Jeremy Jass Prize for Research Excellence in Pathology 2013

Emerging evidence indicates that Cullin 4B (CUL4B), a major component of ubiquitin ligase complexes, is over‐expressed in diverse cancer types with pro‐tumourigenic effects. In this issue of the Journal of Pathology, Yuan and colleagues [6] elucidated the oncogenic activity of CUL4B in hepatocellular carcinoma (HCC) and delineated its role in driving Wnt/β‐catenin signalling. In addition to the stabilization of β‐catenin protein against proteasomal degradation, CUL4B also acts in concert with enhancer of Zeste homologue 2 (EZH2) to concordantly silence multiple Wnt inhibitors. These findings provide significant mechanistic insights into the epigenetic activation of the Wnt/β‐catenin pathway in HCC and shed light on the functional importance of ubiquitination in this intricate regulatory system. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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.     

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.     

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.     

AMPKα2 reduces renal epithelial transdifferentiation and inflammation after injury through interaction with CK2β

TGFβ1/Smad, Wnt/β‐catenin and snail1 are preferentially activated in renal tubular epithelia after injury, leading to epithelial–mesenchymal transition (EMT). The stress response is coupled to EMT and kidney injury; however, the underlying mechanism of the stress response in EMT remains elusive. AMP‐activated protein kinase (AMPK) signalling is responsive to stress and regulates cell energy balance and differentiation. We found that knockdown of AMPKα, especially AMPKα2, enhanced EMT by up‐regulating β‐catenin and Smad3 in vitro. AMPKα2 deficiency enhanced EMT and fibrosis in a murine unilateral ureteral obstruction (UUO) model. AMPKα2 deficiency also increased the expression of chemokines KC and MCP‐1, along with enhanced infiltration of inflammatory cells into the kidney after UUO. CK2β interacted physically with AMPKα and enhanced AMPKα Thr172 phosphorylation and its catalytic activity. Thus, activated AMPKα signalling suppresses EMT and secretion of chemokines in renal tubular epithelia through interaction with CK2β to attenuate renal injury. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Oncogenic KRAS is not necessary for Wnt signalling activation in APC‐associated FAP adenomas

Recent studies have suggested that APC loss alone may be insufficient to promote aberrant Wnt/β‐catenin signalling. Our aim was to comprehensively characterize Wnt signalling components in a set of APC‐associated familial adenomatous polyposis (FAP) tumours. Sixty adenomas from six FAP patients with known pathogenic APC mutations were included. Somatic APC and KRAS mutations, β‐catenin immunostaining, and qRT‐PCR of APC, MYC, AXIN2 and SFRP1 were analysed. Array‐comparative genomic hybridization (aCGH) was also assessed in 26 FAP adenomas and 24 paired adenoma–carcinoma samples. A somatic APC alteration was present in 15 adenomas (LOH in 11 and four point mutations). KRAS mutations were detected in 10% of the cases. APC mRNA was overexpressed in adenomas. MYC and AXIN2 were also overexpressed, with significant intra‐case heterogeneity. Increased cytoplasmic and/or nuclear β‐catenin staining was seen in 94% and 80% of the adenomas. β‐Catenin nuclear staining was strongly associated with MYC levels (p value 0.03) but not with KRAS mutations. Copy number aberrations were rare. However, the recurrent chromosome changes observed more frequently contained Wnt pathway genes (p value 0.012). Based on β‐catenin staining and Wnt pathway target genes alterations the Wnt pathway appears to be constitutively activated in all APC‐FAP tumours, with alterations occurring both upstream and downstream of APC. Wnt aberrations are present at both the DNA and the RNA level. Somatic profiling of APC‐FAP tumours provides new insights into the role of APC in tumourigenesis. Copyright © 2010 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.     

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.     

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.