Localization Of Β-catenin Research Articles

Disruption of Wnt/β-Catenin Signaling Exerts Antileukemia Activity and Sensitizes with Tyrosine Kinase Inhibitors in FLT3 Mutated AML In Vivo

Wnt/β-catenin signaling is associated with pathogenesis of AML and required for establishment of leukemic stem cells. FLT3 mutations are frequently observed in AML and predict poor clinic outcomes. Aberrant activation of FLT3 signaling stabilizes β-catenin and increases its nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKIs) are used to treat FLT3-mutated AML, but their effects are limited due to primary or secondary resistance to TKIs. We previously reported (ASH 2015) that disrupting Wnt/b-catenin signaling by C-82, a selective β-catenin/cAMP binding protein antagonist in combination with FLT3 inhibitors had a synergistic cytotoxicity in vitro in FLT3-mutated AML cells and AML stem/progenitor cells through inhibiting nuclear localization of β-catenin and suppressing the expression of β-catenin target proteins including survivin, CD44, c-Myc, and cyclin D1; several frizzled family receptors/co-receptors; Wnt ligands; and FLT3 downstream signaling proteins. The synergy was also observed in TKI-resistant FLT3 mutated cells. In this study, we evaluated the antileukemia effect of combined inhibition of β-catenin and FLT3 signaling in vivo in immunodeficient mice xenografted with FLT3-ITD mutated cells either from a cell line or from an AML patient.Molm13-GFP/Luc cells were injected into NOD-SCID IL2RγNull (NSG) mice. Once engraftment was confirmed by in vivo imaging, mice were treated with PRI-724 (C-82 pro-drug), sorafenib, or both. Treatment with PRI-724 or sorafenib decreased leukemia burden, and the combination was the most effective as assessed by in vivo imaging, flow cytometric measurement of human CD45+ cells in blood, and bone marrow (BM) and spleen H&E staining. The mice in PRI-724 (19 days, P = 0.025) or sorafenib (28 days, P = 0.0002) treated group had significantly longer median survival time than the control group (17 days), and the combined treatment further prolonged the survival time (30.5 days) (P = 0.0005, combination vs RPI-724; P = 0.0056, combination vs sorafenib). CyTOF and SPADE tree analysis showed a great reduction of human CD45+ cells and decreased expression of β-catenin, CD44, c-Myc, survivin, p-FLT3, p-ERK, p-AKT, and p-STAT5 in BM cells of the combination treated mice.Cells from a FLT3-ITD mutated AML patient sample collected from patient-derived xenograft (spleen) were injected into NOD-SCID IL2RγNull-3/GM/SF (NSGS) mice. After engraftment was confirmed by flow cytometry, mice were treated as above. Leukemia burden was decreased by sorafenib or PRI-724 treatment, as determined by flow cytometry measurement of human CD45+ cells in blood, BM, and spleen samples, but did not reach statistical significance. The combination significantly enhanced the antileukemia effect. PRI-724 (31 days, P = 0.008) or sorafenib (48 days, P = 0.0003) significantly prolonged median survival time compared with control (29 days), and the combination further extended the survival time (54 days) (P = 0.0005, combination vs RPI-724; P = 0.0067, combination vs sorafenib).Our in vivo study further demonstrates that disruption of Wnt/b-catenin signaling exerts antileukemia activity and sensitizes with TKIs in FLT3 mutated AML. These findings provide a rationale for clinic development of combined inhibition of Wnt/β-catenin and FLT3 signaling to overcome resistance and improve outcomes in AML patients with FLT3 mutations. DisclosuresKonopleva:Cellectis: Research Funding; Calithera: Research Funding. Carter:PRISM Pharma/Eisai: Research Funding.

Nuclear expression and/or reduced membranous expression of β-catenin correlate with poor prognosis in colorectal carcinoma: A meta-analysis.

The differential subcellular localizations of β-catenin (including membrane, cytoplasm, and nucleus) play different roles in the progression of colorectal cancer (CRC). However, the correlation between each subcellular localization of β-catenin and the prognosis of CRC patients remains undetermined. Systematic strategies were applied to search for eligible published studies in the PubMed, Embase, and Web of Science databases. The correlation between each subcellular localizations of β-catenin expression and patients' clinicopathological features or prognosis was analyzed. Finally, this meta-analysis, including 6238 cases from 34 studies, revealed that β-catenin overexpression in the nucleus (HR: 1.50[95% CI: 1.08-2.10]) or reduced expression of β-catenin in the membrane (HR: 1.33[95% CI: 1.15-1.54]) significantly correlated with lower 5-year overall survival (OS). Conversely, overexpression of β-catenin in the cytoplasm (HR: 1.00[95% CI: 0.85-1.18]) did not show significant association with 5-year OS. This study suggested that β-catenin overexpression in the nucleus or reduced expression in the membrane, but not its overexpression in cytoplasm, could serve as a valuable prognostic predictor for CRC. However, additional large and well-designed prospective studies are required to verify our results.

The effect of a germline mutation in the APC gene on β-catenin in human embryonic stem cells.

BackgroundMost cases of colorectal cancer (CRC) are initiated by inactivation mutations in the APC gene, which is a negative regulator of the Wnt-β-catenin pathway. Patients with familial adenomatous polyposis (FAP) inherit a germline mutation in one APC allele, and loss of the second allele leads to the development of polyps that will turn malignant if not removed. It is not fully understood which molecular mechanisms are activated by APC loss and when the loss of the second APC allele occurs.MethodsTwo FAP human embryonic stem cell (hESCs) lines were derived from APC mutated embryos following pre-implantation genetic diagnosis (PGD) for FAP. These FAP-hESCs were cultured in vitro and following extended culture: 1) β-catenin expression was analyzed by Western blot analysis; 2) Wnt-β-catenin/TCF-mediated transcription luciferase assay was performed; 3) cellular localization of β-catenin was evaluated by immunoflorecence confocal microscopy; and 4) DNA sequencing of the APC gene was performed.ResultsWe have established a novel human in-vitro model for studying malignant transformation, using hESCs that carry a germline mutation in the APC gene following PGD for FAP. Extended culturing of FAP1 hESCs led to activation of the Wnt signaling pathway, as demonstrated by enhanced β-catenin/TCF-mediated activity. Additionally, β-catenin showed a distinct perinuclear distribution in most (91 %) of the FAP1 hESCs high passage colonies. DNA sequencing of the whole gene detected several polymorphisms in FAP1 hESCs, however, no somatic mutations were discovered in the APC gene. On the other hand, no changes in β-catenin were detected in the FAP2 hESCs, demonstrating the natural diversity of the human FAP population.ConclusionsOur results describe the establishment of novel hESC lines from FAP patients with a predisposition for cancer mutation. These cells can be maintained in culture for long periods of time and may serve as a platform for studying the initial molecular and cellular changes that occur during early stages of malignant transformation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2809-9) contains supplementary material, which is available to authorized users.

Genetic disruption of tubulin acetyltransferase, αTAT1, inhibits proliferation and invasion of colon cancer cells through decreases in Wnt1/β-catenin signaling

Microtubules are required for diverse cellular processes, and abnormal regulation of microtubule dynamics is closely associated with severe diseases including malignant tumors. In this study, we report that α-tubulin N-acetyltransferase (αTAT1), a regulator of α-tubulin acetylation, is required for colon cancer proliferation and invasion via regulation of Wnt1 and its downstream genes expression. Public transcriptome analysis showed that expression of ATAT1 is specifically upregulated in colon cancer tissue. A knockout (KO) of ATAT1 in the HCT116 colon cancer cell line, using the CRISPR/Cas9 system showed profound inhibition of proliferative and invasive activities of these cancer cells. Overexpression of αTAT1 or the acetyl-mimic K40Q α-tubulin mutant in αTAT1 KO cells restored the invasiveness, indicating that microtubule acetylation induced by αTAT1 is critical for HCT116 cell invasion. Analysis of colon cancer-related gene expression in αTAT1 KO cells revealed that the loss of αTAT1 decreased the expression of WNT1. Mechanistically, abrogation of tubulin acetylation by αTAT1 knockout inhibited localization of β-catenin to the plasma membrane and nucleus, thereby resulting in the downregulation of Wnt1 and of its downstream genes including CCND1, MMP-2, and MMP-9. These results suggest that αTAT1-mediated Wnt1 expression via microtubule acetylation is important for colon cancer progression.

Aberrant β-catenin expression in urothelial carcinomas in blackfoot disease-endemic areas

Arsenic is a well-known toxic element and carcinogenic agent. The aim of this study was to investigate p63, E-cadherin, and β-catenin proteins in urothelial carcinoma (UC) in both arsenic contaminated areas [so-called blackfoot disease (BFD) area] and non-BFD areas. The expressions of p63, E-cadherin, and β-catenin proteins in 20 UC cases of blackfoot disease and 22 UC cases in non-BFD areas were detected using immunohistochemical methods. The results revealed a high p63 expression in 20 (47.6%) UC cases and high E-cadherin expression in six (14.3%) UC cases. Expressions of p63 and E-cadherin showed no significant correlations with clinicopathologic parameters. However, all 20 BFD cases and 12 of 22 (54.5%) non-BFD cases showed aberrant β-catenin expression. Ten out of 22 (45.5%) non-BFD cases also had normal membranous immunoreactivity. The β-catenin staining pattern significantly differed between cases in endemic and nonendemic areas of BFD (p=0.001). Tumor sites also significantly correlated with β-catenin expression (p=0.044). In addition, membranous localization of β-catenin was lower in UC from BFD-endemic areas compared with those from non-BFD endemic areas. In conclusion, it is suggested that relocalization of β-catenin from membrane to cytoplasm may be involved in the tumorigenesis of UC from BFD-endemic areas.

MiRNA-194 activates the Wnt/β-catenin signaling pathway in gastric cancer by targeting the negative Wnt regulator, SUFU

Emerging evidence has shown that miRNA-194 is aberrantly upregulated in gastric cancer (GC); however, the biological mechanisms underlying its involvement are largely unknown. Wnt/β-catenin signaling has been implicated in gastric tumorigenesis; we therefore hypothesized that miRNA-194 promotes gastric carcinogenesis by activating Wnt/β-catenin signaling. MiRNA-194 was found to be overexpressed in GC cell lines and 43 paired GC tissues. Overexpression of miRNA-194 promoted cell proliferation and migration, while inhibition of miRNA-194 blocked these processes. Inhibition of miRNA-194 decreased tumor volumes in nude mice. Furthermore, miRNA-194 inhibitors promoted cytoplasmic localization of β-catenin, leading to repression of Wnt signaling. We also discovered that SUFU, a known negative regulator of Hedgehog and Wnt signaling, was a target of miRNA-194. Anti-SUFU siRNAs rescued the inhibitory effects of miRNA-194 antagonists on cell proliferation and migration and on colony formation. We also found that SUFU expression was downregulated in GC tissues and cell lines and negatively correlated with miRNA-194 expression in primary GC tissues. Moreover, SUFU expression was negatively correlated with tumor stage, supporting its potential as a diagnostic or prognostic marker in GC. Taken together, these findings suggest that miRNA-194 is oncogenic and promotes GC cell proliferation and migration by activating Wnt signaling, at least in part, via suppression of SUFU.

CK2α/CSNK2A1 Phosphorylates SIRT6 and Is Involved in the Progression of Breast Carcinoma and Predicts Shorter Survival of Diagnosed Patients

Recently, the roles of sirtuins (SIRTs) in tumorigenesis have been of interest to oncologists, and protein kinase CK2 α1 (CSNK2A1) has been shown to be involved in tumorigenesis by phosphorylating various proteins, including SIRT1. Therefore, we evaluated the roles of CSNK2A1, SIRT6, and phosphorylated SIRT6 and their relationships in breast carcinoma. Nuclear expression of CSNK2A1 and SIRT6 predicted shorter overall survival and relapse-free survival by multivariate analysis. Inhibition of CSNK2A1 decreased the proliferative and invasive activity of cancer cells. In addition, CSNK2A1 was bound to SIRT6 and phosphorylated SIRT6; evidence for this is provided from immunofluorescence staining, co-immunoprecipitation of CSNK2A1 and SIRT6, a glutathione S-transferase pull-down assay, an invitro kinase assay, and transfection of mutant CSNK2A1. Knockdown of SIRT6 decreased the proliferation and invasiveness of cancer cells. Overexpression of SIRT6 increased proliferation, but mutation at the Ser338 phosphorylation site of SIRT6 inhibited the proliferation of MCF7 cells. Moreover, both knockdown of SIRT6 and a mutation at the phosphorylation site of SIRT6 decreased expression of matrix metallopeptidase 9, β-catenin, cyclin D1, and NF-κB. Especially, SIRT6 expression was associated with the nuclear localization of β-catenin. This study demonstrates that CSNK2A1 and SIRT6 are indicators of poor prognosis for breast carcinomas and that CSNK2A1-mediated phosphorylation of SIRT6 might be involved in the progression of breast carcinoma.

β-Catenin Expression Regulates Cell Migration of Human Colonic Adenocarcinoma Cells Through Gelsolin.

β-Catenin is one of the key players in colonic carcinogenesis. Being part of the E-cadherin complex, it regulates cell-cell adhesion and the migratory ability of cells. However, the role of nuclear β-catenin in the cell migration process is poorly understood. Gelsolin is one of the most abundant actin-binding proteins, and is implicated in tumour cell motility and invasiveness. The aim of the present study was to evaluate the potential association between expression of β-catenin and gelsolin, and their influence on the migration ability of colon adenocarcinoma LS180 cells. The colonic adenocarcinoma cell line LS180, its more motile sublines (EB3, 3LNLN, 5W) and β-catenin-knockdown LS180 cells were used to investigate the expression levels and subcellular localization of β-catenin and gelsolin. Increased motility of colonic cancer cells was accompanied by a reduction of β-catenin and up-regulation of gelsolin. β-Catenin seems to be involved in the regulation of gelsolin expression, which in turn affects the migratory ability of colonic cancer cells. Our results could have important implications for the design of new anticancer therapies.

Wnt/β-catenin signaling pathway activation is required for proliferation of chicken primordial germ cells in vitro.

Here, we investigated the role of the Wnt/β-catenin signaling pathway in chicken primordial germ cells (PGCs) in vitro. We confirmed the expression of Wnt signaling pathway-related genes and the localization of β-catenin in the nucleus, revealing that this pathway is potentially activated in chicken PGCs. Then, using the single-cell pick-up assay, we examined the proliferative capacity of cultured PGCs in response to Wnt ligands, a β-catenin-mediated Wnt signaling activator (6-bromoindirubin-3′-oxime [BIO]) or inhibitor (JW74), in the presence or absence of basic fibroblast growth factor (bFGF). WNT1, WNT3A, and BIO promoted the proliferation of chicken PGCs similarly to bFGF, whereas JW74 inhibited this proliferation. Meanwhile, such treatments in combination with bFGF did not show a synergistic effect. bFGF treatment could not rescue PGC proliferation in the presence of JW74. In addition, we confirmed the translocation of β-catenin into the nucleus by the addition of bFGF after JW74 treatment. These results indicate that there is signaling crosstalk between FGF and Wnt, and that β-catenin acts on PGC proliferation downstream of bFGF. In conclusion, our study suggests that Wnt signaling enhances the proliferation of chicken PGCs via the stabilization of β-catenin and activation of its downstream genes.

Valproic acid inhibits irradiation-induced epithelial-mesenchymal transition and stem cell-like characteristics in esophageal squamous cell carcinoma.

Esophageal carcinoma is one of the most aggressive malignancies, and is characterized by poor response to current therapy and a dismal survival rate. In this study we investigated whether irradiation induces epithelial-mesenchymal transition (EMT) in esophageal squamous cell carcinoma (ESCC) TE9 cells and whether the classic histone deacetylase (HDAC) inhibitor valproic acid (VPA) suppresses these changes. First, we showed that 2 Gy irradiation induced spindle cell-like morphologic changes, decreased expression of membranous E-cadherin, upregulated vimentin expression, and altered the localization of β-catenin from its usual membrane-bound location to cytoplasm in TE9 cells. Irradiation induced upregulation of transcription factors including Slug, Snail, and Twist, which regulate EMT. Stimulation by irradiation resulted in increased TGF-β1 and HIF-1α expression and induced Smad2 and Smad3 phosphorylation. Furthermore, irradiation enhanced CD44 expression, indicating acquisition of cancer stem-like cell properties. In addition, irradiation enhanced invasion and migration ability with upregulation of matrix metalloproteinases. These findings indicate that single-dose irradiation can induce EMT in ESCC cells. Second, we found that treatment with 1 mM VPA induced reversal of EMT caused by irradiation in TE9 cells, resulting in attenuated cell invasion and migration abilities. These results suggest that VPA might have clinical value to suppress irradiation-induced EMT. The reversal of EMT by HDAC inhibitors may be a new therapeutic strategy to improve the effectiveness of radiotherapy in ESCC by inhibiting the enhancement of invasion and metastasis.

Wnt signalling modulates transcribed-ultraconserved regions in hepatobiliary cancers

ObjectiveTranscribed-ultraconserved regions (T-UCR) are long non-coding RNAs which are conserved across species and are involved in carcinogenesis. We studied T-UCRs downstream of the Wnt/β-catenin pathway in liver cancer.DesignHypomorphic Apc mice...

Elevated PTH induces endothelial-to-chondrogenic transition in aortic endothelial cells.

Previous studies have shown that increased parathyroid hormone (PTH) attributable to secondary hyperparathyroidism in chronic kidney disease accelerates the arteriosclerotic fibrosis and calcification. Although the underlying mechanisms remain largely unknown, endothelial cells (ECs) have recently been demonstrated to participate in calcification in part by providing chondrogenic cells via the endothelial-to-mesenchymal transition (EndMT). Therefore, this study aimed to investigate whether elevated PTH could induce endothelial-to-chondrogenic transition in aortic ECs and to determine the possible underlying signaling pathway. We found that treatment of ECs with PTH significantly upregulated the expression of EndMT-related markers. Accordingly, ECs treated with PTH exhibited chondrogenic potential. In vivo, lineage-tracing model-subjected mice with endothelial-specific green fluorescent protein fluorescence to chronic PTH infusion showed a marked increase in the aortic expression of chondrocyte markers, and confocal microscopy revealed the endothelial origin of cells expressing chondrocyte markers in the aorta after PTH infusion. Furthermore, this in vitro study showed that PTH enhanced the nuclear localization of β-catenin in ECs, whereas β-catenin siRNA or DKK1, an inhibitor of β-catenin nuclear translocation, attenuated the upregulation of EndMT-associated and chondrogenic markers induced by PTH. In summary, our study demonstrated that elevated PTH could induce the transition of ECs to chondrogenic cells via EndMT, possibly mediated by the nuclear translocation of β-catenin.

Epithelial-mesenchymal transition and nuclear β-catenin induced by conditional intestinal disruption of Cdh1 with Apc is E-cadherin EC1 domain dependent.

Two important protein-protein interactions establish E-cadherin (Cdh1) in the adhesion complex; homophilic binding via the extra-cellular (EC1) domain and cytoplasmic tail binding to β-catenin. Here, we evaluate whether E-cadherin binding can inhibit β-catenin when there is loss of Adenomatous polyposis coli (APC) from the β-catenin destruction complex. Combined conditional loss of Cdh1 and Apc were generated in the intestine, intestinal adenoma and adenoma organoids. Combined intestinal disruption (Cdh1fl/flApcfl/flVil-CreERT2) resulted in lethality, breakdown of the intestinal barrier, increased Wnt target gene expression and increased nuclear β-catenin localization, suggesting that E-cadherin inhibits β-catenin. Combination with an intestinal stem cell Cre (Lgr5CreERT2) resulted in ApcΔ/Δ recombination and adenoma, but intact Cdh1fl/fl alleles. Cultured ApcΔ/ΔCdh1fl/fl adenoma cells infected with adenovirus-Cre induced Cdh1fl/fl recombination (Cdh1Δ/Δ), disruption of organoid morphology, nuclear β-catenin localization, and cells with an epithelial-mesenchymal phenotype. Complementation with adenovirus expressing wild-type Cdh1 (Cdh1-WT) rescued adhesion and β-catenin membrane localization, yet an EC1 specific double mutant defective in homophilic adhesion (Cdh1-MutW2A, S78W) did not. These data suggest that E-cadherin inhibits β-catenin in the context of disruption of the APC-destruction complex, and that this function is also EC1 domain dependent. Both binding functions of E-cadherin may be required for its tumour suppressor activity.

Targeted interference of SIN3A-TGIF1 function by SID decoy treatment inhibits Wnt signaling and invasion in triple negative breast cancer cells.

Cancer cell invasion is an obligatory step for metastatic dissemination that contributes to rapid relapse and a poorer survival in triple negative breast cancer (TNBC) patients. Development of novel therapeutic strategies to block tumor invasion is an unmet need in the treatment of cancer. We reported that the selective inhibition of the PAH2 domain of SIN3A protein function markedly suppressed metastatic dissemination to the lungs in TNBC xenograft bearing mice. Here, we show that TNBC cell lines treated with Sin3 interaction domain (SID) decoy peptides that bind to PAH2 display a strong in vitro inhibition of transwell invasion. This is accompanied by actin cytoskeleton reorganization with increased cortical actin deposition and downregulation of known Wnt target genes that are associated with epithelial to mesenchymal transition (EMT) and cancer cell invasion. Wnt pathway inhibition by SID decoy peptide was confirmed by decreased Wnt reporter activity and altered cytoplasmic localization of nuclear β-catenin. TGIF1, a transcription factor that modulates Wnt signaling and known to interact with the PAH2 domain of SIN3A, can be dissociated from the SIN3A complex by SID decoys. TGIF1 knockdown inhibits WNT target genes and in vitro cell invasion suggesting that TGIF1 might be a key target of the SID decoys to block tumor invasion. Taken together, targeting SIN3 function using SID decoys is a novel strategy to reverse invasion and the EMT program in TNBC translating into the inhibition of metastasis dissemination and eradication of residual disease.

CTNNB1 Mutations and Estrogen Receptor Expression in Neuromuscular Choristoma and Its Associated Fibromatosis.

Neuromuscular choristoma (NMC) is a very rare, developmental malformation characterized by the endoneurial intercalation of mature muscle fibers among peripheral nerve fibers. NMC typically arises in the major proximal peripheral nerves, most commonly the sciatic nerve, and may involve the lumbosacral and brachial plexus. Patients present clinically with progressive neuropathy or plexopathy. NMC is strongly associated with development of a fibromatosis, histologically identical to conventional desmoid-type fibromatosis (NMC-fibromatosis). The development of NMC-fibromatosis is often precipitated by iatrogenic trauma (ie, biopsy). Desmoid-type fibromatosis is characterized by CTNNB1 exon 3 mutations, which result in aberrant nuclear β-catenin localization and dysregulated canonical Wnt signaling. In contrast, the pathogenesis of NMC and NMC-fibromatosis is unknown. Desmoid-type fibromatosis expresses estrogen receptors (ER), specifically the ER-beta isoform (ERβ), and endocrine therapies may be used in surgically unresectable cases. In contrast, the ER expression profile of NMC-fibromatosis is unknown. We evaluated a series of NMC and NMC-fibromatosis for CTNNB1 mutations, β-catenin expression, and ER isoform expression. Five NMCs occurred in 2 female and 3 male patients (median age: 14 y, range <1 to 42 y), as masses involving the sciatic nerve (N=4) or brachial plexus (N=1). Four (of 5) NMCs had CTNNB1 mutations: 3 c.134 C>T (p.S45F) and 1 c.121 A>G (p.T41A). Four patients subsequently developed NMC-fibromatosis, and all 4 cases contained CTNNB1 mutations, including 1 p.T41A and 3 p.S45F mutations. In 3 patients, the NMC and NMC-fibromatosis had identical CTNNB1 mutations. Only 1 NMC had no detectable CTNNB1 mutation; however, the patient's subsequent NMC-fibromatosis had a CTNNB1 p.T41A mutation. All NMC and NMC-fibromatosis showed aberrant nuclear localization of β-catenin, nuclear ERβ expression, and no ERα expression. The presence of CTNNB1 mutations both in NMC and NMC-fibromatosis may be a shared molecular genetic abnormality underlying their pathogenesis.

Glucagon Like Peptide-1 Promotes Adipocyte Differentiation via the Wnt4 Mediated Sequestering of Beta-Catenin.

Glucagon-like peptide-1 (GLP-1) plays a role in the regulation of adipogenesis; however, the precise underlying molecular mechanism has not been fully defined. Wnt was recently identified as an important regulator of adipogenesis. This study aimed to investigate the involvement of the Wnt signaling pathway in the effects of GLP-1 on adipocyte differentiation. 3T3-L1 cells were induced to differentiate. The changes in the expression levels of adipogenic transcription factors and Wnts and the phosphorylation level and subcellular localization of β-catenin were quantified after GLP-1 treatment. GLP-1 stimulated adipocyte differentiation and lipid accumulation, which were accompanied by the expression of adipocyte marker genes. The expression of Wnt4 was upregulated in the process of adipocyte differentiation, which was further enhanced by treatment with GLP-1. β-catenin, an important mediator of the Wnt pathway, was immediately dephosphorylated and translocated from cytoplasm to nucleus when differentiation was induced. In the presence of GLP-1, however, β-catenin was redirected to the cell plasma membrane leading to its decreased accumulation in the nucleus. Knockdown of Wnt4 blocked the effect of GLP-1 on the cellular localization of β-catenin and expression level of adipogenic transcription factors. Our findings showed that GLP-1 promoted adipogenesis through the modulation of the Wnt4/β-catenin signaling pathway, suggesting that the GLP-1-Wntβ-catenin system might be a new target for the treatment of metabolic disease.

Association of FOXM1 expression with tumor histology and prognosis in Wilms tumor: Potential for a new prognostic marker

Wilms tumor (WT) is the most common pediatric renal malignancy. A recent ontogenic model suggests that undifferentiated tumor state, and hence poor prognosis, in WT is determined by stabilization of β-catenin in the nucleus. Forkhead box M1 (FOXM1) is a downstream component of the Wnt pathway and promotes nuclear localization of β-catenin. As elevation of FOXM1 gene expression is prognostic in various types of malignancy, we hypothesized that high FOXM1 expression in WT is associated with undifferentiated histology and thus poor prognosis. In the current study, the expression of FOXM1 mRNA was determined in 46 WT specimens and 11 renal tissue controls from patients undergoing tumor nephrectomy, and these data were assessed with regard to clinicopathological parameters. The results demonstrated an upregulation of FOXM1 in WT by 10-fold compared to normal tissue. Expression differed significantly between controls and tumors of intermediate- and high-risk histopathology (P<0.001, Kruskal-Wallis), and distinguished normal tissue from tumors of good and adverse clinical outcome (P<0.001, Kruskal-Wallis). Notably, FOXM1 expression was significantly lower (P=0.009) in patients that received preoperative doxorubicin. These results suggest that FOXM1 may serve as a companion diagnostic factor for doxorubicin-based therapies in WT.

Histamine prevents radiation-induced mesenchymal changes in breast cancer cells

Radiotherapy is a prime option for treatment of solid tumors including breast cancer though side effects are usually present. Experimental evidence shows an increase in invasiveness of several neoplastic cell types through conventional tumor irradiation. The induction of epithelial to mesenchymal transition is proposed as an underlying cause of metastasis triggered by gamma irradiation. Experiments were conducted to investigate the role of histamine on the ionizing radiation-induced epithelial to mesenchymal transition events in breast cancer cells with different invasive phenotype. We also evaluated the potential involvement of Src phosphorylation in the migratory capability of irradiated cells upon histamine treatment.MCF-7 and MDA-MB-231 mammary tumor cells were exposed to a single dose of 2Gy of gamma radiation and five days after irradiation mesenchymal-like phenotypic changes were observed by optical microscope. The expression and subcellular localization of E-cadherin, β-catenin, vimentin and Slug were determined by immunoblot and indirect immunofluorescence. There was a decrease in the epithelial marker E-cadherin expression and an increase in the mesenchymal marker vimentin after irradiation. E-cadherin and β-catenin were mainly localized in cytoplasm. Slug positive nuclei, matrix metalloproteinase-2 activity and cell migration and invasion were significantly increased. In addition, a significant enhancement in Src phosphorylation/activation could be determined by immunoblot in irradiated cells. MCF-7 and MDA-MB-231 cells also received 1 or 20μM histamine during 24h previous to be irradiated. Notably, pre-treatment of breast cancer cells with 20μM histamine prevented the mesenchymal changes induced by ionizing radiation and also reduced the migratory behavior of irradiated cells decreasing phospho-Src levels.Collectively, our results suggest that histamine may block events related to epithelial to mesenchymal transition in irradiated mammary cancer cells and open a perspective for the potential use of histamine to improve radiotherapy efficacy.

Effects of silver nanoparticles on adult neural stem cell differentiation and beta-catenin signaling.

Silver nanoparticles (AgNP) are distinguished by their unique antimicrobial actions. Since AgNPs inhibit the growth of bacteria, they are incorporated into common consumer products such as clothing, electronics and medicine. In concentrations as high as 50-100μg/mL, AgNP cause cytotoxicity in neural cells; yet in low concentrations of 30μg/mL and below, cytotoxicity decreased and cell viability remained. Most people will not be exposed to toxic doses of AgNP, but rather a sub-lethal exposure which may cause subtle effects. AgNP can disrupt the blood brain barrier, cross cell membranes, and accumulate in the brain, resulting in chronic low-level exposure. My project used cultured adult neural stem cells (NCS) to test the effects of AgNP exposure at 1μg/mL, well below cytotoxic levels. NCS play a key role in learning, memory and repair and provide a cell culture model for brain cells. We previously found that low level AgNP exposure disrupted the cytoskeleton structure in cultured NSC, resulting in inhibition of neurite dynamics and the formation of f-actin puncta. This research aims to investigate β-catenin signaling pathways as a potential target of AgNP effects on NSC cytoskeleton function. We found that β-catenin and f-actin puncta co-localize after AgNP treatment. Also, the subcellular localization of β-catenin in differentiating NCS is altered after exposure to AgNP. This suggests an interaction between AgNP and β-catenin, resulting in the formation of cytoskeletal disruptions during NCS differentiation. Consequently, as β-catenin signaling contributes to healthy neural physiology and development, disruption by low-level AgNP could affect proper growth and function.

Abstract 2276: Sorafenib/Refametinib potently inhibits Wnt/β-catenin in vitro and patient-derived xenograft models of human hepatocellular carcinoma

Abstract Background: Mutations affecting the Wnt/β-catenin pathway have been found in 26-40% of HCC samples. Aberrant constitutive activation of this pathway leads to uncontrolled cell proliferation growth and survival of tumor cells. Thus, identification of Wnt/β-catenin pathway inhibitors might lead to a new treatment option for a large subset of HCC patients. In the present study, we aim to investigate the effects of Sorafenib plus the allosteric MEK1/2 inhibitor Refametinib (Sorafenib/Refametinib) on Wnt/β-catenin signaling in vitro and in PDX models of HCC. Methods: We treated 15 patient-derived HCC xenograft models with i) 10 mg/kg Sorafenib QD, ii) 15 mg/kg Refametinib QD, and iii) Sorafenib/Refametinib. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant Wnt/β-catenin signaling. Apoptosis, cell proliferation and localization of β-catenin were analyzed by immunohistochemistry. In-vitro explant culture from 3 patient-derived HCC lines was also tested. Results: In both wild-type and activated-mutated β-catenin HCC primary cells, Refametinib and Sorafenib/Refametinib potently inhibited cell proliferation and caused cell death, which were correlated with downregulation of Dvl3 and β-catenin target genes (Axin2, c-myc, cyclin D1 and survivin). Inhibition of p-LRP6 at Ser1490 and p-β-catenin at Tyr142 and elevation of E-cadherin were also observed. These changes were not observed in Sorafenib-treated samples. In addition, the Sorafenib/Refametinib combination also suppressed Wnt3a-induced p-LRP6 at Ser1490 and β-catenin accumulation in cells. In vivo, significant reductions in p-LRP6 at Ser1490 and p-β-catenin at Tyr142 were observed in tumor samples from the Sorafenib/Refametinib treatment group and to a lesser extent, samples from the Refametinib. Although the overall protein levels of β-catenin were not significantly changed, non-phosphorylated (active) β-catenin Ser33/37/Thr41 and WNT/β-catenin target genes in Refametinib- and Sorafenib/Refametinib-treated tumors were significantly reduced. While vehicle-treated β-catenin-mutated tumors showed strong cytoplasmatic and nuclear β-catenin localization, most of β-catenin in Sorafenib/Refametinib- and, to a lesser extent, Refametinib-treated tumors was accumulated at the membrane. Conclusion: Sorafenib/Refametinib and, to a lesser extent, Refametinib inhibit the growth of patient derived explant cultures and PDX models. They also inactivate β-catenin signaling. Since activating mutations in the Wnt/β-catenin pathway are detected in a high proportion of HCC, combination of Sorafenib and Refametinib may represent an alternative treatment for β-catenin-dependent HCC. Citation Format: Huynh T. Hung, Richard Ong, Florian Puehler, Arne Scholz, Oliver Politz, Dieter Zopf, Dominik Mumberg, Karl Ziegelbauer. Sorafenib/Refametinib potently inhibits Wnt/β-catenin in vitro and patient-derived xenograft models of human hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2276.