Wnt3a but not CDX-2 expression is associated with differentiated thyroid cancer.

OBJECTIVE: Thyroid neoplasm incidence has increased worldwide, mostly due to the advancements in medical imaging and screening rates. The aberrant Wnt/ß-catenin pathway has been identified as a key mechanism, and it has also been related to the metastatic activity of differentiated thyroid cancer. We aimed to verify the difference in the expression of Wnt3a, a canonical activator of the ß-catenin signaling, and CDX-2, a transcription factor upregulated by Wnt/ß-catenin pathway, in multinodular goiter and differentiated thyroid cancer and to determine their prognostic value. METHODS: We included 194 thyroid tissue surgical specimen and their clinicopathological data: study group (differentiated thyroid cancer, n=154) and control group (multinodular goiter, n=40). Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tissue by the primary antibodies Wnt3a and CDX-2. RESULTS: High Wnt3a expression was significantly associated with differentiated thyroid cancer (p=0.031). CDX-2 was negative in all differentiated thyroid cancer cases (100%) and also in multinodular goiter. Wnt3a expression was significantly associated with tumors ≤20 mm (p=0.044) and with the absence of capsule invasion (p=0.031). The multivariate analyses suggested that older age (≥55), independent of capsular invasion and tumor size, was an independent prognostic factor for Wnt3a expression (p=0.058). CONCLUSIONS: Wnt3a expression but not CDX-2 is correlated with differentiated thyroid cancer samples in comparison to multinodular goiter. Although its prognostic value was limited to tumor size and capsule invasion, a combined model in a panel of immune markers can add accuracy in the classification of challenging thyroid follicular-derived lesions.

The Glypican proteoglycans show intrinsic interactions with Wnt-3a in human prostate cancer cells that are not always associated with cascade activation.

BACKGROUND: Prostate cancer occurs through multiple steps until advanced metastasis. Signaling pathways studies can result in the identification of targets to interrupt cancer progression. Glypicans are cell surface proteoglycans linked to the membrane through glycosylphosphatidylinositol. Their interaction with specific ligands has been reported to trigger diverse signaling, including Wnt. In this study, prostate cancer cell lines PC-3, DU-145, and LNCaP were compared to normal prostate RWPE-1 cell line to investigate glypican family members and the activation of the Wnt signaling pathway. RESULTS: Glypican-1 (GPC1) was highly expressed in all the examined cell lines, except for LNCaP, which expressed glypican-5 (GPC5). The subcellular localization of GPC1 was detected on the cell surface of RWPE-1, PC-3, and DU-145 cell lines, while GPC5 suggested cytoplasm localization in LNCaP cells. Besides glypican, flow cytometry analysis in these prostate cell lines confirmed the expression of Wnt-3a and unphosphorylated ß-catenin. The co-immunoprecipitation assay revealed increased levels of binding between Wnt-3a and glypicans in cancer cells, suggesting a relationship between these proteoglycans in this pathway. A marked increase in nuclear ß-catenin was observed in tumor cells. However, only PC-3 cells demonstrated activation of canonical Wnt signaling, according to the TOPFLASH assay. CONCLUSIONS: GPC1 was the majorly expressed gene in all the studied cell lines, except for LNCaP, which expressed GPC5. We assessed by co-immunoprecipitation that these GPCs could interact with Wnt-3a. However, even though nuclear ß-catenin was found increased in the prostate cancer cells (i.e., PC-3, DU-145 and LNCaP), activation of Wnt pathway was only found in PC-3 cells. In these PC-3 cells, GPC1 and Wnt-3a revealed high levels of colocalization, as assessed by confocal microscopy studies. This suggests a localization at the cellular surface, where Frizzled receptor is required for downstream activation. The interaction of Wnt-3a with GPCs in DU-145 and LNCaP cells, which occurs in absence of Wnt signaling activation, requires further studies. Once non-TCF-LEF proteins can also bind ß-catenin, another signaling pathway may be involved in these cells with regulatory function.

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.

LncRNA-599554 sponges miR-15a-5p to contribute inductive ability of dermal papilla cells through positive regulation of the expression of Wnt3a in cashmere goat

BACKGROUND: Long non-coding RNAs (lncRNAs), as post-transcriptional regulators, were thought to function in the inductive property of dermal papilla cells (DPCs) in cashmere goat. Previously, lncRNA-599554 was identified in secondary hair follicle (SHF) of cashmere goat, but its functional significance is unknown. RESULTS: In the present investigation, we verified that lncRNA-599554 had significantly higher expression at the anagen dermal papilla of cashmere goat SHF than that at telogen. Based on overexpression and knockdown techniques, we found that lncRNA-599554 contributes the inductive property of DPCs of cashmere goat, which was assessed by detecting the changes in the expression of several typical indictor genes in DPCs including ET-1, SCF, Versican, ALP, Lef1 and Ptc-1. Based on RNA pull-down assay, we verified that lncRNA-599554 directly interacted with chi-miR-15a-5p. Also, we showed that lncRNA-599554 positively regulated the Wnt3a expression in DPCs but which did not appear to involve its modulating of promoter methylation. Based on the use of Dual-luciferase reporter assays, our data indicated that lncRNA-599554 regulated the Wnt3a expression through chi-miR-15a-5p-mediated post-transcriptional level. CONCLUSIONS: We showed that lncRNA-599554 contributes the inductive property of DPCs in cashmere goat which might be achieved through sponging chi-miR-15b-5p to promote the Wnt3a expression. The results from the present investigation provided a novel insight into the functional mechanism of lncRNA-599554 in the SHF regeneration of cashmere goat along with the formation and growth of cashmere fiber.

GBM-Derived Wnt3a Induces M2-Like Phenotype in Microglial Cells Through Wnt/ß-Catenin Signaling.

Glioblastoma is an extremely aggressive and deadly brain tumor known for its striking cellular heterogeneity and capability to communicate with microenvironment components, such as microglia. Microglia-glioblastoma interaction contributes to an increase in tumor invasiveness, and Wnt signaling pathway is one of the main cascades related to tumor progression through changes in cell migration and invasion. However, very little is known about the role of canonical Wnt signaling during microglia-glioblastoma crosstalk. Here, we show for the first time that Wnt3a is one of the factors that regulate interactions between microglia and glioblastoma cells. Wnt3a activates the Wnt/ß-catenin signaling of both glioblastoma and microglial cells. Glioblastoma-conditioned medium not only induces nuclear translocation of microglial ß-catenin but also increases microglia viability and proliferation as well as Wnt3a, cyclin-D1, and c-myc expression. Moreover, glioblastoma-derived Wnt3a increases microglial ARG-1 and STI1 expression, followed by an upregulation of IL-10 mRNA levels, and a decrease in IL1ß gene expression. The presence of Wnt3a in microglia-glioblastoma co-cultures increases the formation of membrane nanotubes accompanied by changes in migration capability. In vivo, tumors formed from Wnt3a-stimulated glioblastoma cells presented greater microglial infiltration and more aggressive characteristics such as growth rate than untreated tumors. Thus, we propose that Wnt3a belongs to the arsenal of factors capable of stimulating the induction of M2-like phenotype on microglial cells, which contributes to the poor prognostic of glioblastoma, reinforcing that Wnt/ß-catenin pathway can be a potential therapeutic target to attenuate glioblastoma progression.

Wnt3a ligand facilitates autophagy in hippocampal neurons by modulating a novel GSK-3ß-AMPK axis.

BACKGROUND: In the adult central nervous system (CNS), Wnt signaling regulates dendritic structure and synaptic plasticity. The Wnt signaling pathway can be divided into the canonical (ß-catenin-dependent) and non-canonical pathways. In the canonical pathway, the binding of canonical ligands such as Wnt3a to the Frizzled receptor induces inactivation of glycogen synthase kinase-3ß (GSK-3ß), which stabilizes ß-catenin and allows its translocation to the nucleus. However, to date, few studies have focused on ß-catenin-independent Wnt signaling or explained the underlying mechanisms connecting Wnt signaling to cellular energy metabolism. A recent study demonstrated negative regulation of 5' adenosine monophosphate-activated protein kinase (AMPK), a major target of GSK-3ß that regulates cellular metabolism under diverse conditions. Mainly based on these observations, we evaluated whether Wnt3a ligand modulates autophagy by regulating the GSK-3ß/AMPK axis. METHODS: Cultured primary hippocampal neurons and slices of the CA1 region of rat hippocampus were used. GSK-3ß inhibition, AMPK activation, PP2Ac expression, and LC3 processing were examined by western blotting. Autophagic compartments were studied using the CYTO-ID® fluorescent probe, and mature autophagosomes were observed via transmission electron microscopy (TEM). RESULTS: Wnt3a ligand, acting through the Frizzled receptor, promotes the rapid activation of AMPK by inactivating GSK-3ß. Biochemical analysis of downstream targets indicated that Wnt3a ligand modulates autophagy in hippocampal neurons. CONCLUSIONS: Our results revealed new aspects of Wnt signaling in neuronal metabolism. First, AMPK is an additional target downstream of the Wnt cascade, suggesting a molecular mechanism for the metabolic effects previously observed for Wnt signaling. Second, this mechanism is independent of ß-catenin, suggesting a relevant role for non-genomic activity of the Wnt pathway in cellular metabolism. Finally, these results have new implications regarding the role of Wnt signaling in the modulation of autophagy in neurons, with a possible role in the removal of accumulated intracellular proteins.

Dishevelled stability is positively regulated by PKCζ-mediated phosphorylation induced by Wnt agonists.

Dishevelled (Dvl) proteins are central mediators of both canonical and non-canonical Wnt signaling. It is well known that, upon Wnt stimulation, Dvl becomes phosphorylated. However, how Wnt-induced phosphorylation of Dvl is regulated and its consequences are poorly understood. Here we found that Dvl proteins are overexpressed in colon cancer cells. In addition, we found that Wnt3a treatment rapidly induces hyperphosphorylation and stabilization of Dvl2 and Dvl3. The latter can be blocked by inhibition of Protein Kinase C (PKC)α, PKCδ, and PKCζ isoforms. We also found that Wnt3a-induced phosphorylation of Dvl3 by PKCζ is required to avoid Dvl3 degradation via proteasome. This demonstrated, to our knowledge for the first time, that hyperphosphorylation of Dvl by PKCζ results in Dvl stabilization. This is clear contrast with the consequences reported to date of CK1δ/ε-mediated Dvl phosphorylation upon Wnt treatment. Mapping the interaction domain between PKCζ and Dvl3 indicated that, although the Dvl-DIX domain is required to stabilize PKCζ-phosphorylated Dvl, it is not the region phosphorylated by this kinase. Our data show that the Dvl-DEP domain, required for specific interaction with PKCζ, is the site phosphorylated by this kinase, and also probably the Dvl-C terminus. Our findings suggest a model of positive regulation of PKCζ-mediated Dvl signaling activity, to produce a strong and sustained response to Wnt3a treatment by stabilizing Dvl protein levels.

Wnt Signaling Prevents the Aß Oligomer-Induced Mitochondrial Permeability Transition Pore Opening Preserving Mitochondrial Structure in Hippocampal Neurons.

Alzheimer's disease (AD) is a neurodegenerative disorder mainly known for synaptic impairment and neuronal cell loss, affecting memory processes. Beside these damages, mitochondria have been implicated in the pathogenesis of AD through the induction of the mitochondrial permeability transition pore (mPTP). The mPTP is a non-selective pore that is formed under apoptotic conditions, disturbing mitochondrial structure and thus, neuronal viability. In AD, Aß oligomers (Aßos) favor the opening of the pore, activating mitochondria-dependent neuronal cell death cascades. The Wnt signaling activated through the ligand Wnt3a has been described as a neuroprotective signaling pathway against amyloid-ß (Aß) peptide toxicity in AD. However, the mechanisms by which Wnt signaling prevents Aßos-induced neuronal cell death are unclear. We proposed here to study whether Wnt signaling protects neurons earlier than the late damages in the progression of the disease, through the preservation of the mitochondrial structure by the mPTP inhibition. To study specific events related to mitochondrial permeabilization we performed live-cell imaging from primary rat hippocampal neurons, and electron microscopy to analyze the mitochondrial morphology and structure. We report here that Wnt3a prevents an Aßos-induced cascade of mitochondrial events that leads to neuronal cell death. This cascade involves (a) mPTP opening, (b) mitochondrial swelling, (c) mitochondrial membrane potential loss and (d) cytochrome c release, thus leading to neuronal cell death. Furthermore, our results suggest that the activation of the Wnt signaling prevents mPTP opening by two possible mechanisms, which involve the inhibition of mitochondrial GSK-3ß and/or the modulation of mitochondrial hexokinase II levels and activity. This study suggests a possible new approach for the treatment of AD from a mitochondrial perspective, and will also open new lines of study in the field of Wnt signaling in neuroprotection.

Activation of Wnt Signaling in Cortical Neurons Enhances Glucose Utilization through Glycolysis.

The Wnt signaling pathway is critical for a number of functions in the central nervous system, including regulation of the synaptic cleft structure and neuroprotection against injury. Deregulation of Wnt signaling has been associated with several brain pathologies, including Alzheimer's disease. In recent years, it has been suggested that the Wnt pathway might act as a central integrator of metabolic signals from peripheral organs to the brain, which would represent a new role for Wnt signaling in cell metabolism. Energy metabolism is critical for normal neuronal function, which mainly depends on glucose utilization. Brain energy metabolism is important in almost all neurological disorders, to which a decrease in the capacity of the brain to utilize glucose has been linked. However, little is known about the relationship between Wnt signaling and neuronal glucose metabolism in the cellular context. In the present study, we found that acute treatment with the Wnt3a ligand induced a large increase in glucose uptake, without changes in the expression or localization of glucose transporter type 3. In addition, we observed that Wnt3a treatment increased the activation of the metabolic sensor Akt. Moreover, we observed an increase in the activity of hexokinase and in the glycolytic rate, and both processes were dependent on activation of the Akt pathway. Furthermore, we did not observe changes in the activity of glucose-6-phosphate dehydrogenase or in the pentose phosphate pathway. The effect of Wnt3a was independent of both the transcription of Wnt target genes and synaptic effects of Wnt3a. Together, our results suggest that Wnt signaling stimulates glucose utilization in cortical neurons through glycolysis to satisfy the high energy demand of these cells.

Sustained behavioral effects of lithium exposure during early development in zebrafish: involvement of the Wnt-ß-catenin signaling pathway.

Lithium has been the paradigmatic treatment for bipolar disorder since 1950s, offering prophylactic and acute efficacy against maniac and depressive episodes. Its use during early pregnancy and the perinatal period remains controversial due to reports of negative consequences on the newborn including teratogenic and neurobehavioral effects generally referred as Floppy baby syndrome. The mechanisms underlying lithium therapeutic action are still elusive but exacerbation of Wnt signaling pathway due to GSK-3 inhibition is believed to represent its main effect. In this study we evaluated the impact of lithium exposure during zebrafish embryonic and early development including behavioral and molecular characterization of Wnt-ß-catenin pathway components. Wild-type zebrafish embryos were individually treated for 72 hpf with LiCl at 0.05, 0.5 and 5mM. No significant teratogenic and embryotoxic effects were observed. At the end of treatment period western blot analysis of selected Wnt-ß-catenin system components showed increased ß-catenin and decreased N-cadherin protein levels, without significant changes in Wnt3a, supporting GSK-3 inhibition as lithium's main target. At 10 dpf 0.5 and 5mM lithium-treated larvae showed a dose-dependent decrease in locomotion among other exploratory parameters, resembling lithium-induced Floppy baby syndrome neurobehavioral symptoms in humans. At this later period previously altered proteins returned to control levels in treated groups, suggesting that the neurobehavioral effects are a lasting consequence of lithium exposure during early development. RT-qPCR analysis of ß-catenin and N-cadherin gene expression showed no effects of lithium at 3 or 10 dpf, suggesting that protein fluctuations were likely due to post-transcriptional events. Other Wnt target genes were evaluated and only discrete alterations were observed. These results suggest that zebrafish may be a valuable model for investigation of early effects of lithium that may be mediated by effects on the Wnt signaling pathway.

WNT3A gene polymorphisms are associated with bone mineral density variation in postmenopausal mestizo women of an urban Mexican population: findings of a pathway-based high-density single nucleotide screening.

Osteoporosis (OP) is a common skeletal disorder characterized by low bone mineral density (BMD) and is a common health problem in Mexico. To date, few genes affecting BMD variation in the Mexican population have been identified. The aim of this study was to investigate the association of single nucleotide polymorphisms (SNPs) located in genes of the Wnt pathway with BMD variation at various skeletal sites in a cohort of postmenopausal Mexican women. A total of 121 SNPs in or near 15 Wnt signaling pathway genes and 96 ancestry informative markers were genotyped in 425 postmenopausal women using the Illumina GoldenGate microarray SNP genotyping method. BMD was measured by dual-energy X-ray absorptiometry in total hip, femoral neck, Ward's triangle, and lumbar spine. Associations were tested by linear regression for quantitative traits adjusting for possible confounding factors. SNP rs752107 in WNT3A was strongly associated with decreased total hip BMD showing the highest significance under the recessive model (P = 0.00012). This SNP is predicted to disrupt a binding site for microRNA-149. In addition, a polymorphism of the Wnt antagonist DKK2 was associated with BMD in femoral neck under a recessive model (P = 0.009). Several LRP4, LRP5, and LRP6 gene variants showed site-specific associations with BMD. In conclusion, this is the first report associating Wnt pathway gene variants with BMD in the Mexican population.

Effect of the Jianpi Bushen Prescription on expressions of the Wnt3a and Cyclin D1 genes in radiation-damaged mice.

The effects of the traditional Chinese drug Jianpi Bushen Prescription (JBP) were investigated on expressions of Wnt3a and Cyclin D1 genes in radiation-damaged mice. The radiation damage model was induced in Kumming mice by single total body irradiation treatment for 9 days. Mice were divided into the radiation group, low-dose (100%) JBP group, high-dose (200%) JBP group, or batyl alcohol group (positive control), which were administered twice a day for 9 days. mRNA and protein expressions of Wnt3a were detected in bone marrow mononuclear cells by real-time polymerase chain reaction and Western blot, whereas Cyclin D1 mRNA was detected by in situ hybridization. Wnt3a expressions were significantly downregulated in the radiation damage model group compared with all other groups (P < 0.05). The positive cell rate of Cyclin D1 mRNA expression and the number of granulocyte macrophage colonies were significantly decreased in the radiation damage model group relative to all other groups (P < 0.05). Furthermore, mRNA and protein expressions of Wnt3a, the positive cell rate of Cyclin D1 mRNA expression in bone marrow cells, and the number of granulocyte macrophage colonies were all significantly higher in the low-dose JBP group than in the high-dose JBP group (P < 0.05). In summary, JBP plays a protective role on radiation-induced bone marrow through the activation of the Wnt3a signaling pathway, and promotes the transcription and expression of Cyclin D1.

Protein kinase C delta negatively modulates canonical Wnt pathway and cell proliferation in colon tumor cell lines.

The tumor suppressor Adenomatous Polyposis coli (APC) gene is mutated or lost in most colon cancers. Alterations in Protein kinase C (PKC) isozyme expression and aberrant regulation also comprise early events in intestinal carcinomas. Here we show that PKCδ expression levels are decreased in colon tumor cell lines with respect to non-malignant cells. Reciprocal co-immunoprecipitation and immunofluorescence studies revealed that PKCδ interacts specifically with both full-length (from non-malignant cells) and truncated APC protein (from cancerous cells) at the cytoplasm and at the cell nucleus. Selective inhibition of PKCδ in cancer SW480 cells, which do not possess a functional ß-catenin destruction complex, did not affect ß-catenin-mediated transcriptional activity. However, in human colon carcinoma RKO cells, which have a normal ß-catenin destruction complex, negatively affected ß-catenin-mediated transcriptional activity, cell proliferation, and the expression of Wnt target genes C-MYC and CYCLIN D1. These negative effects were confirmed by siRNA-mediated knockdown of PKCδ and by the expression of a dominant negative form of PKCδ in RKO cells. Remarkably, the PKCδ stably depleted cells exhibited augmented tumorigenic activity in grafted mice. We show that PKCδ functions in a mechanism that involves regulation of ß-catenin degradation, because PKCδ inhibition induces ß-catenin stabilization at the cytoplasm and its nuclear presence at the C-MYC enhancer even without Wnt3a stimulation. In addition, expression of a dominant form of PKCδ diminished APC phosphorylation in intact cells, suggesting that PKCδ may modulate canonical Wnt activation negatively through APC phosphorylation.