Β-catenin Accumulation In Nucleus Research Articles

Acylglycerol kinase promotes the stemness of nasopharyngeal carcinoma cells by promoting β-catenin translocation to the nucleus through activating PI3K/Akt pathway.

Recent evidences show that acylglycerol kinase (AGK) expression is related to the occurrence and development of various human cancers. However, its roles in nasopharyngeal carcinoma (NPC) progression are still unclear. This work aims to explore the roles of AGK in NPC cell stemness. It was shown that AGK expression was higher in NPC tissues compared to the adjacent tissues. Online dataset analysis revealed that AGK expression was negatively correlated with the overall survival of NPC patients. Gain and loss of functional experiments demonstrated that AGK positively regulated the stemness of NPC cells, as evident by the change of the tumor sphere-formation ability, ALDH1 activity and expression of stemness critical regulators. KEGG analysis were performed to determine the potential pathways of AGK involved in NPC cell stemness and showed that the PI3K/Akt pathway exhibited the most correlation with AGK expression. Further mechanistic studies confirmed that AGK promoted the stemness of NPC cells through activating the PI3K/Akt pathway, and thus enhancing β-catenin accumulation in nucleus. This study demonstrates a novel AGK/PI3K/Akt/β-catenin axis involving in NPC cell stemness.

Indonesian Ginger (Bangle) Extract Promotes Neurogenesis of Human Neural Stem Cells through WNT Pathway Activation.

Indonesian ginger (Zingiber purpureum Rosc.), also known as Bangle, exhibits neurotrophic effects on cultured murine cortical neurons and in the adult mouse brain, but the underlying mechanisms remain unknown. Here, using human fetal neural stem cells (hfNSCs) as a model system for in vitro human neurogenesis, we show that Bangle extracts activate canonical WNT/β-catenin signaling. Bangle extract-treatment of hfNSCs not only promoted neuronal differentiation, but also accelerated neurite outgrowth from immature neurons. Furthermore, Bangle extracts induced expression of neurogenic genes and WNT signaling-target genes, and facilitated the accumulation of β-catenin in nuclei of hfNSC. Interestingly, altered histone modifications were also observed in Bangle-treated hfNSCs. Together, these findings demonstrate that Bangle contributes to hfNSC neurogenesis by WNT pathway and epigenetic regulation.

FEZF1-AS1 is a key regulator of cell cycle, epithelial-mesenchymal transition and Wnt/β-catenin signaling in nasopharyngeal carcinoma cells.

Background: Accumulating studies discloses that long non-coding RNAs (lncRNAs) serve important roles in human tumorigenesis, including nasopharyngeal carcinoma (NPC). The purpose of the present study was to determine the role of lncRNA FEZF1-AS1 in NPC.Materials and methods: The expression levels of FEZF1-AS1 in NPC tissues and cell lines were detected by RT-qPCR analysis. MTT assay was performed to investigate the proliferation of NPC cells in vitro, whereas the migration and invasion of NPC cells were determined by wound healing assay and transwell assay. A nude mouse tumor model was established to study the role of FEZF1-AS1 in NPC tumorigenesis in vivo. The expression levels of proteins were detected by Western blot assay.Results: The results showed that FEZF1-AS1 expression was increased in the NPC tissues and cell lines, and the higher expression of FEZF1-AS1 was closely associated with poor prognosis of NPC patients. We further observed that knockdown of FEZF1-AS1 inhibited the proliferation of NPC cells in vitro and suppressed NPC xenograft growth in vivo through inducing G2/M cell cycle arrest. The migratory and invasive abilities of NPC cells were also reduced upon FEZF1-AS1 knockdown. Moreover, we demonstrated that inhibition of FEZF1-AS1 remarkably suppressed epithelial–mesenchymal transition (EMT) and reduced β-catenin accumulation in nucleus in NPC cells.Conclusions: Collectively, we showed that FEZF1-AS1 might be a key regulator of cell cycle, EMT and Wnt/β-catenin signaling in NPC cells, which may be helpful for understanding of pathogenesis of NPC.

Microcystin-LR promotes migration via the cooperation between microRNA-221/PTEN and STAT3 signal pathway in colon cancer cell line DLD-1

Previous researches have reported that microcystin-LR (MC-LR) contributes to the progression of multiple types of carcinomas including colon cancer; however, the underlying molecular mechanisms remain unclear and require in-depth investigation. Here, the colon cell line DLD-1 was arranged for the analysis by the microRNA microarray which was associated with the cancer metastasis after MC-LR exposure. 31 human microRNAs were differentially expressed, including miR-221, which targeted 3′-UTR of PTEN mRNA and PTEN level was down-regulated by MC-LR treatment. Besides, MC-LR also induced the phosphorylation of STAT3, which can be reversed by adding miR-221 inhibitor and PTEN expression plasmid. Furthermore, miR-221 inhibitor, STAT3 siRNA and PTEN expression plasmid could reverse the effects of MC-LR induced migration with the accumulation of β-catenin in nuclei. In conclusion, our study suggested that MC-LR promoted the progression of colon carcinoma, at least in part, by regulating the expression miR-221, PTEN and STAT3 phosphorylation, which offers a novel perspective to understand the connection between MC-LR and colon cancer.

NANOGP8 is the key regulator of stemness, EMT, Wnt pathway, chemoresistance, and other malignant phenotypes in gastric cancer cells.

BackgroundAccumulating evidence demonstrated that NANOG1, the key transcription factor for embryonic stem cells, is associated with human cancers. NANOGP8, one of the pseudogenes in NANOG gene family, contains an intact open reading frame and also said to be expressed in cancer tissues. Therefore, a systematic study is greatly needed to address the following questions: among NANOG1 and NANOGP8, which gene is the main contributor for NANOG expression in cancer cells and which one is the key regulator responsible for stemness, epithelial-mesenchymal transition (EMT), metastasis, chemoresistance and other malignant phenotypes. Here we try to explore these issues with gastric adenocarcinoma cell lines in vitro using variety of molecular and cellular techniques.MethodsSpecial primers were designed to distinguish PCR products from NANOG1 and NANOGP8. Sphere-forming cells were cultured with serum-free and selective medium. A stable cell line was established with infection of lentivirus containing NANOGP8. qPCR was performed to measure NANOGP8 expression and its association with stemness, EMT and CSC markers in adherent cells and sphere-forming cells. Western blot analysis was deployed to confirm results of the transcript analysis. Experiments of cell proliferation, migration, invasion, clonogenic assay, sphere cell growth assays, cell cycle analysis, β-catenin accumulation and translocation in nucleus, and drug resistance were conducted to measure the impact of NANOGP8 on malignant statuses of gastric cancer cells. Immunofluorescence staining was used to analyze cell subpopulations with different markers.ResultsNANOGP8 is mainly responsible for NANOG expression in sphere-forming (stem cell-like) cells derived from gastric cancer cell lines regardless their differentiation status. Ectopic expression of NANOGP8 significantly up-regulates stemness transcription factors, EMT inducers, and cancer stem cell markers (CSC) including Lgr5. NANOGP8 also promotes expression of the signature genes vimentin and N-caderin for mesenchymal cells and down-regulates the signature gene E-caderin for epithelial cells whereby confer the cells with mesenchymal cell phenotype. In NANOGP8 over-expressed adherent and sphere-forming cells, Lgr5+ cells are significantly increased. Ectopic expression of NANOGP8 endows gastric cells with enhanced proliferation, migration, invasion, sphere-forming and clonogenic capacity, and chemoresistance. NANOGP8 expression also enhances β-catenin accumulation in nucleus and strengthens Wnt signal transduction.ConclusionNANOGP8 is the main regulator of gastric cancer stem cells. It is closely associated with EMT, stemness, and CSC marker as well as Wnt signal pathway. NANOGP8 is correlated with cell proliferation, migration, invasion, clonogenic capacity, β-catenin accumulation in nucleus, and chemoresistance in gastric cancer. NANOGP8 is a promising molecular target for clinical intervention of gastric cancer.

MicroRNA-146a inhibits cancer metastasis by downregulating VEGF through dual pathways in hepatocellular carcinoma.

Growing evidence indicates that miR-146a is involved in carcinogenesis and tumor progression in several human malignancies. However, the molecular details underlying miR-146a mediated regulation of its target genes and its precise biological function in cancer, especially in hepatocellular carcinoma (HCC) remains unclear.MethodsThe expression levels of genes including miR-146a, APC, VEGF and HAb18G were examined in HCC cell lines and patient specimens were compared with control levels using quantitative reverse transcription-PCR. The functions of miR-146a and HAb18G in migration/invasion and liver metastasis formation were determined by transwell and spleen injection assays, respectively. miR-146a related genes were determined by PCR array. The potential regulatory targets of miR-146a were determined by bioinformatics and prediction tools, correlation with target protein expression, and luciferase reporter assay. DNA methylation status of miR-146a promoter were performed by PCR analysis of bisulfite-modified genomic DNA.ResultsWe demonstrated that miR-146a expression was markedly downregulated in hepatoma cells and hepatoma tissues compared to immortalized normal liver epithelial cells and normal hepatic tissues. DNA methylation of miR-146a promoter correlated with its downexpression and with liver cancer metastasis. The restoration of miR-146a dramatically suppressed HCC cell invasion and metastasis by repressing VEGF expression through upregulating APC, which inhibits β-catenin accumulation in nucleus, and downregulating NF-κB p65 by targeting HAb18G. In human HCC, miR-146a expression was negative correlated with increased HAb18G, VEGF, NF-κB p65 and beneficial prognosis.ConclusionThis study identified a novel target of miR-146a and defined miR-146a as a crucial tumor suppressor in human HCC that acts through multiple pathways and mechanisms to suppress HCC invasion or metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-14-5) contains supplementary material, which is available to authorized users.

Loss of VHL expression contributes to epithelial–mesenchymal transition in oral squamous cell carcinoma

Loss of Von Hippel-Lindau (VHL) gene expression has been implicated in the development of human cancers. However, its function in oral squamous cell carcinoma (OSCC) remains undefined. The aim of this study was to clarify the VHL expression in OSCC and to explore the underlying mechanisms of VHL in modulating the epithelial-mesenchymal transition (EMT) in OSCC. The expression of VHL, HIF-1α and EMT related proteins in OSCC tissues were evaluated by immunohistochemistry. The correlation of VHL with clinico-pathological characteristics, prognosis and EMT related proteins were analyzed. The roles of VHL on the cell morphology, proliferation, migration, and invasion were determined by MTT, scratch and transwell invasion assay in Tscca and Tca8113P160 cells. The EMT related proteins were determined by Western blot and immunofluorescence (IF) methods. Loss of VHL expression was closely associated with pathologic grading, lymph node metastasis, poor prognosis, and EMT in OSCC. After re-expression of VHL, there was a cell morphologic change and motivation, proliferation, invasion of the cells were inhibited. The expression of Snail, N-cadherin and MMP-2/9, HIF-1α and VEGF were down-regulated in both the cell lines after transfection with VHL plasmid, while E-cadherin was up-regulated. Moreover, the effect of VHL suppressing β-catenin accumulation in nucleus was proved by Western blot and IF. VHL was significantly correlated with EMT process of OSCC. β-Catenin was an important downstream gene of VHL besides HIF-1α, which could induce the EMT process in OSCC.

The Integrator Complex Subunit 6 (Ints6) Confines the Dorsal Organizer in Vertebrate Embryogenesis

Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of β-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express vox and ved, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.

Autotaxin/Lpar3 signaling regulates Kupffer's vesicle formation and left-right asymmetry in zebrafish

Left-right (L-R) patterning is essential for proper organ morphogenesis and function. Calcium fluxes in dorsal forerunner cells (DFCs) are known to regulate the formation of Kupffer's vesicle (KV), a central organ for establishing L-R asymmetry in zebrafish. Here, we identify the lipid mediator lysophosphatidic acid (LPA) as a regulator of L-R asymmetry in zebrafish embryos. LPA is produced by Autotaxin (Atx), a secreted lysophospholipase D, and triggers various cellular responses through activation of specific G protein-coupled receptors (Lpar1-6). Knockdown of Atx or LPA receptor 3 (Lpar3) by morpholino oligonucleotides perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ L-R asymmetries, whereas overexpression of lpar3 partially rescued those defects in both atx and lpar3 morphants. Similar defects were observed in embryos treated with the Atx inhibitor HA130 and the Lpar1-3 inhibitor Ki16425. Knockdown of either Atx or Lpar3 impaired calcium fluxes in DFCs during mid-epiboly stage and compromised DFC cohesive migration, KV formation and ciliogenesis. Application of LPA to DFCs rescued the calcium signal and laterality defects in atx morphants. This LPA-dependent L-R asymmetry is mediated via Wnt signaling, as shown by the accumulation of β-catenin in nuclei at the dorsal side of both atx and lpar3 morphants. Our results suggest a major role for the Atx/Lpar3 signaling axis in regulating KV formation, ciliogenesis and L-R asymmetry via a Wnt-dependent pathway.

Dimerization of Laforin Is Required for Its Optimal Phosphatase Activity, Regulation of GSK3β Phosphorylation, and Wnt Signaling

Epilepsy of progressive myoclonus type 2 gene A (EPM2A) encodes a dual specificity protein phosphatase called Laforin. Laforin is also a tumor suppressor that dephosphorylates GSK3beta at the critical Ser9 position and regulates Wnt signaling. The epilepsy-causing mutations have a deleterious effect on phosphatase activity, regardless of whether they locate in the carbohydrate-binding domain (CBD) at the N terminus or the dual specificity phosphatase domain (DSPD) at the C terminus. How mutations outside the DSPD reduce the phosphatase activity of Laforin remains unexplained. Here we report that Laforin expressed in mammalian cells forms dimers that are highly resistant to SDS treatment. Deleting CBD completely abolished the dimerization and phosphatase activity of Laforin. Moreover, all of the naturally occurring Laforin mutations tested impaired laforin GSK3beta dephosphorylation at Ser9 dimerization, and beta-catenin accumulation in nucleus. Our results demonstrate a critical role of dimerization in Laforin function and suggest an important new dimension in protein phosphatase function and in molecular pathogenesis of Lafora's disease.

Nuclear beta-catenin is required to specify vegetal cell fates in the sea urchin embryo.

Beta-catenin is thought to mediate cell fate specification events by localizing to the nucleus where it modulates gene expression. To ask whether beta-catenin is involved in cell fate specification during sea urchin embryogenesis, we analyzed the distribution of nuclear beta-catenin in both normal and experimentally manipulated embryos. In unperturbed embryos, beta-catenin accumulates in nuclei that include the precursors of the endoderm and mesoderm, suggesting that it plays a role in vegetal specification. Using pharmacological, embryological and molecular approaches, we determined the function of beta-catenin in vegetal development by examining the relationship between the pattern of nuclear beta-catenin and the formation of endodermal and mesodermal tissues. Treatment of embryos with LiCl, a known vegetalizing agent, caused both an enhancement in the levels of nuclear beta-catenin and an expansion in the pattern of nuclear beta-catenin that coincided with an increase in endoderm and mesoderm. Conversely, overexpression of a sea urchin cadherin blocked the accumulation of nuclear beta-catenin and consequently inhibited the formation of endodermal and mesodermal tissues including micromere-derived skeletogenic mesenchyme. In addition, nuclear beta-catenin-deficient micromeres failed to induce a secondary axis when transplanted to the animal pole of uninjected host embryos, indicating that nuclear beta-catenin also plays a role in the production of micromere-derived signals. To examine further the relationship between nuclear beta-catenin in vegetal nuclei and micromere signaling, we performed both transplantations and deletions of micromeres at the 16-cell stage and demonstrated that the accumulation of beta-catenin in vegetal nuclei does not require micromere-derived cues. Moreover, we demonstrate that cell autonomous signals appear to regulate the pattern of nuclear beta-catenin since dissociated blastomeres possessed nuclear beta-catenin in approximately the same proportion as that seen in intact embryos. Together, these data show that the accumulation of beta-catenin in nuclei of vegetal cells is regulated cell autonomously and that this localization is required for the establishment of all vegetal cell fates and the production of micromere-derived signals.