Cytoplasmic Accumulation Of Β-catenin Research Articles

Activation of the Wnt/β-catenin signaling pathway and CTNNB1 mutations in canine intestinal epithelial proliferative lesions.

Nuclear expression of β-catenin has been reported in canine intestinal epithelial tumors (IETs) and colorectal inflammatory polyps (CIPs) with dysplastic epithelia. However, the role of the Wnt/β-catenin signaling pathway in these lesions remains unclear. To investigate the association between the nuclear β-catenin expression and the activation of the Wnt/β-catenin signaling pathway, immunohistochemistry and mutation analyses were conducted on 64 IETs and 20 CIPs. IETs and CIPs with β-catenin nuclear and/or cytoplasm immunolabeling were classified as β-catenin (+). The immunostaining scores of c-Myc and Cyclin D1 and Ki-67 index were significantly higher in β-catenin (+) cases than in β-catenin (-) cases. Identical APC mutations (p.E154D and p.K155X) were detected in 6/41 β-catenin (+) IETs; all 6 of IETs with APC mutations were Jack Russell Terriers. CTNNB1 mutations were detected in 29/42 β-catenin (+) IETs, 3/11 β-catenin (+) CIPs, and 2/22 β-catenin (-) IETs, most of which were hotspots associated with human colorectal carcinoma. In one Miniature Dachshund diagnosed with a CIP that subsequently developed into an IET, the same CTNNB1 mutation was detected in both lesions. The immunohistochemical results suggest that cell proliferative activity in β-catenin (+) cases may be associated with activation of the Wnt/β-catenin signaling pathway. The mutation analysis results suggest that CTNNB1 mutations may be associated with cytoplasmic β-catenin accumulation in IET and CIP. Furthermore, the dysplastic epithelium in CIP may progress to IET through the activation of the Wnt/β-catenin signaling pathway by the CTNNB1 mutation.

Tideglusib enhances odontogenic differentiation in human dental pulp stem cells in vitro.

Tideglusib is a small molecule agonist of the canonical Wnt pathway. The present study investigated the influence of Tideglusib on human dental pulp stem cell (hDPSC) proliferation, apoptosis, migration and odonto/osteogenic differentiation. hDPSCs were treated with 50, 100 nM or 200 nM Tideglusib. β-catenin accumulation was detected by immunofluorescence staining. Colony-forming unit ability was assessed by staining with Coomassie blue. Cell cycle progression and cell apoptosis were investigated using flow cytometry. Cell migration was examined using an in vitro wound-healing assay. Osteogenic differentiation was examined using alkaline phosphatase (ALP) staining, alizarin red S staining and osteogenic-related gene expression. The gene expression profile was examined using a high-throughput RNA sequencing technique. All experiments were repeated using cells derived from at least four different donors (n=4). The Mann-Whitney U-test was used to identify significant differences between two independent group comparisons. For three or more group comparisons, statistical differences were assessed using the Kruskal-Wallis test followed by pairwise comparison. The significance level was set at 5% (p < .05). Tideglusib activated the Wnt signalling pathway in hDPSCs as demonstrated by an increase in cytoplasmic β-catenin accumulation and nuclear translocation. Tideglusib did not affect hDPSC proliferation, cell cycle progression, cell apoptosis or cell migration. In contrast, 50 and 100 nM Tideglusib significantly enhanced mineralization and osteogenic marker gene expression (RUNX2, ALP, BMP2 and DSPP; p < .05). Tideglusib enhanced the odonto/osteogenic differentiation of hDPSCs. Therefore, incorporating this bioactive molecule in a pulp-capping material could be a promising strategy to promote dentine repair.

Mutational landscape of non-functional adrenocortical adenomas

Adrenal incidentalomas are the most frequent human neoplasms. Recent genomic investigations on functional adrenocortical tumors have demonstrated that somatic mutations in PRKACA and KCNJ5 responsible for the development of adrenocortical adenomas (ACAs) are associated with hypercortisolism and aldosteronism, respectively. Several studies have identified CTNNB1 mutations in ACAs and have been mostly involved in the tumorigenesis of non-functional ACA (NFACA). However, integrated genomic characterization of NFACAs is lacking. In the current study, we utilized pan-genomic methods to comprehensively analyze 60 NFACA samples. A total of 1264 somatic mutations in coding regions among the 60 samples were identified, with a median of 15 non-silent mutations per tumor. Twenty-two NFACAs (36.67%) had genetic alterations in CTNNB1. We also identified several somatic mutations in genes of the cAMP/PKA pathway and KCNJ5. Histone modification genes (KMT2A, KMT2C, and KMT2D) were altered in 10% of cases. Germline mutations of MEN1 and RET were also found. Finally, by comparison of our transcriptome data with those available in the TCGA, we illustrated the molecular characterization of NFACA. We revealed the genetic profiling and molecular landscape of NFACA. Wnt/β-catenin pathway activation as shown ssby nuclear and/or cytoplasmic β-catenin accumulation is frequent, occurring in about one-third of ACA cases. cytochrome P450 enzymes could be markers to reveal the functional status of adrenocortical tumors. These observations strongly suggest the involvement of the Wnt/β-catenin pathway in benign adrenal tumorigenesis and possibly in the regulation of steroid secretion.

The uPA/uPAR system in astrocytic wound healing.

The repair of injured tissue is a highly complex process that involves cell proliferation, differentiation, and migration. Cell migration requires the dismantling of intercellular contacts in the injured zone and their subsequent reconstitution in the wounded area. Urokinase-type plasminogen activator (uPA) is a serine proteinase found in multiple cell types including endothelial cells, smooth muscle cells, monocytes, and macrophages. A substantial body of experimental evidence with different cell types outside the central nervous system indicates that the binding of uPA to its receptor (uPAR) on the cell surface prompts cell migration by inducing plasmin-mediated degradation of the extracellular matrix. In contrast, although uPA and uPAR are abundantly found in astrocytes and uPA binding to uPAR triggers astrocytic activation, it is unknown if uPA also plays a role in astrocytic migration. Neuronal cadherin is a member of cell adhesion proteins pivotal for the formation of cell-cell contacts between astrocytes. More specifically, while the extracellular domain of neuronal cadherin interacts with the extracellular domain of neuronal cadherin in neighboring cells, its intracellular domain binds to β-catenin, which in turn links the complex to the actin cytoskeleton. Glycogen synthase kinase 3β is a serine-threonine kinase that prevents the cytoplasmic accumulation of β-catenin by inducing its phosphorylation at Ser33, Ser37, and Ser41, thus activating a sequence of events that lead to its proteasomal degradation. The data discussed in this perspective indicate that astrocytes release uPA following a mechanical injury, and that binding of this uPA to uPAR on the cell membrane induces the detachment of β-catenin from the intracellular domain of neuronal cadherin by triggering its extracellular signal-regulated kinase 1/2-mediated phosphorylation at Tyr650. Remarkably, this is followed by the cytoplasmic accumulation of β-catenin because uPA-induced extracellular signal-regulated kinase 1/2 activation also phosphorylates lipoprotein receptor-related protein 6 at Ser1490, which in turn, by recruiting glycogen synthase kinase 3β to its intracellular domain abrogates its effect on β-catenin. The cytoplasmic accumulation of β-catenin is followed by its nuclear translocation, where it induces the expression of uPAR, which is required for the migration of astrocytes from the injured edge into the wounded area.

The role of E-cadherin and β-catenin in laryngeal cancer.

Epithelial cadherins with catenins form the E-cadherin-catenin complex that acts on cell-to-cell adhesion. The loss of these complex lead to the reduction or absence of epithelial cadherin expression in the cell membrane, cytoplasmic accumulation of β-catenin and its translocation to the nucleus, contributing to carcinogenic events. The objective of this study was to evaluate the expression of epithelial cadherin and β-catenin in patients with laryngeal tumor. A retrospective study of 52 patients with glottic or supraglottic squamous cell carcinoma was conducted and evaluated according to the tumor site, histological differentiation, TNM stage, survival analysis and compared with the immunohistochemical expression of epithelial cadherin and β-catenin. We observed statistically significant association between the epithelial cadherin expression reduction and supraglottic localization of the lesion, the presence of cervical metastasis, poorly differentiated tumors and locally advanced tumors when in glottic topography. Related to the expression of β-catenin, statistical significance was also found to the presence of cervical metastasis and tumor of low differentiation with the decreased expression of this marker. Regarding survival analysis, the low expression of β-catenin is related to worse overall survival and the reduction of expression of both markers to worse disease-free survival. We concluded that the reduction in expression of the markers studied leads to a prognostic impact as they are related to tumors with greater local aggressiveness and presence of cervical metastasis.

High-frequency deregulated expression of Wnt signaling pathway members in breast carcinomas.

PurposeBreast carcinoma is the most common malignancy and leading cause of cancer-related deaths in women worldwide including Saudi Arabia. Breast cancer in Saudi women develops at a much early age with median age of onset of 49 years compared to 62 years observed in patients from USA. Aberrations in wingless and integration site growth factor (Wnt) signaling pathway have been pathologically implicated in development of breast cancers and hence its role was examined in Saudi patients.Materials and methodsWe immunohistochemically examined various components of Wnt signaling pathway including β-catenin, tumor suppressor proteins, adenomatous polyposis coli (APC), and Axin, expression of naturally occurring pathway antagonists such as Dickkopf Wnt signaling pathway inhibitor 3 (DKK3), FRP2, and WIF1, as well as Wnt target cyclin D1 and c-Myc to establish if the pathway is constitutively activated in breast cancers arising in Saudi women.ResultsCytoplasmic β-catenin, indicative of activation of the pathway, was observed in 24% of cases. Expression of APC and Axin, which are components of β-catenin destruction complex, was lost in 5% and 10% of tumors, respectively. Additionally, Wnt signaling inhibitors DKK3, FRP2, and Wnt inhibitory factor 1 (WIF1) were not expressed in 8%, 14%, and 5% breast tumors, respectively. Overall, accumulation of cytoplasmic β-catenin and downregulation of other Wnt pathway proteins (APC/Axin/DKK3/FRP2/WIF1) were found in approximately half of the breast cancers (47%) in our cohort. Consistent with this, analysis of Wnt target genes demonstrated moderate-to-strong expression of c-Myc in 58% and cyclin D1 in 50% of breast cancers. Deregulation of Wnt pathway was not associated with age of onset of the disease, tumor grade, and triple-negative status of breast cancers.ConclusionsHigh level of deregulated expression of Wnt pathway proteins suggests its important role in pathogenesis of breast cancers arising in Saudi women who may benefit from development of therapeutic drugs targeting this pathway.

Evodiamine Exerts an Anti-Hepatocellular Carcinoma Activity through a WWOX-Dependent Pathway.

Evodiamine is one of the main components isolated from Evodia rutaecarpa, and it has been reported to exert inhibitory effects on cancers by anti-proliferative and apoptosis-inducing activities. Although the anti-cancer activity of evodiamine has been identified, the precise mechanisms of this action remain obscure. While previous studies indicated that evodiamine exerts anti-tumor effects through inhibiting β-catenin activity, and WW domain-containing oxidoreductase (WWOX) regulates β-catenin accumulation in cytoplasm, the effects of evodiamine on the expression of WWOX are still unknown. In this study, we provide evidence that evodiamine dose- and time-dependently inhibits both Mus musculus and Homo sapiens hepatocellular carcinoma (HCC) cells, as well as Hepa1-6 and HepG2 cell proliferation. We further tested the therapeutic effects of evodiamine in Hepa1-6 hepatoma-bearing mice, and we found that treatment of evodiamine by oral gavage significantly decreased the tumor size of the mice. Moreover, the expressions of WWOX were dose-dependently increased in HCC cell lines as well as in Hepa1-6 hepatoma-bearing mice after the treatment with evodiamine. Knockdown of WWOX in HepG2 and Hepa1-6 cells diminished the effects of evodiamine on the inhibitory effect of cancer cell growth, indicating that evodiamine induced anti-cancer activity through a WWOX-dependent pathway. As such, evodiamine activated WWOX to exert an anti-HCC activity, and might be a potential therapeutic or preventive candidate for HCC treatment.

MicroRNA-21 promotes osteogenic differentiation of mesenchymal stem cells by the PI3K/β-catenin pathway.

Osteogenesis of mesenchymal stem cells (MSCs) is essential for bone repair. Recently, microRNAs have been proven to play an important role in the regulation of MSC differentiation, including osteogenesis. Here, the function of microRNA-21 (miR-21) in the osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUMSCs) was investigated. Briefly, the miR-21 mimics (m-miR-21) and the antisense miR-21 (as-miR-21) were transfected to hUMSCs, and the capacity of miR-21 for the osteogenic differentiation of hUMSCs was evaluated by the expression of osteogenic markers encoding alkaline phosphatase (ALP), runt-related gene-2 (RUNX-2) and osteocalcin (OCN), as well as by Alizarin red S staining. The results indicated that the overexpression of miR-21 elevated the expression level of the osteogenesis-related genes of hUMSCs. During this process, the PI3K-AKT signaling pathway activity had an increasing tendency responding to miR-21 up-regulation. This enhancement promoted the phosphorylation of GSK-3β, leading to the stabilization and high concentration accumulation of β-catenin in cytoplasm to activate the transcription of RUNX-2, and finally increased the osteogenesis of hUMSCs. This work demonstrated that miR-21 and its target PI3K-AKT-GSK3β pathway played an important role in the osteogenic differentiation of hUMSCs by stabilizing β-catenin.

Wnt/β-catenin signaling pathway in hepatocellular carcinomas cases from Colombia

Background and aim. Hepatocellular carcinoma (HCC) is the most common primary liver cancer diagnosed worldwide. Deregulation of Wnt/β-catenin pathway has been associated with the development of HCC in a substantial number of cases in Europe and far less in Asia. Nothing is known about this pathway in HCC cases from South America. This study aimed to investigate the frequency of mutations in β-catenin gene (CTNNB1) and the subcellular localization of β-catenin in HCC cases from Colombia.Material and methods. We determine by direct sequencing the frequency of mutations in exon 3 of CTNNB1 gene and by immunohistochemistry the subcellular localization of β-catenin in 54 samples of HCC obtained from three pathology units in Bogota and Medellin cities.Results. Only three HCC cases (5.6%) were found mutated at residues (G34E, S45P, P44S, T41I) important for phosphorylation and ubiquitination of β-catenin protein. Strikingly, nuclear or cytoplasmic accumulation of β-catenin, hallmark of Wnt pathway activation, was found in 42.6% HCC cases (23/54). Interestingly, β-catenin accumulation was significantly more frequent in young patients and hepatitis B virus-related HCC.Conclusions. Although, CTNNB1 exon 3 mutations are not frequent in HCC from Colombian patients, our findings indicate that Wnt/β-catenin signaling is activated in 42.6% of HCC samples. Furthermore, Wnt signaling was demonstrated in HCC cases associated of HBV infection, one of the most important HCC risk factors in Colombia.

Cytoplasmic and/or nuclear expression of β-catenin correlate with poor prognosis and unfavorable clinicopathological factors in hepatocellular carcinoma: a meta-analysis.

BackgroundThe β-catenin is an important effector in WNT/β-catenin signaling pathway, which exerts a crucial role in the development and progression of hepatocellular carcinoma (HCC). Some researchers have suggested that the overexpression of β-catenin in cytoplasm and/or nucleus was closely correlated to metastasis, poor differentiation and malignant phenotype of HCC while some other researchers hold opposite point. So far, no consensus was obtained on the prognostic and clinicopathological significance of cytoplasmic/nuclear β-catenin overexpression for HCCs.MethodsSystematic strategies were applied to search eligible studies in all available databases. Subgroup analyses, sensitivity analyses and multivariate analysis were performed. In this meta-analysis, we utilized either fixed- or random-effects model to calculate the pooled odds ratios (OR) and its 95% confidence intervals (CI).ResultsA total of 22 studies containing 2334 cases were enrolled in this meta-analysis. Pooled data suggested that accumulation of β-catenin in cytoplasm and/or nucleus significantly correlated with poor 1-, 3- and 5-year OS and RFS. Moreover, nuclear accumulation combined with cytoplasmic accumulation of β-catenin tended to be associated with dismal metastasis and vascular invasion while cytoplasmic or nuclear expression alone showed no significant effect. Besides, no significant association was observed between cytoplasmic and/or nuclear β-catenin expression and poor differentiation grade, advanced TNM stage, liver cirrhosis, tumor size, tumor encapsulation, AFP and etiologies. Additional subgroup analysis by origin suggested that the prognostic value and clinicopathological significance of cytoplasmic and/or nuclear β-catenin expression was more validated in Asian population. Multivariate analyses of factors showed that cytoplasmic and/or nuclear β-catenin expression, as well as TNM stage, metastasis and tumor size, was an independent risk factors for OS and RFS.ConclusionsCytoplasmic and/or nuclear accumulation of β-catenin, as an independent prognostic factor, significantly associated with poor prognosis and deeper invasion of HCC, and could serve as a valuable prognostic predictor for HCC.

Abstract 4844: Extracellular hyaluronan accumulation by hyaluronan synthase 3 promotes pancreatic cancer growth and modulates tumor microenvironment via epithelial-mesenchymal transition

Abstract Pancreatic cancer is one of most deadly cancers with a 5-year survival rate of 6%. Accumulation of hyaluronan (HA) is found in about 87% of human pancreatic adenocarcinomas, and removal of HA suppresses tumor growth in HA-rich preclinical models. In a transgenic pancreatic cancer mouse model (LSL-KrasG12D/+;LSLTrp53R172H/+;Pdx-1-Cre, KPC), removal of HA by pegylated human recombinant PH20 hyaluronidase (PEGPH20) inhibits tumor growth and increases survival in combination with gemcitabine compared to gemcitabine monotherapy. In this study, we explored the role of HA synthesizing (HAS) enzymes HAS2 and HAS3 and HA accumulation in pancreatic cancer tumor growth and remodeling of tumor microenvironment. HAS2 and HAS3 were overexpressed in BxPC3 human pancreatic cancer cells using lentiviral vectors. Stable HAS2 and HAS3 overexpressing pancreatic cancer cell lines secreted more HA to culture medium and produced larger pericellular HA matrices than parental BxPC3 cells. In vivo, overexpression of HAS2 or HAS3 led to an increase in BxPC3 xenograft tumor growth (peritibial i.m. tumor model) compared to parental cells. Interestingly, overexpression of HAS3 was more effective to enhance tumor growth than overexpression of HAS2. In addition, massive accumulation of extracellular HA was found in HAS3 overexpressing tumors while HAS2 overexpressing tumors contained both extracellular and intracellular HA. Treatment with PEGPH20 removed the majority of extracellular HA and induced a 87% reduction of tumor volume in BxPC3 HAS3 model (p&amp;lt;0.001) but had weaker effect on BxPC3 HAS2 (33%, p&amp;lt;0.001) and BxPC3 tumors (36%, p&amp;lt;0.01). Accumulation of extracellular HA was associated with enriched tumor stroma, loss of membranous E-cadherin and accumulation of cytoplasmic β-catenin in pancreatic cancer cells, suggesting HA-induced epithelial-mesenchymal transition (EMT). Removal of HA by PEGPH20 reversed the remodeling of the tumor stroma and induced translocation of E-cadherin and β-catenin to the plasma membrane.Translocation of E-cadherin was also observed in the KPC pancreatic tumors after PEGPH20 treatment. In conclusion, accumulation of extracellular HA by HAS3 overexpression favors tumor growth and leads to a strong response to PEGPH20 in a pancreatic cancer xenograft model. Deposition of extracellular HA is associated with optimization of the tumor microenvironment and EMT. Depletion of HA by PEGPH20 reverses changes in the tumor stroma and induces translocation of epithelial markers to the plasma membrane. Citation Format: Anne Kultti, Chunmei Zhao, Susan Zimmerman, Ryan J. Osgood, Yanling Chen, Rebecca Symons, Ping Jiang, Curtis B. Thompson, David A. Tuveson, Gregory I. Frost, H Michael Shepard, Zhongdong Huang. Extracellular hyaluronan accumulation by hyaluronan synthase 3 promotes pancreatic cancer growth and modulates tumor microenvironment via epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4844. doi:10.1158/1538-7445.AM2014-4844

Platelet-Derived Growth Factor BB Mimics Serum-Induced Dispersal of Pancreatic Epithelial Cell Clusters

We showed previously that proliferating human islet-derived de-differentiated cells (DIDs) exhibit many characteristics of mesenchymal stem cells. Dispersed DIDs can be induced by serum deprivation to undergo mesenchymal-to-epithelial transition and aggregate into epithelial cell clusters (ECCs). Conversely, ECCs can be induced to disperse and undergo epithelial-to-mesenchymal transition (EMT) by re-addition of mammalian sera. In this study, we show that platelet-derived growth factor BB (PDGF-BB) mimics and mediates serum-induced ECCs' dispersal accompanied by accumulation of cytoplasmic β-catenin and a decrease in the levels of insulin and glucagon mRNAs. Moreover, we show that PDGF-BB-induced dispersal of ECCs is a more general phenomenon that occurs also with bone marrow mesenchymal stem cells (BM-MSCs) and dermal fibroblasts (DFs). In DIDs, BM-MSCs, and DFs, PDGF decreased the levels of DKK1 mRNA, suggesting involvement of the Wnt signaling pathway. PDGF-BB stimulated a significant increase in S473 phosphorylation of Akt and the PI3K specific inhibitor (PIP828) partially inhibited PDGF-BB-induced ECC dispersal. Lastly, the PDGF-receptor (PDGF-R) antagonist JNJ-10198409 inhibited both PDGF-BB--and serum-induced ECC dispersal. Epidermal growth factor (EGF), which shares most of the PDGF signaling pathway, did not induce dispersal and only weakly stimulated Akt phosphorylation. Our data suggest that PDGF-BB mediates serum-induced DIDs dispersal, correlated with the activation of the PI3K-Akt pathway.

Accumulation of extracellular hyaluronan by hyaluronan synthase 3 promotes tumor growth and modulates the pancreatic cancer microenvironment.

Extensive accumulation of the glycosaminoglycan hyaluronan is found in pancreatic cancer. The role of hyaluronan synthases 2 and 3 (HAS2, 3) was investigated in pancreatic cancer growth and the tumor microenvironment. Overexpression of HAS3 increased hyaluronan synthesis in BxPC-3 pancreatic cancer cells. In vivo, overexpression of HAS3 led to faster growing xenograft tumors with abundant extracellular hyaluronan accumulation. Treatment with pegylated human recombinant hyaluronidase (PEGPH20) removed extracellular hyaluronan and dramatically decreased the growth rate of BxPC-3 HAS3 tumors compared to parental tumors. PEGPH20 had a weaker effect on HAS2-overexpressing tumors which grew more slowly and contained both extracellular and intracellular hyaluronan. Accumulation of hyaluronan was associated with loss of plasma membrane E-cadherin and accumulation of cytoplasmic β-catenin, suggesting disruption of adherens junctions. PEGPH20 decreased the amount of nuclear hypoxia-related proteins and induced translocation of E-cadherin and β-catenin to the plasma membrane. Translocation of E-cadherin was also seen in tumors from a transgenic mouse model of pancreatic cancer and in a human non-small cell lung cancer sample from a patient treated with PEGPH20. In conclusion, hyaluronan accumulation by HAS3 favors pancreatic cancer growth, at least in part by decreasing epithelial cell adhesion, and PEGPH20 inhibits these changes and suppresses tumor growth.

PI3K/Akt Signaling Interacts With Wnt/β-Catenin Signaling But Does Not Induce An Accumulation Of β-Catenin In The Nucleus Of Acute Lymphoblastic Leukemia Cell Lines

Signaling pathways play essential roles in biological processes as development, cell proliferation and homeostasis. The accurate modulation of signaling pathways, their adapted interaction and their time- and tissue-specific adjusted regulation are required for normal cell development. PI3K/Akt and Wnt/β-Catenin signaling pathways act as key regulators in cell proliferation, differentiation and growth. Both signaling pathways include GSK3β as a common protein, which may mediate an interaction and cross-talk between the pathways. Aberrant activation of PI3K/Akt signaling has been linked to different types of leukemia while Wnt/β-Catenin signaling is known to be deregulated in some solid tumors. However, a potential role of Wnt/β-Catenin signaling for pathogenesis of acute lymphoblastic leukemia (ALL) has not yet been analyzed.In our study we analyzed both signaling pathways in different B- and T-ALL cell lines (RS4;11, SEM, REH, CEM, Jurkat, MOLT-4), thereby focusing mainly on their potential interaction via the protein GSK3β. Western Blot experiments were performed to evaluate the expression of specific PI3K/Akt and Wnt/β-Catenin key proteins. To evaluate the activation status of Wnt signaling immunofluorescence and protein fractionation experiments were performed, analyzing the activation linked nucleic localization of β-Catenin. The effect of pathway activation and inhibition on cell proliferation via chemical compounds was analyzed by WST-1 test.High pAkt levels were detected in B-ALL cell line SEM and T-ALL cell line CEM, indicating a hyperactive PI3K/Akt signaling, whereas other analyzed cell lines diplayed lower pAkt status. Among all cell lines analyzed SEM and CEM also showed the highest cytoplasmic β-Catenin levels, indicating a direct interaction of both signaling pathways. However, immunofluorescence and fractionation experiments revealed that a translocation of β-Catenin into the nucleus did not occur. To further investigate the role and interaction of PI3K/Akt and Wnt/β-Catenin signaling, pathway inhibiting and stimulating experiments were performed. Treatment of cells with Wnt3a led to activation of the Wnt/β-Catenin signaling cascade, characterized by nuclear β-Catenin accumulation. Inhibition of cell proliferation was detected after treatment with high concentrations Wnt3a (≥ 500 ng/ml). PI3K inhibition by LY294002 led to decreased phosphorylation of GSK3β at Ser9 and an increased decay of β-Catenin. Stimulation of PI3K/Akt signaling using activating ligand FLT3L induced GSK3β phosphorylation at Ser9 and accumulation of cytoplasmic β-Catenin. However a translocation of β-Catenin into the nucleus seems not to occur.In summary our results indicate that PI3K/Akt and Wnt/β-Catenin signaling can interact through their common protein GSK3β, but stimulation of the PI3K/Akt signaling pathway by addition of PI3K/Akt specific activators does not fully activate Wnt/β-Catenin signaling in ALL cells. Complete activation of the Wnt cascade characterized by translocation of β-Catenin into the nucleus can only be induced by use of specific Wnt effectors. Disclosures:No relevant conflicts of interest to declare.

Papillary Carcinoma of the Thyroid Gland with Nodular Fasciitis-like Stroma

Papillary thyroid carcinoma with nodular fasciitis-like stroma (PTC-NFS) is a rare variant of PTC. The term 'PTC with fibromatosis-like stroma' has been used as a synonym to describe this variant. It is characterized by extensive proliferation of fibroblasts and myofibroblasts in the tumor stroma, which occurs in up to 80% of the tumors. We herein describe a case of PTC-NFS which developed in a 49-year-old woman with the demonstration of findings of ultrasonography, fine needle aspiration cytology and histological examination of the lesion. To characterize the stromal components, we investigated the expression of several immunohistochemical markers which have been shown to be expressed differently in nodular fasciitis (NF) and fibromatosis (FM). The immunostaining results demonstrated nuclear and cytoplasmic accumulation of β-catenin, cytoplasmic transforming growth factor-β expression and nuclear Smad expression in the stromal cells, suggesting that the stromal cells in this case have similar molecular profiles to those of FM rather than NF.

Anti-apoptotic effect of claudin-1 on TNF-α-induced apoptosis in human breast cancer MCF-7 cells

Accumulating evidence reveals that aberrant expression of claudins manifests in various tumors; however, their biological functions are poorly understood. Here, we report on the elevated expression of claudin-1 in human breast cancer MCF-7 cells under tumor necrosis factor (TNF)-α treatment. Interestingly, the increased expression of claudin-1 contributes to an anti-apoptotic role in TNF-α-induced apoptosis. In line with this, upon TNF-α stimulus, downregulation of claudin-1 by siRNA knockdown results in a significant increase in cleavage of caspase-8 and poly (ADP-ribose) polymerase, a decrease of cyclinD1 expression, and DNA fragmentation. Consistently, TdT-mediated dUTP nick end labeling assay also shows that loss of claudin-1 increases the susceptibility of MCF-7 cells to TNF-α-induced apoptosis. However, there is no obvious effect on the expression of Bax and p53 after the treatment aforementioned. In addition, TNF-α increases the amount of claudin-1 and the cytoplasmic accumulation of β-catenin, while claudin-1 siRNA increases the amount of β-catenin in the cell membrane as well as the amount of E-cadherin in the cytoplasm. In conclusion, our data reveal a novel role of claudin-1 in regulating apoptosis in MCF-7 cells.

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.

Aberrant activation of Wnt/β-catenin signaling pathway contributes to the sequential progression of DMBA-induced HBP carcinomas

Wnt signaling pathway mediated via interactions between β-catenin and members of the TCF/LEF-1 family of transcription factors plays a central role in the regulation of epithelial cell proliferation, apoptosis, differentiation, adhesion, epithelial-mesenchymal transition, and invasion. Aberrant activation of the Wnt/β-catenin signaling pathway with overexpression of Wnt and Fz, mutations of APC, β-catenin, and axin 1, and cytoplasmic accumulation of β-catenin have been frequently reported in a broad spectrum of human malignancies including oral squamous cell carcinomas (OSCCs). However, changes in the components of the Wnt signaling pathway have not been documented during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis, a paradigm for oral oncogenesis and chemointervention. In this study, we evaluated the role of β-catenin accumulation and Wnt ligands, Wnt signaling members (Fz, Dvl, APC, GSK-3β, axin, and WIF) and the downstream targets of Wnt (cyclin D1, MMP-2, and MMP-9) during the sequential progression of DMBA-induced HBP carcinomas by semi-quantitative RT-PCR and western blot analyses. Our data reveal a correlation between β-catenin accumulation and activation of Wnt signaling, and its downstream effector molecules during the sequential development of HBP carcinomas from hyperplasia to invasive carcinoma through dysplasia. Our data also support a pivotal role for β-catenin in the malignant transition of the HBP. Aberrant Wnt signaling may be a hallmark of progression to malignancy during DMBA-induced HBP carcinogenesis and could be a potential preventive and therapeutic target for suppression of OSCC.

Retained Cell–Cell Adhesion in Serrated Neoplastic Pathway as Opposed to Conventional Colorectal Adenomas

The molecular features of serrated polyps of colorectum remain to be elucidated. The expression pattern of adhesive molecules (E-cadherin, α-catenin, and β-catenin) has not been examined in serrated neoplastic pathway. The expression of E-cadherin, α-catenin, and β-catenin were analyzed by immunohistochemistry in 32 hyperplastic polyps (HPs), 28 sessile serrated adenomas (SSAs), 37 traditional serrated adenomas (TSAs), 51 traditional adenomas (TAs), and 10 normal colonic tissues (NCs). Retained membranous expression for E-cadherin, α-catenin, and β-catenin was more frequent in HPs, SSAs, and TSAs than that in TAs (p < 0.001). Nuclear labeling of β-catenin was detected in 19.6% of TAs, but in none of HPs, SSAs, and TSAs (p < 0.001). Cytoplasmic accumulation of β-catenin was found in 3.1% of HPs, 3.6% of SSAs, and 21.6% of TSAs, significantly lower than that in TAs (60.8%, p < 0.001). The membranous co-expression of E-cadherin, α-catenin, and β-catenin was more frequent in HPs (68.8%), SSAs (60.7%), and TSAs (37.8%) than that in TAs (7.8%, p < 0.001). Cell adhesion function is retained in serrated neoplastic pathway. Wnt signaling pathway plays a less active role in the development of colorectal serrated polys than in TAs.

The identification of a novel Paneth cell-associated antigen in a familial adenomatous polyposis mouse model

Wnt signaling is important for the differentiation of the Paneth cell lineage in the small intestine. However, abnormal Wnt signaling predisposes to intestinal tumorigenesis in the familial adenomatous polyposis (FAP) mouse model. Vaccination with dendritic cells fused with tumor cells from FAP mice, in which Wnt signaling is constitutively activated, induced humoral immunity and suppressed intestinal tumor development. We identified the novel antigen Apa1 (Adenomatous polyposis antigen 1) recognized by antibodies in vaccinated mouse serum. Apa1 was localized in the Paneth cell-like tumor cells showing cytoplasmic β-catenin accumulation and also in normal Paneth cells at the bottom of the crypts. Phospholipase A2 (Pla2g2a), known to act as an anti-bacterial agent and a major suppressor of intestinal tumors, was also expressed in the Paneth cells. These results suggest that Apa1 might be involved in anti-microbial defense and could influence tumor development in FAP mice via modulation of commensal microbiota.