Wnt/β‐Catenin Signaling Pathway Upregulates c‐Myc Expression to Promote Cell Proliferation of P19 Teratocarcinoma Cells

Aberrant activation of the Wnt/β‐catenin signaling pathway is a common event in human tumor progression. Wnt signaling has also been implicated in maintaining a variety of adult and embryonic stem cells by imposing a restraint to differentiation. To understand the function and mechanism of Wnt/β‐catenin signaling on the pathogenesis of teratocarcinoma, we used the mouse teratocarcinoma P19 cell line as a model in vitro. Gsk3β specific inhibitor (SB216763) was used to activate Wnt/β‐catenin signaling. All trans‐retinoic acid (RA) was used to induce P19 cell differentiation. At different culture times, gene expression was examined by immunofluorescence staining, quantitative real‐time PCR, and Western‐blotting; BrdU incorporation assays were performed to measure P19 cell proliferation. Small interference RNA technology was used to downregulate c‐myc expression. The results showed that SB216763 induced the nuclear translocation of β‐catenin, upregulated the expression of c‐myc and pluripotency related genes, oct4, sox2 and nanog, and blocked cell differentiation induced by all trans‐RA. The proliferation of P19 cells was significantly enhanced by SB216763, as well as c‐myc overexpression. C‐myc downregulation inhibited P19 cell proliferation caused by activation of Wnt/β‐catenin signaling and induced P19 cell differentiation. In conclusion, activation of the Wnt/β‐catenin pathway could promote the proliferation and inhibit the differentiation of mouse teratocarcinoma cells by upregulation of c‐myc expression. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

SOX1 inhibits breast cancer cell growth and invasion through suppressing the Wnt/β‐catenin signaling pathway

Abnormal activation of the Wnt/β‐catenin signaling pathway is common in human cancers. Several studies have demonstrated that SRY (sex‐determining region Y)‐box (SOX) family genes serve as either tumor suppressor genes or oncogenes by regulating the Wnt signaling pathway in different cancers. However, the role of SOX1 in breast cancer and the underlying mechanism is still unclear. The aim of this study was to explore the effect and mechanism of SOX1 on the breasted cancer cell growth and invasion. In this study, we established overexpressed SOX1 and investigated its function by in vitro experiments. SOX1 was down‐regulated in breast cancer tissues and cell lines. Overexpression of SOX1 inhibited cell proliferation and invasion in vitro, and it promoted cell apoptosis. Furthermore, SOX1 inhibited the expression of β‐catenin, cyclin D1, and c‐Myc in breast cancer cells. Taken together, these data suggest that SOX1 can function as a tumor suppressor partly by interfering with Wnt/β‐catenin signaling in breast cancer.     

Serine‐arginine protein kinase 1 promotes a cancer stem cell‐like phenotype through activation of Wnt/β‐catenin signalling in NSCLC

Cancer stem cells (CSCs) are commonly associated with cancer recurrence and metastasis that occurs in up to 30–55% of non‐small‐cell lung carcinoma (NSCLC) patients. Herein, we showed that serine‐arginine protein kinase 1 (SRPK1) was highly expressed at both the mRNA and the protein levels in human NCSLC. SRPK1 was associated with the clinical features of human NSCLC, including clinical stage (p < 0.001) and T (p = 0.001), N (p = 0.007), and M (p = 0.001) classifications. Ectopic overexpression of SRPK1 promoted the acquisition of a stem cell‐like phenotype in human NSCLC cell lines cultured in vitro. Overexpression of SRPK1 increased sphere formation and the proportion of side‐population cells that exclude Hoechst dye. Conversely, SRPK1 silencing reduced the number of spheres and the proportion of side‐population cells. Mouse studies indicated that SRPK1 promoted NSCLC cell line tumour growth and SRPK1 overexpression reduced the number of tumour cells required to initiate tumourigenesis in vivo. Mechanistically, gene set enrichment analysis showed that Wnt/β‐catenin signalling correlated with SRPK1 mRNA levels and this signalling pathway was hyperactivated by ectopic SRPK1 expression in NSCLC cell lines. Immunofluorescence demonstrated that SRPK1 enhanced β‐catenin accumulation in the nuclei of NSCLC cell lines, and inhibition of β‐catenin signalling abrogated the SRPK1‐induced stem cell‐like phenotype. Together, our findings suggest that SRPK1 promotes a stem cell‐like phenotype in NSCLC via Wnt/β‐catenin signalling. Moreover, SRPK1 may represent a novel target for human NSCLC diagnosis and therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Activation of canonical Wnt/β‐catenin signaling stimulates proliferation in neuromasts in the zebrafish posterior lateral line

Background: The posterior lateral line in zebrafish develops from a migrating primordium that deposits clusters of cells that differentiate into neuromasts at regular intervals along the trunk. The deposition of these neuromasts is known to be coordinated by Wnt and FGF signals that control the proliferation, migration, and organization of the primordium. However, little is known about the control of proliferation in the neuromasts following their deposition. Results: We show that pharmacological activation of the Wnt/β‐catenin signaling pathway with 1‐azakenpaullone upregulates proliferation in neuromasts post‐deposition. This results in increased size of the neuromasts and overproduction of sensory hair cells. We also show that activation of Wnt signaling returns already quiescent supporting cells to a proliferative state in mature neuromasts. Additionally, activation of Wnt signaling increases the number of supporting cells that return to the cell cycle in response to hair cell damage and the number of regenerated hair cells. Finally, we show that inhibition of Wnt signaling by overexpression of dkk1b suppresses proliferation during both differentiation and regeneration. Conclusions: These data suggest that Wnt/β‐catenin signaling is both necessary and sufficient for the control of proliferation of lateral line progenitors during development, ongoing growth of the neuromasts, and hair cell regeneration. Developmental Dynamics 242:832–846, 2013. © 2013 Wiley Periodicals, Inc.     

Twist1 contributes to cranial bone initiation and dermal condensation by maintaining wnt signaling responsiveness

Background: Specification of cranial bone and dermal fibroblast progenitors in the supraorbital arch mesenchyme is Wnt/β‐catenin signaling‐dependent. The mechanism underlying how these cells interpret instructive signaling cues and differentiate into these two lineages is unclear. Twist1 is a target of the Wnt/β‐catenin signaling pathway and is expressed in cranial bone and dermal lineages. Results: Here, we show that onset of Twist1 expression in the mouse cranial mesenchyme is dependent on ectodermal Wnts and mesenchymal β‐catenin activity. Conditional deletion of Twist1 in the supraorbital arch mesenchyme leads to cranial bone agenesis and hypoplastic dermis, as well as craniofacial malformation of eyes and palate. Twist1 is preferentially required for cranial bone lineage commitment by maintaining Wnt responsiveness. In the conditional absence of Twist1, the cranial dermis fails to condense and expand apically leading to extensive cranial dermal hypoplasia with few and undifferentiated hair follicles. Conclusions: Thus, Twist1, a target of canonical Wnt/β‐catenin signaling, also functions to maintain Wnt responsiveness and is a key effector for cranial bone fate selection and dermal condensation. Developmental Dynamics 245:144–156, 2016. © 2015 Wiley Periodicals, Inc.     

De Novo ARID1B mutations cause growth delay associated with aberrant Wnt/β–catenin signaling

Haploinsufficiency of ARID1B (AT‐rich interaction domain 1B) has been involved in autism spectrum disorder, nonsyndromic and syndromic intellectual disability, and corpus callosum agenesis. Growth impairment is a major clinical feature caused by ARID1B mutations; however, the mechanistic link has not been elucidated. Here, we confirm that growth delay is a common characteristic of patients with ARID1B mutations, which may be associated with dysregulation of the Wnt/β–catenin signaling pathway. An analysis of patients harboring pathogenic variants of ARID1B revealed that nearly half had short stature and nearly all had below‐average height. Moreover, the percentage of patients with short stature increased with age. Knockdown of arid1b in zebrafish embryos markedly reduced body length and perturbed the expression of both chondrogenic and osteogenic genes including sox9a, col2a1a, runx2b, and col10a1. Knockout of Arid1b in chondrogenic ATDC5 cells inhibited chondrocyte proliferation and differentiation. Finally, Wnt/β–catenin signaling was perturbed in Arid1b‐depleted zebrafish embryos and Arid1b knockout ATDC5 cells. These data indicate that ARID1B modulates bone growth possibly via regulation of the Wnt/β–catenin pathway, and may be an appropriate target for gene therapy in disorders of growth and development.     

Heterogeneity in readouts of canonical wnt pathway activity within intestinal crypts

Background: Canonical Wnt pathway signaling is necessary for maintaining the proliferative capacity of mammalian intestinal crypt base columnar stem cells (CBCs). Furthermore, dysregulation of the Wnt pathway is a major contributor to disease, including oncogenic transformation of the intestinal epithelium. Given the critical importance of this pathway, numerous tools have been used as proxy measures for Wnt pathway activity, yet the relationship between Wnt target gene expression and reporter allele activity within individual cells at the crypt base remains unclear. Conclusions: Wnt target genes and reporter alleles can vary greatly in their cell‐type specificity, demonstrating that these proxies cannot be used interchangeably. Furthermore, Axin2‐CreERT2‐tdTomato is a robust marker of both active and reserve intestinal stem cells and is thus useful for understanding the intestinal stem cell compartment. Developmental Dynamics 245:822–833, 2016. © 2016 Wiley Periodicals, Inc.     

Inactive Wnt/β‐catenin pathway in conventional high‐grade osteosarcoma

Osteosarcoma is the most common malignant bone tumour, with a peak incidence in children and young adolescents, suggesting a role of rapid bone growth in its pathogenesis. The Wnt/β‐catenin pathway plays a crucial role in skeletal development and is indispensable for osteoblasts' lineage determination. Previous studies suggesting an oncogenic role for the Wnt/β‐catenin pathway in osteosarcoma were based on cytoplasmic staining of β‐catenin or the detection of one component of this pathway. However, those approaches are inappropriate to address whether the Wnt/β‐catenin pathway is functionally active. Therefore, in this study, we examined nuclear β‐catenin expression in 52 human osteosarcoma biopsies, 15 osteoblastomas (benign bone tumours), and four human osteosarcoma cell lines by immunohistochemistry. Furthermore, we modulated Wnt/β‐catenin pathway activity using a GIN (GSK3β inhibitor) and evaluated its effect on cell growth and osteogenic differentiation. Absence of nuclear β‐catenin staining was found in 90% of the biopsies and all osteosarcoma cell lines, whereas strong nuclear β‐catenin staining was observed in all osteoblastomas. Wnt‐luciferase activity was comparable to the negative control in all osteosarcoma cell lines. GIN stimulated the Wnt/β‐catenin pathway, as shown by translocation of β‐catenin into the nucleus and increased Wnt‐luciferase activity as well as mRNA expression of AXIN2, a specific downstream target gene. Stimulation of the Wnt/β‐catenin pathway by GIN significantly reduced cell proliferation in the cell lines MG‐63 and U‐2‐OS and enhanced differentiation in the cell lines HOS and SJSA‐1, as shown by an increase in alkaline phosphatase (ALP) activity and mineralization. In contrast with the oncogenic role of the Wnt/β‐catenin pathway in osteosarcoma as previous studies suggested, here we demonstrate that this pathway is inactivated in osteosarcoma. Moreover, activation of the Wnt/β‐catenin pathway inhibits cell proliferation or promotes osteogenic differentiation in osteosarcoma cell lines. Our data suggest that loss of Wnt/β‐catenin pathway activity, which is required for osteoblast differentiation, may contribute to osteosarcoma development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Demethylation of Specific Wnt/β‐Catenin Pathway Genes and its Upregulation in Rat Brain Induced by Prenatal Valproate Exposure

Valproate (VPA) has been used for decades in the treatment of epilepsy and migraine. However, maternal administration of VPA during pregnancy increases susceptibility to autism spectrum disorders (ASDs) in the offspring. The aim of this study was to investigate the methylation modification and its effects on the activity of Wnt/β‐catenin pathway in the rat brain prenatally exposed to VPA. We exposed the rats in early pregnancy to VPA and found that the prenatal VPA exposure, in comparison with the prenatal vehicle exposure, induced demethylation in the promoter regions of wnt1 and wnt2, but not in those of Wnt inhibitory factor‐1 and Dickkopf 1, in the prefrontal cortexes and hippocampi of the offspring. Consequently, both mRNA and protein expression of wnt1 and wnt2 were increased. Furthermore, the activity of Wnt/β‐catenin pathway was upregulated, as indicated by the increased levels of β‐catenin, hence the growing expression of its target genes. This work suggested an epigenetic action via which VPA, when administered in early pregnancy, induced dysregulation of signaling pathway, further facilitating susceptibility to ASDs. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Sebox regulates mesoderm formation in early amphibian embryos

Background: Mix/Bix genes are important regulators of mesendoderm formation during vertebrate embryogenesis. Sebox, an additional member of this gene family, has been implicated in endoderm formation during early embryogenesis in zebrafish. However, it remains unclear whether Sebox plays a unique role in early Xenopus embryos. Results: In this study, we provide evidence that Sebox is uniquely required for the formation of mesoderm during early Xenopus embryogenesis. Sebox is dynamically expressed in the involuted mesoderm during gastrulation. It is activated by Nodal/Activin signaling and modulated by zygotic Wnt/β‐catenin signaling. Overexpression of Sebox perturbs movements during convergent extension and inhibits the expression of mesodermal, but not endodermal, genes induced by Nodal/Activin signaling. Depletion of Sebox using a specific morpholino increases the expression of noncanonical wnt5a, wnt5b, and wnt11b. Depletion of Sebox also up‐regulates the expression of pcdh8.2, a paraxial mesoderm‐specific protocadherin, in a Wnt11B‐dependent manner. Sebox morphants display reduced development of the head and notochord. Conclusions: Our findings illustrate that Sebox, a unique member of the Mix/Bix gene family, functions downstream of Nodal/Activin signaling and is required for the proper expression of noncanonical Wnt ligands and the normal development of mesoderm in Xenopus. Developmental Dynamics 244:1415–1426, 2015. © 2015 Wiley Periodicals, Inc.     

Critical role of Frizzled1 in age‐related alterations of Wnt/β‐catenin signal in myogenic cells during differentiation

Activation of Wnt/β‐catenin signal in muscle satellite cells (mSCs) of aged mice during myogenic differentiation has been appreciated as an important age‐related feature of the skeletal muscles, resulting in impairment of their regenerative ability following muscle injury. However, it remains elusive about molecules involved in this age‐related alteration of Wnt/β‐catenin signal in myogenic cells. To clarify this issue, we carried out expression analyses of Wnt receptor genes using real‐time RT‐PCR in mSCs isolated from the skeletal muscles of young and aged mice. Here, we show that expression of Frizzled1 (Fzd1) was detected at high levels in mSCs of aged mice. Higher expression levels of Fzd1 were also detected in mSC‐derived myogenic cells from aged mice and associated with activation of Wnt/β‐catenin signal during their myogenic differentiation in vitro. We also provide evidence that suppressed expression of Fzd1 in myogenic cells from aged mice results in a significant increase in myogenic differentiation, and its forced expression in those from young mice results in its drastic inhibition. These findings indicate the critical role of Fzd1 in altered myogenic differentiation associated with aging.     

Investigation of Frizzled‐5 during embryonic neural development in mouse

Recent studies revealed that the Wnt receptor Frizzled‐5 (Fzd5) is required for eye and retina development in zebrafish and Xenopus, however, its role during mammalian eye development is unknown. In the mouse embryo, Fzd5 is prominently expressed in the pituitary, distal optic vesicle, and optic stalk, then later in the progenitor zone of the developing retina. To elucidate the role of Fzd5 during eye development, we analyzed embryos with a germline disruption of the Fzd5 gene at E10.25, just before embryos die due to defects in yolk sac angiogenesis. We observed severe defects in optic cup morphogenesis and lens development. However, in embryos with conditional inactivation of Fzd5 using Six3‐Cre, we observed no obvious early eye defects. Analysis of Axin2 mRNA expression and TCF/LEF‐responsive reporter activation demonstrate that Fzd5 does not regulate the Wnt/β‐catenin pathway in the eye. Thus, the function of Fzd5 during eye development appears to be species‐dependent. Developmental Dynamics 237:1614–1626, 2008. © 2008 Wiley‐Liss, Inc.     

Beta‐catenin status in paediatric medulloblastomas: correlation of immunohistochemical expression with mutational status,genetic profiles,and clinical characteristics

Medulloblastoma is the most frequent malignant paediatric brain tumour. The activation of the Wnt/β‐catenin pathway occurs in 10‐15% of medulloblastomas and has been recently described as a marker for favourable patient outcome. We report a series of 72 paediatric medulloblastomas evaluated for β‐catenin protein expression, CTNNB1 mutations, and comparative genomic hybridization. Gene expression profiles were also available in a subset of 40 cases. Immunostaining of β‐catenin showed extensive nuclear staining (>50% of the tumour cells) in six cases and focal nuclear staining (<10% of cells) in three cases. The other cases either exhibited a signal strictly limited to the cytoplasm (58 cases) or were negative (five cases). CTNNB1 mutations were detected in all β‐catenin extensively nucleopositive cases. The expression profiles of these cases documented strong activation of the Wnt/β‐catenin pathway. Remarkably, five out of these six tumours showed a complete loss of chromosome 6. In contrast, cases with focal nuclear β‐catenin staining, as well as tumours with negative or cytoplasmic staining, never demonstrated CTNNB1 mutation, Wnt/β‐catenin pathway activation or chromosome 6 loss. Patients with extensive nuclear staining were significantly older at diagnosis and were in continuous complete remission after a mean follow‐up of 75.7 months (range 27.5–121.2 months) from diagnosis. All three patients with focal nuclear staining of β‐catenin died within 36 months from diagnosis. Altogether, these data confirm and extend previous observations that CTNNB1‐mutated tumours represent a distinct molecular subgroup of medulloblastomas with favourable outcome, indicating that therapy de‐escalation should be considered. International consensus on the definition criteria of this distinct medulloblastoma subgroup should be achieved. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.