaling pathway. Secreted frizzled-related protein (SFRP)-5 acts as a decoy receptor that binds and sequesters WNT5A, preventing activation of frizzled receptors and attenuating the noncanonical Wnt signaling. The aim of the study was to evaluate the involvement of WNT5A and SFRP5 in obesity and obesity-related comorbidities as well as to explore their effect in visceral adipose tissue inflammation. Samples obtained from 90 subjects were used. Circulating and gene expression levels of WNT5A and SFRP5 were analyzed in different metabolic tissues. The effect of TNF-α and lipopolysaccharide on the transcript levels of WNT5A and SFRP5 in adipocytes was explored. We also investigated whether WNT5A itself can activate an inflammatory response. Increased circulating levels of WNT5A in obese patients (P < .05) were decreased (P < .001) after gastric bypass. In this line, WNT5A mRNA in visceral adipose tissue was increased (P < .05) in obese patients with gene expression levels of SFRP5 being down-regulated (P < .05). WNT5A mRNA expression was significantly enhanced (P < .01) by lipopolysaccharide and TNF-α treatment, whereas no effects were found in SFRP5 gene expression levels. Furthermore, exogenous WNT5A induced (P < .05) IL-6, IL1B, MMP2, MMP9, and SSP1 mRNA expression in human adipocyte cultures. Activation of noncanonical Wnt signaling through the up-regulation of WNT5A and down-regulation of SFRP5 may promote a proinflammatory state in visceral adipose tissue contributing to the development of obesity-associated comorbidities.

Matrix Metalloproteinase-9 Inhibition Improves Proliferation and Engraftment of Myogenic Cells in Dystrophic Muscle of mdx Mice

Duchenne muscular dystrophy (DMD) caused by loss of cytoskeletal protein dystrophin is a devastating disorder of skeletal muscle. Primary deficiency of dystrophin leads to several secondary pathological changes including fiber degeneration and regeneration, extracellular matrix breakdown, inflammation, and fibrosis. Matrix metalloproteinases (MMPs) are a group of extracellular proteases that are involved in tissue remodeling, inflammation, and development of interstitial fibrosis in many disease states. We have recently reported that the inhibition of MMP-9 improves myopathy and augments myofiber regeneration in mdx mice (a mouse model of DMD). However, the mechanisms by which MMP-9 regulates disease progression in mdx mice remain less understood. In this report, we demonstrate that the inhibition of MMP-9 augments the proliferation of satellite cells in dystrophic muscle. MMP-9 inhibition also causes significant reduction in percentage of M1 macrophages with concomitant increase in the proportion of promyogenic M2 macrophages in mdx mice. Moreover, inhibition of MMP-9 increases the expression of Notch ligands and receptors, and Notch target genes in skeletal muscle of mdx mice. Furthermore, our results show that while MMP-9 inhibition augments the expression of components of canonical Wnt signaling, it reduces the expression of genes whose products are involved in activation of non-canonical Wnt signaling in mdx mice. Finally, the inhibition of MMP-9 was found to dramatically improve the engraftment of transplanted myoblasts in skeletal muscle of mdx mice. Collectively, our study suggests that the inhibition of MMP-9 is a promising approach to stimulate myofiber regeneration and improving engraftment of muscle progenitor cells in dystrophic muscle.

Activation of Wnt Planar cell polarity (PCP) signaling promotes growth plate column formation in vitro

Disrupting the Wnt Planar Cell Polarity (PCP) signaling pathway in vivo results in loss of columnar growth plate architecture, but it is unknown whether activation of this pathway in vitro is sufficient to promote column formation. We hypothesized that activation of the Wnt PCP pathway in growth plate chondrocyte cell pellets would promote columnar organization in these cells that are normally oriented randomly in culture. Rat growth plate chondrocytes were transfected with plasmids encoding the Fzd7 cell-surface Wnt receptor, a Fzd7 deletion mutant lacking the Wnt-binding domain, or Wnt receptor-associated proteins Ror2 or Vangl2, and then cultured as three-dimensional cell pellets in the presence of recombinant Wnt5a or Wnt5b for 21 days. Cellular morphology was evaluated using histomorphometric measurements. Activation of Wnt PCP signaling components promoted the initiation of columnar morphogenesis in the chondrocyte pellet culture model, as measured by histomorphometric analysis of the column index (ANOVA p = 0.01). Activation of noncanonical Wnt signaling through overexpression of both the cell-surface Wnt receptor Fzd7 and receptor-associated protein Ror2 with addition of recombinant Wnt5a promotes the initiation of columnar architecture of growth plate chondrocytes in vitro, representing an important step toward growth plate regeneration.

Differential Dependence On Wnt Signaling Allows Chemical Mediated Eradication of Human Acute Leukemia without Affecting Normal Blood Stem Cells

AbstractAbstract 3278Specific targets of signaling pathways that control self-renewal and survival of acute myeloid leukemic stem cells (LSCs) vs. normal hematopoietic stem cells (HSCs) are largely unknown. Here, using a small molecule (CWP232228) derived from a parent compound that inhibits Wnt/TCF targets, we demonstrate reduction of primary human AML-blast growth and clonogenic capacity ex vivo, without effects on normal hematopoietic progenitors. Upon establishment of AML or normal hematopoiesis in immune-deficient recipients, in vivo administration of CWP232228 reduced leukemic disease and abolished LSC self-renewal, with no effect on normal HSC function. In vivo gene profiling and ex vivo molecular studies revealed that CWP232228 induces apoptosis and differentiation of AML-blasts via inhibition of Wnt/b-catenin signaling and activation of non-canonical Wnt signaling which phenocopies the effects of this small molecule. Our study reveals an in vivo differential dependence of AML on canonical vs. non-canonical Wnt signaling that allows therapeutic targeting of LSCs whilst sparing normal HSCs. Disclosures:Chung:Choongwae Pharma Corporation: Employment.

Wnt Signaling Molecules in Left Ventricular Remodeling

Under acute or chronic stresses, the adult heart undergoes a remodeling process that involves cardiomyocyte hypertrophy accompanied by apoptosis, necrosis, and fibrosis that lead to impaired cardiac contractility. The role of endogenous regeneration in this process is currently under investigation. Sustained deleterious stimuli will lead to a decompensated form of hypertrophy often culminating in heart failure.1 This form of hypertrophy is often referred to as “maladaptive.” When dealing with hypertrophy, it appears important to distinguish between the term being used on the cellular and molecular level (enlargement of individual cardiomyocytes and re-expression of fetal/embryonic genes) and the organ level (increased heart weight, left ventricular wall thickness, and functional diastolic and systolic impairment). In our view, these processes are certainly linked but not identical. Hypertrophy on the organ levels summarizes several independent cellular and molecular processes (see below), where cardiomyocyte growth is not necessarily the most important. Independent of its origin, cardiac hypertrophy is associated with alterations in cardiac geometry, mass, architecture, and function controlled by a complex network of interconnected and abundant signal-transduction pathways.2 New signaling molecules are emerging as possible targets to specifically attenuate maladaptive hypertrophy. Pathological, stress-induced growth of cardiomyocytes was shown to depend on Wnt/β-catenin nuclear signaling rather than its adhesive function in cell adhesion. However, the specificity of the cell type and the molecular mechanisms governing the Wnt signaling–dependent changes are currently unknown.3 In this issue of the Hypertension , the study by Malekar et al4 provides new evidences concerning the …

Activation of Non-Canonical Wnt Pathway in Human Mesenchymal Cells Affects Osteogenic Differentiation: A Potential Target in Multiple Myeloma Microenvironment

Osteoblast suppression is the hallmark of Multiple Myeloma (MM) osteolytic bone lesions mainly due to the capacity of MM cells to inhibit the osteogenic differentiation of bone marrow (BM) mesenchymal cells (MSC). Many evidences suggest that Wnt signaling is critically involved in the regulation of osteoblast formation. Recently, in murine osteoprogenitor cells and in MM mouse models it has been shown that activation of canonical Wnt pathway stimulate osteoblast formation and blunts MM-induced bone destruction. In this study we have investigated whether modulation of both canonical and noncanonical Wnt signaling pathway may affect osteogenic differentiation of human MSC and counterbalance the suppressive effect of MM cells. First we checked the potential expression of Wnt activators and inhibitors by human MSC and osteoprogenitor cells (PreOB) by gene arrays. We found that both cells expressed the activator of non-canonical Wnt pathways Wnt5a but lack of express the main activators of canonical Wnt signaling as Wnt1, Wnt3a and Wnt8. The presence of the Wnt5a receptor FZD2 and FZD5 was also detected in both cells as well as that of FZD3, FZD6 and FDZ7 and the Wnt canonical co-receptors LRP5 and LRP6. On the other hand we found that both inhibitors of canonical and non-canonical Wnt pathways DKK-1 and sFRP-1 were expressed by MSC. Secondly, activation of either canonical or non-canonical Wnt signaling pathway by Wnt3a and Wnt5a treatment respectively was performed in human MSC to evaluate the effect on osteogenic differentiation and the expression of osteoblast related markers (Collagen I, Osteocalcin and Alkaline Phosphatase). We found that Wnt5a treatment but not Wnt3a significantly increased the early osteogenic differentiation and the expression of alkaline phosphatase in MSC. Consistently in a co-culture system with MM cells Wnt5a treatment blunted, at least in part, the inhibitory effect of MM cells on alkaline phosphatase expression by MSC and PreOB. To go further inside, we evaluated in both primary human BM MSC and the human MSC cell line HS-5 the effect of either the activation of non-canonical Wnt signaling by Wnt5a overexpression using a lentivirus vector or the Wnt5a suppression using siRNA. Wnt5a over-expression in MSC induced the activation of Wnt/Ca++ non-canonical pathway as demonstrated by the increase of Wnt5a secretion and phospho-PKC expression detected by westernblot analysis. Consequently to non-canonical Wnt signal activation we found a significant increase of alkaline phosphatase expression by MSC cells as well as of their osteogenic differentiation. Interestingly, analyzing the gene expression profile by microarray, we found that Wnt5a overexpression in MSC also affects the expression of chemokines, inflammatory cytokines and pro-angiogenic molecules. In conclusion our data indicate that activation of non-canonical Wnt signal pathway may represent a potential target in MM microenvironment to counterbalance the inhibitory effect of MM cells on osteogenic differentiation of human MSC.

Diversin regulates heart formation and gastrulation movements in development

Canonical and noncanonical Wnt signaling regulate crucial events in the development of vertebrates and invertebrates. In this work we show that vertebrate Diversin, a potential orthologue of Drosophila Diego, controls fusion of heart precursors and gastrulation movements in zebrafish embryogenesis. These events are regulated by noncanonical Wnt signaling, which is independent of beta-catenin. We found that Diversin directly interacts with Dishevelled and that this interaction is necessary and sufficient to mediate signals of the noncanonical Wnt pathway to downstream effectors like Rho family GTPases and Jun N-terminal kinase. The ankyrin repeats of Diversin are required for the interaction with Dishevelled, for the activation of noncanonical Wnt signaling, and for the biological responses. The mutation K446M in the DEP domain of vertebrate Dishevelled, which mimics a classical Drosophila loss of function mutation, prevents functional interaction with Diversin's ankyrin repeats. Diversin also affects planar cell polarity in Drosophila, which is controlled by the noncanonical Wnt signaling pathway. Our data thus demonstrate that Diversin and Dishevelled function together in a mutually dependent fashion in zebrafish gastrulation and organ formation.