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.     

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.     

The expression and significance of WWOX and β‐catenin in hepatocellular carcinoma

WW domain‐containing oxidoreductase (WWOX) is a novel tumor suppressor gene, and its expression is reduced in various cancers, including hepatocellular carcinoma (HCC). WWOX has been reported to be downregulated in HCC cell lines as well as in primary HCC tissues. It has been suggested that WWOX is implicated in Wnt/β‐catenin pathway, which is frequently affected in HCC. The aim of this study was to evaluate the expression of WWOX, β‐catenin and T‐cell factor 4 (TCF4) in HCC. Our result showed that downregulation of WWOX in HCC was correlated with cytoplasmic accumulation of β‐catenin. In addition, strong nuclear TCF4 expression was associated with tumor grade and stage in HCC. In conclusion, our result implied that downregulation of WWOX might lead to accumulation of cytoplasmic β‐catenin and the subsequent activation of Wnt/β‐catenin signaling pathway in HCC.     

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.     

Wnt/β‐catenin and sonic hedgehog pathways interact in the regulation of the development of the dorsal mesenchymal protrusion

Background: The dorsal mesenchymal protrusion (DMP) is a second heart field (SHF) derived tissue involved in cardiac septation. Molecular mechanisms controlling SHF/DMP development include the Bone Morphogenetic Protein and Wnt/β‐catenin signaling pathways. Reduced expression of components in these pathways leads to inhibition of proliferation of the SHF/DMP precursor population and failure of the DMP to develop. While the Sonic Hedgehog (Shh) pathway has also been demonstrated to be critically important for SHF/DMP development and atrioventricular septation, its role in the regulation of SHF proliferation is contentious. Results: Tissue‐specific deletion of the Shh receptor Smoothened from the SHF resulted in compromised DMP formation and atrioventricular septal defects (AVSDs). Immunohistochemical analysis at critical stages of DMP development showed significant proliferation defect as well as reduction in levels of the Wnt/β‐catenin pathway‐intermediates β‐catenin, Lef1, and Axin2. To determine whether the defects seen in the conditional Smoothened knock‐out mouse could be attributed to reduced Wnt/β‐catenin signaling, LiCl, a pharmacological activator of this Wnt/β‐catenin pathway, was administered. This resulted in restoration of proliferation and partial rescue of the AVSD phenotype. Conclusions: The data presented suggest that the Wnt/β‐catenin pathway interact with the Shh pathway in the regulation of SHF/DMP‐precursor proliferation and, hence, the development of the DMP. Developmental Dynamics 245:103–113, 2016. © 2015 Wiley Periodicals, Inc.     

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.     

TUSC3 promotes colorectal cancer progression and epithelial–mesenchymal transition (EMT) through WNT/β‐catenin and MAPK signalling

Colorectal cancer (CRC) is one of the most common malignancies and is the second leading cause of cancer death in humans. Tumour suppressor candidate 3 (TUSC3) plays an important role in embryogenesis and metabolism. Deletion of TUSC3 often causes non‐syndromic mental retardation. Even though TUSC3 deregulation is frequently observed in epithelial cancers, the function of TUSC3 in CRC has remained unknown. In this study, we observed greater expression of TUSC3 at the mRNA and protein level in clinical colorectal tumour samples compared with paired normal tissues. Gain‐ and loss‐of‐function analyses were performed to evaluate the functional significance of TUSC3 in CRC initiation and progression. Immunoblotting, immunofluorescence, and co‐immunoprecipitation analyses were used to identify potential pathways with which TUSC3 might be involved. Overexpression of TUSC3 in CRC cells induced epithelial–mesenchymal transition (EMT) in CRC cells, accompanied by down‐regulation of the epithelial marker, E‐cadherin, and up‐regulation of the mesenchymal marker, vimentin. Increased proliferation, migration, and invasion, as well as accelerated xenograft tumour growth, were observed in TUSC3‐overexpressing CRC cells, while opposite effects were achieved in TUSC3‐silenced cells. In conclusion, our study demonstrated the oncogenic role of TUSC3 in CRC and showed that TUSC3 may be responsible for alternations in the proliferation ability, aggressiveness, and invasive/metastatic potential of CRC through regulating the MAPK, PI3K/Akt, and Wnt/β‐catenin signalling pathways. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Circular RNA ITCH mediates H2O2‐induced myocardial cell apoptosis by targeting miR‐17‐5p via wnt/β‐catenin signalling pathway

Cardiovascular disease is a severe threat health worldwide, and circRNAs have been shown to be correlated with the development of cardiovascular disease. Expression of circ‐ITCH and miR‐17a‐5p was evaluated by RT‐qPCR. Cell viability was measured using CCK‐8. Flow cytometry was applied to measure apoptosis rate. Binding between miR‐17‐5p and circ‐ITCH was detected via luciferase reporter assays. Levels of ATP in cells were examined with ATP testing. Western blot was used to evaluate apoptosis‐related proteins and proteins in Wnt/β‐catenin signalling pathway. H₂O₂ induced apoptosis of H9c2 cells and lowered cell viability as well as ATP levels and circ‐ITCH expression. After overexpression, circ‐ITCH enhanced cell viability and ATP concentration. Meanwhile, apoptosis was inhibited. MiR‐17‐5p was the target of circ‐ITCH as evidenced by luciferase report assays, with higher expression in H₂O₂‐induced H9c2 cells. Knockdown of miR‐17‐5p could promote cell viability and level of ATP and curb apoptosis and p53 and PARP expression. Moreover, overexpressed miR‐17‐5p could reverse the function of upregulated circ‐ITCH. Wnt3a, Wnt5a and β‐catenin in Wnt/β‐catenin signalling pathway were increased after H₂O₂ induction. Suppression of Wnt/β‐catenin signalling pathway could initiate the process of injury in H9c2 cells. Circ‐ITCH could protect myocardial cells from injuries caused by H₂O₂ by suppressing apoptosis while miR‐17‐5p played a reverse role, which could upregulate apoptosis and inhibit cell viability via Wnt/β‐catenin signalling pathway.     

Focal β‐catenin mutation identified on formalin‐fixed and paraffin‐embedded inflammatory hepatocellular adenomas

The identification of hepatocellular adenoma (HCA) with mutation in exon 3 of the CTNNB1 gene encoding for β‐catenin is clinically relevant due to a higher risk of malignant transformation. Inflammatory HCA (IHCA) can exhibit β‐catenin activation (β‐IHCA). We report two cases with multiple IHCA in which focal β‐catenin activation has been found in one of the IHCA. In both cases, the diagnosis of IHCA was confirmed on the resected nodules by routine stains, immunohistochemical detection of C‐reactive protein (CRP) and molecular biology on frozen material. An additional molecular analysis was performed on formalin‐fixed paraffin‐embedded (FFPE) material that showed focal glutamine synthetase (GS) staining, the surrogate marker of β‐catenin activation. In case 1, it was a 1.8‐cm area within the 7.5 cm IHCA, and in case 2 a small 0.3‐cm area within a 1.8 cm resected IHCA located close to a larger IHCA, negative for GS. In both cases, nuclear β‐catenin expression and decreased reticulin network were observed in the GS expressing foci, together with cholestasis and diffuse CD34 expression in case 1. Molecular analysis by pyrosequencing on FFPE material using the GS‐stained slides as reference to select areas with/without positive staining revealed a CTNNB1 exon 3 mutation restricted to the areas exhibiting both positive GS and CRP expression, whereas wild‐type CTNNB1 was found in areas showing only CRP staining. These two cases illustrate focal β‐catenin activation that can occur within IHCAs. Additional data are needed to determine if β‐catenin mutation is a secondary event in IHCA.