Wnt/β-Catenin Signaling Contributes to Paclitaxel Resistance in Bladder Cancer Cells with Cancer Stem Cell-Like Properties

The Wnt/β-catenin pathway plays an important role in tumor progression and chemotherapy resistance and seems to be essential for the maintenance of cancer stem cells (CSC) in several tumor types. However, the interplay of these factors has not been fully addressed in bladder cancer. Here, our goal was to analyze the role of the Wnt/β-catenin pathway in paclitaxel resistance and to study the therapeutic efficacy of its inhibition in bladder cancer cells, as well as to determine its influence in the maintenance of the CSC-like phenotype in bladder cancer. Our results show that paclitaxel-resistant HT1197 cells have hyperactivation of the Wnt/β-catenin pathway and increased CSC-like properties compared with paclitaxel-sensitive 5637 cells. Paclitaxel sensitivity diminishes in 5637 cells after β-catenin overexpression or when they are grown as tumorspheres, enriched for the CSC-like phenotype. Additionally, downregulation of β-catenin or inhibition with XAV939 sensitizes HT1197 cells to paclitaxel. Moreover, a subset of muscle-invasive bladder carcinomas shows aberrant expression of β-catenin that associates with positive expression of the CSC marker ALDH1A1. In conclusion, we demonstrate that Wnt/β-catenin signaling contributes to paclitaxel resistance in bladder cancer cells with CSC-like properties.     

Wnt-Signaling Inhibitor Wnt-C59 Suppresses the Cytokine Upregulation in Multiple Organs of Lipopolysaccharide-Induced Endotoxemic Mice via Reducing the Interaction between β-Catenin and NF-κB

Sepsis is characterized by multiple-organ dysfunction caused by the dysregulated host response to infection. Until now, however, the role of the Wnt signaling has not been fully characterized in multiple organs during sepsis. This study assessed the suppressive effect of a Wnt signaling inhibitor, Wnt-C59, in the kidney, lung, and liver of lipopolysaccharide-induced endotoxemic mice, serving as an animal model of sepsis. We found that Wnt-C59 elevated the survival rate of these mice and decreased their plasma levels of proinflammatory cytokines and organ-damage biomarkers, such as BUN, ALT, and AST. The Wnt/β-catenin and NF-κB pathways were stimulated and proinflammatory cytokines were upregulated in the kidney, lung, and liver of endotoxemic mice. Wnt-C59, as a Wnt signaling inhibitor, inhibited the Wnt/β-catenin pathway, and its interaction with the NF-κB pathway, which resulted in the inhibition of NF-κB activity and proinflammatory cytokine expression. In multiple organs of endotoxemic mice, Wnt-C59 significantly reduced the β-catenin level and interaction with NF-κB. Our findings suggest that the anti-endotoxemic effect of Wnt-C59 is mediated via reducing the interaction between β-catenin and NF-κB, consequently suppressing the associated cytokine upregulation in multiple organs. Thus, Wnt-C59 may be useful for the suppression of the multiple-organ dysfunction during sepsis.     

Neuroinflammation in Schizophrenia: The Key Role of the WNT/β-Catenin Pathway

Schizophrenia is a very complex syndrome involving widespread brain multi-dysconnectivity. Schizophrenia is marked by cognitive, behavioral, and emotional dysregulations. Recent studies suggest that inflammation in the central nervous system (CNS) and immune dysfunction could have a role in the pathogenesis of schizophrenia. This hypothesis is supported by immunogenetic evidence, and a higher incidence rate of autoimmune diseases in patients with schizophrenia. The dysregulation of the WNT/β-catenin pathway is associated with the involvement of neuroinflammation in schizophrenia. Several studies have shown that there is a vicious and positive interplay operating between neuroinflammation and oxidative stress. This interplay is modulated by WNT/β-catenin, which interacts with the NF-kB pathway; inflammatory factors (including IL-6, IL-8, TNF-α); factors of oxidative stress such as glutamate; and dopamine. Neuroinflammation is associated with increased levels of PPARγ. In schizophrenia, the expression of PPAR-γ is increased, whereas the WNT/β-catenin pathway and PPARα are downregulated. This suggests that a metabolic-inflammatory imbalance occurs in this disorder. Thus, this research’s triptych could be a novel therapeutic approach to counteract both neuroinflammation and oxidative stress in schizophrenia.     

Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid β-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal β-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid β-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid β-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.     

The PPARβ/δ-AMPK Connection in the Treatment of Insulin Resistance

The current treatment options for type 2 diabetes mellitus do not adequately control the disease in many patients. Consequently, there is a need for new drugs to prevent and treat type 2 diabetes mellitus. Among the new potential pharmacological strategies, activators of peroxisome proliferator-activated receptor (PPAR)β/δ show promise. Remarkably, most of the antidiabetic effects of PPARβ/δ agonists involve AMP-activated protein kinase (AMPK) activation. This review summarizes the recent mechanistic insights into the antidiabetic effects of the PPARβ/δ-AMPK pathway, including the upregulation of glucose uptake, muscle remodeling, enhanced fatty acid oxidation, and autophagy, as well as the inhibition of endoplasmic reticulum stress and inflammation. A better understanding of the mechanisms underlying the effects resulting from the PPARβ/δ-AMPK pathway may provide the basis for the development of new therapies in the prevention and treatment of insulin resistance and type 2 diabetes mellitus.     

Cannabidiol and the Canonical WNT/β-Catenin Pathway in Glaucoma

Glaucoma is a progressive neurodegenerative disease which constitutes the main frequent cause of irreversible blindness. Recent findings have shown that oxidative stress, inflammation and glutamatergic pathway play key roles in the causes of glaucoma. Recent studies have shown a down regulation of the WNT/β-catenin pathway in glaucoma, associated with overactivation of the GSK-3β signaling. WNT/β-catenin pathway is mainly associated with oxidative stress, inflammation and glutamatergic pathway. Cannabidiol (CBD) is a non-psychotomimetic phytocannabinoid derived from Cannabis sativa plant which possesses many therapeutic properties across a range of neuropsychiatric disorders. Since few years, CBD presents an increased interest as a possible drug in anxiolytic disorders. CBD administration is associated with increase of the WNT/β-catenin pathway and decrease of the GSK-3β activity. CBD has a lower affinity for CB1 but can act through other signaling in glaucoma, including the WNT/β-catenin pathway. CBD downregulates GSK3-β activity, an inhibitor of WNT/β-catenin pathway. Moreover, CBD was reported to suppress pro-inflammatory signaling and neuroinflammation, oxidative stress and glutamatergic pathway. Thus, this review focuses on the potential effects of cannabidiol, as a potential therapeutic strategy, on glaucoma and some of the presumed mechanisms by which this phytocannabinoid provides its possible benefit properties through the WNT/β-catenin pathway.     

Crosstalk between β-Catenin and CCL2 Drives Migration of Monocytes towards Glioblastoma Cells

Isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) is a fast growing and highly heterogeneous tumor, often characterized by the presence of glioblastoma stem cells (GSCs). The plasticity of GSCs results in therapy resistance and impairs anti-tumor immune response by influencing immune cells in the tumor microenvironment (TME). Previously, β-catenin was associated with stemness in GBM as well as with immune escape mechanisms. Here, we investigated the effect of β-catenin on attracting monocytes towards GBM cells. In addition, we evaluated whether CCL2 is involved in β-catenin crosstalk between monocytes and tumor cells. Our analysis revealed that shRNA targeting β-catenin in GBMs reduces monocytes attraction and impacts CCL2 secretion. The addition of recombinant CCL2 restores peripheral blood mononuclear cells (PBMC) migration towards medium (TCM) conditioned by shβ-catenin GBM cells. CCL2 knockdown in GBM cells shows similar effects and reduces monocyte migration to a similar extent as β-catenin knockdown. When investigating the effect of CCL2 on β-catenin activity, we found that CCL2 modulates components of the Wnt/β-catenin pathway and alters the clonogenicity of GBM cells. In addition, the pharmacological β-catenin inhibitor MSAB reduces active β-catenin, downregulates the expression of associated genes and alters CCL2 secretion. Taken together, we showed that β-catenin plays an important role in attracting monocytes towards GBM cells in vitro. We hypothesize that the interactions between β-catenin and CCL2 contribute to maintenance of GSCs via modulating immune cell interaction and promoting GBM growth and recurrence.     

The Interaction between microRNAs and the Wnt/β-Catenin Signaling Pathway in Osteoarthritis

Osteoarthritis (OA) is a chronic disease affecting the whole joint, which still lacks a disease-modifying treatment. This suggests an incomplete understanding of underlying molecular mechanisms. The Wnt/β-catenin pathway is involved in different pathophysiological processes of OA. Interestingly, both excessive stimulation and suppression of this pathway can contribute to the pathogenesis of OA. microRNAs have been shown to regulate different cellular processes in different diseases, including the metabolic activity of chondrocytes and osteocytes. To bridge these findings, here we attempt to give a conclusive overview of microRNA regulation of the Wnt/β-catenin pathway in bone and cartilage, which may provide insights to advance the development of miRNA-based therapeutics for OA treatment.     

Hyperforin and Myrtucommulone Derivatives Act as Natural Modulators of Wnt/β-Catenin Signaling in HCT116 Colon Cancer Cells

The therapeutic activities of natural plant extracts have been well known for centuries. Many of them, in addition to antiviral and antibiotic effects, turned out to have anti-tumor activities by targeting different signaling pathways. The canonical Wnt pathway represents a major tumorigenic pathway deregulated in numerous tumor entities, including colon cancer. Here, we investigated the acylphloroglucinols hyperforin (HF) from St. John’s wort (Hypericum perforatum L.) and myrtucommulone A (MC A) from myrtle (Myrtus communis) and semi-synthetic derivatives thereof (HM 177, HM 297, HM298) for their effects on Wnt/β-catenin signaling. None of these substances revealed major cytotoxicity on STF293 embryonic kidney and HCT116 colon carcinoma cells at concentrations up to 10 μM. At this concentration, HF and HM 177 showed the strongest effect on cell proliferation, whereas MC A and HM 177 most prominently inhibited anchorage-independent growth of HCT116 cells. Western blot analyses of active β-catenin and β-catenin/TCF reporter gene assays in STF293 cells revealed inhibitory activities of HF, MC A and HM 177. In line with this, the expression of endogenous Wnt target genes, Axin and Sp5, in HCT116 cells was significantly reduced. Our data suggest that the acylphloroglucinols hyperforin, myrtucommulone A and its derivative HM 177 represent potential new therapeutic agents to inhibit Wnt/β-catenin signaling in colon cancer.     

Inhibition of β-Catenin Activity Abolishes LKB1 Loss-Driven Pancreatic Cystadenoma in Mice

Pancreatic cancer (PC) is the seventh leading cause of cancer death worldwide, and remains one of our most recalcitrant and dismal diseases. In contrast to many other malignancies, there has not been a significant improvement in patient survival over the past decade. Despite advances in our understanding of the genetic alterations associated with this disease, an incomplete understanding of the underlying biology and lack of suitable animal models have hampered efforts to develop more effective therapies. LKB1 is a tumor suppressor that functions as a primary upstream kinase of adenine monophosphate-activated protein kinase (AMPK), which is an important mediator in the regulation of cell growth and epithelial polarity pathways. LKB1 is mutated in a significant number of Peutz–Jeghers syndrome (PJS) patients and in a small proportion of sporadic cancers, including PC; however, little is known about how LKB1 loss contributes to PC development. Here, we report that a reduction in Wnt/β-catenin activity is associated with LKB1 tumor-suppressive properties in PC. Remarkably, in vivo functional analyses of β-catenin in the Pdx-1-Cre LKB1^L/L β-catenin^L/L mouse model compared to LKB1 loss-driven cystadenoma demonstrate that the loss of β-catenin impairs cystadenoma development in the pancreas of Pdx-1Cre LKB1^L/L mice and dramatically restores the normal development and functions of the pancreas. This study further determined the in vivo and in vitro therapeutic efficacy of the β-catenin inhibitor FH535 in suppressing LKB1 loss-driven cystadenoma and reducing PC progression that delineates the potential roles of Wnt/β-catenin signaling in PC harboring LKB1 deficiency.     

Targeting of Deregulated Wnt/β-Catenin Signaling by PRI-724 and LGK974 Inhibitors in Germ Cell Tumor Cell Lines

The majority of patients with testicular germ cell tumors (GCTs) can be cured with cisplatin-based chemotherapy. However, for a subset of patients present with cisplatin-refractory disease, which confers a poor prognosis, the treatment options are limited. Novel therapies are therefore urgently needed to improve outcomes in this challenging patient population. It has previously been shown that Wnt/β-catenin signaling is active in GCTs suggesting that its inhibitors LGK974 and PRI-724 may show promise in the management of cisplatin-refractory GCTs. We herein investigated whether LGK-974 and PRI-724 provide a treatment effect in cisplatin-resistant GCT cell lines. Taking a genoproteomic approach and utilizing xenograft models we found the increased level of β-catenin in 2 of 4 cisplatin-resistant (CisR) cell lines (TCam-2 CisR and NCCIT CisR) and the decreased level of β-catenin and cyclin D1 in cisplatin-resistant NTERA-2 CisR cell line. While the effect of treatment with LGK974 was limited or none, the NTERA-2 CisR exhibited the increased sensitivity to PRI-724 in comparison with parental cell line. Furthermore, the pro-apoptotic effect of PRI-724 was documented in all cell lines. Our data strongly suggests that a Wnt/β-catenin signaling is altered in cisplatin-resistant GCT cell lines and the inhibition with PRI-724 is effective in NTERA-2 CisR cells. Further evaluation of Wnt/β-catenin pathway inhibition in GCTs is therefore warranted.     

Dickkopf-1 Inhibition Reactivates Wnt/β-Catenin Signaling in Rhabdomyosarcoma,Induces Myogenic Markers In Vitro and Impairs Tumor Cell Survival In Vivo

The Wnt/β-catenin signaling pathway plays a pivotal role during embryogenesis and its deregulation is a key mechanism in the origin and progression of several tumors. Wnt antagonists have been described as key modulators of Wnt/β-catenin signaling in cancer, with Dickkopf-1 (DKK-1) being the most studied member of the DKK family. Although the therapeutic potential of DKK-1 inhibition has been evaluated in several diseases and malignancies, little is known in pediatric tumors. Only a few works have studied the genetic inhibition and function of DKK-1 in rhabdomyosarcoma. Here, for the first time, we report the analysis of the therapeutic potential of DKK-1 pharmaceutical inhibition in rhabdomyosarcoma, the most common soft tissue sarcoma in children. We performed DKK-1 inhibition via shRNA technology and via the chemical inhibitor WAY-2626211. Its inhibition led to β-catenin activation and the modulation of focal adhesion kinase (FAK), with positive effects on in vitro expression of myogenic markers and a reduction in proliferation and invasion. In addition, WAY-262611 was able to impair survival of tumor cells in vivo. Therefore, DKK-1 could constitute a molecular target, which could lead to novel therapeutic strategies in RMS, especially in those patients with high DKK-1 expression.     

Co-Incubation with PPARβ/δ Agonists and Antagonists Modeled Using Computational Chemistry: Effect on LPS Induced Inflammatory Markers in Pulmonary Artery

Peroxisome proliferator activated receptor beta/delta (PPARβ/δ) is a nuclear receptor ubiquitously expressed in cells, whose signaling controls inflammation. There are large discrepancies in understanding the complex role of PPARβ/δ in disease, having both anti- and pro-effects on inflammation. After ligand activation, PPARβ/δ regulates genes by two different mechanisms; induction and transrepression, the effects of which are difficult to differentiate directly. We studied the PPARβ/δ-regulation of lipopolysaccharide (LPS) induced inflammation (indicated by release of nitrite and IL-6) of rat pulmonary artery, using different combinations of agonists (GW0742 or L−165402) and antagonists (GSK3787 or GSK0660). LPS induced release of NO and IL-6 is not significantly reduced by incubation with PPARβ/δ ligands (either agonist or antagonist), however, co-incubation with an agonist and antagonist significantly reduces LPS-induced nitrite production and Nos2 mRNA expression. In contrast, incubation with LPS and PPARβ/δ agonists leads to a significant increase in Pdk−4 and Angptl−4 mRNA expression, which is significantly decreased in the presence of PPARβ/δ antagonists. Docking using computational chemistry methods indicates that PPARβ/δ agonists form polar bonds with His287, His413 and Tyr437, while antagonists are more promiscuous about which amino acids they bind to, although they are very prone to bind Thr252 and Asn307. Dual binding in the PPARβ/δ binding pocket indicates the ligands retain similar binding energies, which suggests that co-incubation with both agonist and antagonist does not prevent the specific binding of each other to the large PPARβ/δ binding pocket. To our knowledge, this is the first time that the possibility of binding two ligands simultaneously into the PPARβ/δ binding pocket has been explored. Agonist binding followed by antagonist simultaneously switches the PPARβ/δ mode of action from induction to transrepression, which is linked with an increase in Nos2 mRNA expression and nitrite production.     

WNT/β-Catenin Signaling Promotes TGF-β-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-β and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-β-activated human cardiac fibroblasts. We found that WNT3a and TGF-β induced a β-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-β triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the β-catenin pathway with the GSK3β inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-β, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-β-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-β and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-β-induced production of collagen I. In conclusion, WNT/β-catenin signaling promotes TGF-β-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.     

Cardioprotective Effects of PPARβ/δ Activation against Ischemia/Reperfusion Injury in Rat Heart Are Associated with ALDH2 Upregulation,Amelioration of Oxidative Stress and Preservation of Mitochondrial Energy Production

Accumulating evidence support the cardioprotective properties of the nuclear receptor peroxisome proliferator activated receptor β/δ (PPARβ/δ); however, the underlying mechanisms are not yet fully elucidated. The aim of the study was to further investigate the mechanisms underlying PPARβ/δ-mediated cardioprotection in the setting of myocardial ischemia/reperfusion (I/R). For this purpose, rats were treated with PPARβ/δ agonist GW0742 and/or antagonist GSK0660 in vivo and hearts were subjected to ex vivo global ischemia followed by reperfusion. PPARβ/δ activation improved left ventricular developed pressure recovery, reduced infarct size (IS) and incidence of reperfusion-induced ventricular arrhythmias while it also up-regulated superoxide dismutase 2, catalase and uncoupling protein 3 resulting in attenuation of oxidative stress as evidenced by the reduction in 4-hydroxy-2-nonenal protein adducts and protein carbonyl formation. PPARβ/δ activation also increased both mRNA expression and enzymatic activity of aldehyde dehydrogenase 2 (ALDH2); inhibition of ALDH2 abrogated the IS limiting effect of PPARβ/δ activation. Furthermore, upregulation of PGC-1α and isocitrate dehydrogenase 2 mRNA expression, increased citrate synthase activity as well as mitochondrial ATP content indicated improvement in mitochondrial content and energy production. These data provide new mechanistic insight into the cardioprotective properties of PPARβ/δ in I/R pointing to ALDH2 as a direct downstream target and suggesting that PPARβ/δ activation alleviates myocardial I/R injury through coordinated stimulation of the antioxidant defense of the heart and preservation of mitochondrial function.     

Leptin,Acting at Central Level,Increases FGF21 Expression in White Adipose Tissue via PPARβ/δ

The altered function of adipose tissue can result in obesity, insulin resistance, and its metabolic complications. Leptin, acting on the central nervous system, modifies the composition and function of adipose tissue. To date, the molecular changes that occur in epididymal white adipose tissue (eWAT) during chronic leptin treatment are not fully understood. Herein we aimed to address whether PPARβ/δ could mediate the metabolic actions induced by leptin in eWAT. To this end, male 3-month-old Wistar rats, infused intracerebroventricularly (icv) with leptin (0.2 μg/day) for 7 days, were daily co-treated intraperitoneally (ip) without or with the specific PPARβ/δ receptor antagonist GSK0660 (1 mg/kg/day). In parallel, we also administered GSK0660 to control rats fed ad libitum without leptin infusion. Leptin, acting at central level, prevented the starvation-induced increase in circulating levels of FGF21, while induced markedly the endogenous expression of FGF21 and browning markers of eWAT. Interestingly, GSK0660 abolished the anorectic effects induced by icv leptin leading to increased visceral fat mass and reduced browning capacity. In addition, the pharmacological inhibition of PPARβ/δ alters the immunomodulatory actions of central leptin on eWAT. In summary, our results demonstrate that PPARβ/δ is involved in the up-regulation of FGF21 expression induced by leptin in visceral adipose tissue.     

Tankyrase Regulates Neurite Outgrowth through Poly(ADP-ribosyl)ation-Dependent Activation of β-Catenin Signaling

Poly(ADP-ribosyl)ation is a post-translational modification of proteins by transferring poly(ADP-ribose) (PAR) to acceptor proteins by the action of poly(ADP-ribose) polymerase (PARP). Two tankyrase (TNKS) isoforms, TNK1 and TNK2 (TNKS1/2), are ubiquitously expressed in mammalian cells and participate in diverse cellular functions, including wnt/β-catenin signaling, telomere maintenance, glucose metabolism and mitosis regulation. For wnt/β-catenin signaling, TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin, a key component of the β-catenin degradation complex, which allows Axin’s ubiquitination and subsequent degradation, thereby activating β-catenin signaling. In the present study, we focused on the functions of TNKS1/2 in neuronal development. In primary hippocampal neurons, TNKS1/2 were detected in the soma and neurites, where they co-localized with PAR signals. Treatment with XAV939, a selective TNKS1/2 inhibitor, suppressed neurite outgrowth and synapse formation. In addition, XAV939 also suppressed norepinephrine uptake in PC12 cells, a rat pheochromocytoma cell line. These effects likely resulted from the inhibition of β-catenin signaling through the stabilization of Axin, which suggests TNKS1/2 enhance Axin degradation by modifying its poly(ADP-ribosyl)ation, thereby stabilizing wnt/β-catenin signaling and, in turn, promoting neurite outgrowth and synapse formation.