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.     

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.     

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.     

Prognostic Significance of Ror2 and Wnt5a Expression in Medulloblastoma

Medulloblastoma (MB) is a clinically and biologically heterogeneous group of tumors, and currently classified into four molecular subgroups (Wnt, Shh, Group 3 and Group 4). Intracellular signaling of the Wnt pathway has been divided into two classes: the “canonical” and the “non‐canonical” signaling pathway. The canonical signaling pathway is a well‐established, β‐catenin‐dependent signaling pathway in MB. In contrast, very little research about the non‐canonical WNT signaling pathway in MB exists. In order to identify the roles of Wnt‐5a and Ror2, two non‐canonical WNT pathway‐related genes, we studied 76 cases of MB with immunohistochemistry and quantitative real‐time PCR and correlated the results with clinicopathological and other molecular parameters and prognosis. Wnt5a and Ror2 were immunopositive in 20 (29.4%) and 35 (51.5%) of 68 cases, respectively. There were positive associations among protein expressions of Wnt5a, Ror2 and β‐catenin. Ror2 mRNA levels were well correlated with immunoexpression. Ror2 mRNA expression was significantly associated with CTNNB1 mutation. High Ror2 mRNA expression was an independent favorable prognostic factor. In conclusion, our study demonstrates the first attempt to identify Wnt5a and Ror2 as additional mechanisms contributing to dysregulation of the non‐canonical WNT signaling pathway in MB. Ror2 may play a role as an oncosuppressor in MB.     

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.     

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.     

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.     

To β or not to β: Canonical β‐catenin signaling pathway and ovarian development

The mammalian embryonic gonad is a unique organ primordium in that it can adopt two different developmental fates—namely, differentiate as either a testis or an ovary—with dramatic consequences for an individual. While a molecular cascade culminating in testis development is well characterized, the ovarian pathways still remain enigmatic. The canonical Wnt/β‐catenin signaling implements a conserved mechanism of regulating gene expression that is integral to development of all metazoans. In this review, we summarize the recent evidence that suggests a central role for this signaling pathway in the development of the mammalian female. Developmental Dynamics 237:3672–3680, 2008. © 2008 Wiley‐Liss, Inc.     

β‐catenin mutation in ovarian solid pseudopapillary neoplasm

Primaly solid pseudopapillary neoplasm (SPN) of the ovary is a rare tumor; recently 6 cases have been reported. Its pathogenesis, however, remains largely unclear. We report an additional case of primary ovarian SPN of an 18‐year‐old girl. The aim of this study is to define the difference between pancreatic and ovarian SPN by histological and molecular examination. Microscopically the tumor predominantly showed a solid pattern and focally a pseudopapillary pattern. The tumor cells showed two patterns of abundant eosinophilic cytoplasm and intracytoplasmic vacuoles. Immunohistochemistry of the tumor was positive for β‐catenin (nuclear and cytoplasmic reactivity), α1‐antitrypsin, vimentin, CD56, synaptophysin (focal weak), CD10. Mutation analyses revealed a point mutation, c.110C >T, in exon 3 of the the β‐catenin gene (CTNNB1), which causes the replacement of serine with phenylalanine at codon 37. A Ser37 point mutation is known to be one of the oncogenic somatic mutations in pancreatic SPN and the major oncogenic β‐catenin mutation. Ovarian SPN of our case was similar to pancreatic SPN histologicaly and had the same genomic characteristics. We expected that both ovarian and pancreatic SPNs shared the same oncogenesis related to Wnt/β‐catenin pathway for tumorgenesis.     

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.