Regulating Β-catenin Stability Research Articles

Retraction Note: Integrin-β5, a miR-185-targeted gene, promotes hepatocellular carcinoma tumorigenesis by regulating β-catenin stability

Retraction Note: Integrin-β5, a miR-185-targeted gene, promotes hepatocellular carcinoma tumorigenesis by regulating β-catenin stability

Renal interstitial cells promote nephron regeneration by secreting prostaglandin E2.

In organ regeneration, progenitor and stem cells reside in their native microenvironment, which provides dynamic physical and chemical cues essential to their survival, proliferation, and differentiation. However, the types of cells that form the native microenvironment for renal progenitor cells (RPCs) have not been clarified. Here, single-cell sequencing of zebrafish kidney reveals fabp10a as a principal marker of renal interstitial cells (RICs), which can be specifically labeled by GFP under the control of fabp10a promoter in the fabp10a:GFP transgenic zebrafish. During nephron regeneration, the formation of nephrons is supported by RICs that form a network to wrap the RPC aggregates. RICs that are in close contact with RPC aggregates express cyclooxygenase 2 (Cox2) and secrete prostaglandin E2 (PGE2). Inhibiting PGE2 production prevents nephrogenesis by reducing the proliferation of RPCs. PGE2 cooperates with Wnt4a to promote nephron maturation by regulating β-catenin stability of RPC aggregates. Overall, these findings indicate that RICs provide a necessary microenvironment for rapid nephrogenesis during nephron regeneration.

EPB41 suppresses the Wnt/β-catenin signaling in non-small cell lung cancer by sponging ALDOC.

Despite advancements in therapeutic options, the overall prognosis for non-small-cell lung cancer (NSCLC) remains poor. Further exploration of the etiology and targets for novel treatments is crucial for managing NSCLC. In this study, we revealed the significant potential of EPB41 for inhibiting NSCLC proliferation, invasion and metastasis in vitro and in vivo. Consistent with its tumor suppressor role in NSCLC, the expression of EPB41 in NSCLC specimens evidently decreased compared to that in normal tissues, and low EPB41 expression was associated with poor prognoses for NSCLC patients. We further demonstrated the importance of EPB41 protein as a novel inhibitor of the Wnt signaling, which regulates β-Catenin stability, and elucidated the crucial role of the EPB41/ALDOC/GSK3β/β-Catenin axis in NSCLC. Suppression of EPB41 expression in cancer cells elevated the levels of free ALDOC protein released from the EPB41-ALDOC complex, leading to disassembly of the β-catenin destruction complex, reduced proteolytic degradation of β-catenin, elevated cytoplasmic accumulation and nuclear translocation of β-catenin, thereby activating the expression of multiple oncogenes and, thus, NSCLC pathogenesis. Our study highlights the potential of EPB41 as a future therapeutic target for lung cancer.

TMEPAI/PMEPA1 inhibits Wnt signaling by regulating β-catenin stability and nuclear accumulation in triple negative breast cancer cells

Transmembrane prostate androgen-induced protein (TMEPAI) is a type I transmembrane protein induced by several intracellular signaling pathways such as androgen, TGF-β, EGF, and Wnt signaling. It has been reported that TMEPAI functions to suppress TGF-β and androgen signaling but here, we report a novel function of TMEPAI in Wnt signaling suppression. First, we show that TMEPAI significantly inhibits TCF/LEF transcriptional activity stimulated by Wnt3A, LiCl, and β-catenin. Mechanistically, TMEPAI overexpression prevented β-catenin accumulation in the nucleus and TMEPAI knockout in triple negative breast cancer cell lines promoted β-catenin stability and nuclear accumulation together with increased mRNA levels of Wnt target genes AXIN2 and c-MYC. The presence of TGF-β type I receptor kinase inhibitor did not affect the enhanced mRNA expression of AXIN2 in TMEPAI knockout cells. These data suggest that TMEPAI suppresses Wnt signaling by interfering with β-catenin stability and nuclear translocation in a TGF-β signaling-independent manner.

RETRACTED ARTICLE: Integrin-β5, a miR-185-targeted gene, promotes hepatocellular carcinoma tumorigenesis by regulating β-catenin stability

BackgroundThe tumour microenvironment is essential for cancer progress and metastasis. Integrin-β5 (ITGB5), a member of the integrin family, has been implicated to mediate the interactions of cells with the extracellular matrix (ECM) and promote tumorigenesis in several malignancies. However, the role of ITGB5 in hepatocellular carcinoma (HCC) is still unknown.MethodsThe biological function of ITGB5 in HCC was investigated using migration, colony formation assays. The potential molecular mechanism of ITGB5 in regulating HCC tumorigenesis and β-catenin stabilization was investigated by western blotting, co-immunoprecipitation and ubiquitination assays. The expression level of ITGB5 mediated by miR-185 was confirmed by bioinformatic analysis, luciferase assay. The clinical significance of ITGB5 was based on human tissue microarray (TMA) analysis.ResultsHere, we found that the expression of ITGB5 is increased in HCC tissues. Elevated ITGB5 markedly facilitates HCC cell migration and tumorigenesis in vitro and in vivo. Further mechanistic studies revealed that ITGB5, as a partner of β-catenin, directly interacts with β-catenin and inhibits its degradation, thus leading to WNT/β-catenin activity. Subsequently, we also found that ITGB5 is a direct targeted gene of miR-185. The downregulation of miR-185 in HCC cells promotes an increase in ITGB5. An additional increase of ITGB5 is associated with β-catenin upregulation and a miR-185 decrease in HCC tissues.ConclusionsOur data reveal that the miR-185-ITGB5-β-catenin pathway plays an important role in HCC tumorigenesis, and ITGB5 may be a promising specific target for HCC therapy.

Phosphorylations of Serines 21/9 in Glycogen Synthase Kinase 3α/β Are Not Required for Cell Lineage Commitment or WNT Signaling in the Normal Mouse Intestine.

The WNT signalling pathway controls many developmental processes and plays a key role in maintenance of intestine renewal and homeostasis. Glycogen Synthase Kinase 3 (GSK3) is an important component of the WNT pathway and is involved in regulating β-catenin stability and expression of WNT target genes. The mechanisms underpinning GSK3 regulation in this context are not completely understood, with some evidence suggesting this occurs through inhibitory N-terminal serine phosphorylation in a similar way to GSK3 inactivation in insulin signaling. To investigate this in a physiologically relevant context, we have analysed the intestinal phenotype of GSK3 knockin mice in which N-terminal serines 21/9 of GSK3α/β have been mutated to non-phosphorylatable alanine residues. We show that these knockin mutations have very little effect on overall intestinal integrity, cell lineage commitment, β-catenin localization or WNT target gene expression although a small increase in apoptosis at villi tips is observed. Our results provide in vivo evidence that GSK3 is regulated through mechanisms independent of N-terminal serine phosphorylation in order for β-catenin to be stabilised.

CD147 modulates androgen receptor activity through the Akt/Gsk-3β/β-catenin/AR pathway in prostate cancer cells.

The androgen signaling pathway serves an important role in the development of prostate cancer. β-Catenin is an androgen receptor (AR) cofactor and augments AR signaling. Glycogen synthase kinase-3β (GSK-3β), a target of phosphorylated serine/threonine protein kinase B (p-Akt), regulates β-catenin stability. In addition, β-catenin, a coregulator of AR, physically interacts with AR and enhances AR-mediated target gene transcription. The multifunctional glycoprotein cluster of differentiation (CD) 147 is highly expressed on the cell surface of the majority of cancer cells, and it promotes tumor invasion, metastasis and growth. In the present study, the molecular effects of CD147 on the Akt/GSK-3β/β-catenin/AR signaling network were investigated in LNCaP cells. Using short hairpin-mediated RNA knockdown of CD147 in LNCaP cells, it was demonstrated that downregulation of CD147 resulted in inhibitory phosphorylation of GSK-3β, and then promoted degeneration of β-catenin and reduced nuclear accumulation of β-catenin. In addition, immunoprecipitation studies demonstrated that CD147 downregulation decreased the formation of a complex between β-catenin and AR. It was shown that CD147 knockdown suppressed the expression of the AR target gene prostate-specific antigen and the growth of AR-positive LNCaP cells. Furthermore, inhibition of PI3K/Akt with LY294002 augmented CD147-mediated function. The present study indicates that the PI3K/Akt pathway may facilitate CD147-mediated activation of the AR pathway.

Nek2 phosphorylates and stabilizes β-catenin at mitotic centrosomes downstream of Plk1

β-Catenin is a multifunctional protein with critical roles in cell-cell adhesion, Wnt signaling, and the centrosome cycle. Whereas the regulation of β-catenin in cell-cell adhesion and Wnt signaling are well understood, how β-catenin is regulated at the centrosome is not. NIMA-related protein kinase 2 (Nek2), which regulates centrosome disjunction/splitting, binds to and phosphorylates β-catenin. Using in vitro and cell-based assays, we show that Nek2 phosphorylates the same regulatory sites in the N-terminus of β-catenin as glycogen synthase kinase 3β (GSK3β), which are recognized by a specific phospho-S33/S37/T41 antibody, as well as additional sites. Nek2 binding to β-catenin appears to inhibit binding of the E3 ligase β-TrCP and prevents β-catenin ubiquitination and degradation. Thus β-catenin phosphorylated by Nek2 is stabilized and accumulates at centrosomes in mitosis. We further show that polo-like kinase 1 (Plk1) regulates Nek2 phosphorylation and stabilization of β-catenin. Taken together, these results identify a novel mechanism for regulating β-catenin stability that is independent of GSK3β and provide new insight into a pathway involving Plk1, Nek2, and β-catenin that regulates the centrosome cycle.

Tubulin polymerization promoting protein 1 (TPPP1) increases β-catenin expression through inhibition of HDAC6 activity in U2OS osteosarcoma cells

The Rho-associated coiled-coil kinase (ROCK) family of proteins, including ROCK1 and ROCK2, are key regulators of actin and intermediate filament morphology. The newly discovered ROCK substrate Tubulin polymerization promoting protein 1 (TPPP1) promotes microtubule polymerization and inhibits the activity of Histone deacetylase 6 (HDAC6). The effect of TPPP1 on HDAC6 activity is inhibited by ROCK signaling. Moreover, it was recently demonstrated that ROCK activity increases the cellular expression of the oncogene β-catenin, which is a HDAC6 substrate. In this study, we investigated the interplay between ROCK-TPPP1-HDAC6 signaling and β-catenin expression. We demonstrate that β-catenin expression is increased with ROCK signaling activation and is reduced with increased TPPP1 expression in U2OS cells. Further investigation revealed that ROCK-mediated TPPP1 phosphorylation, which prevents its binding to HDAC6, negates TPPP1-mediated reduction in β-catenin expression. We also show that increased HDAC6 activity resulting from ROCK signaling activation reduced β-catenin acetylation at Lys-49, which was also accompanied by its decreased phosphorylation by Caesin kinase 1 (CK1) and Glycogen synthase kinase 3β (GSK3β), thus preventing its proteasomal degradation. Overall, our results suggest that ROCK regulates β-catenin stability in cells via preventing TPPP1-mediated inhibition of HDAC6 activity, to reduce its acetylation and degradation via phosphorylation by CK1 and GSK3β.

Abstract 1000: Rad6B induced β-catenin stabilization requires intact Wnt signaling

Abstract Aberrant activation of Wnt signaling results from stabilization and transcriptional activation of β-catenin and is often associated with carcinogenesis. Clinical evidence suggests that β-catenin accumulation occurs in breast cancer although activating mutations are rare. We have previously shown that Rad6B positively regulates β-catenin stability and transcriptional activity in breast cancer cells by inducing stable polyubiquitin modifications. Here we show that Rad6B physically interacts with β-catenin within the β-catenin amino acid segment containing the first armadillo repeat, which is also a binding site for casein kinase 1 (CK1). β-catenin phosphorylation by GSK3β and subsequent ubiquitination and proteasomal degradation is dependent upon CK1-mediated initiating phosphorylation of β-catenin at Ser45. In vitro kinase assays showed that Rad6B inhibits Ser45 phosphorylation by CK1, and Ser45 phosphorylated β-catenin is decreased in Rad6B overexpressing breast cancer cells. In vitro and in vivo mapping, and site-directed mutation analysis identified K394 of β-catenin as the predominant site for ubiquitination by Rad6B. MCF-7 breast cancer cells engineered to express myc-tagged K394R-β-catenin showed decreased steady-state levels and β-catenin transcriptional activity as compared to cells expressing wild type β-catenin. We also determined whether Rad6B-mediated β-catenin stabilization occurs independently of the canonical Wnt signaling pathway. MDA-MB-231 breast cancer cells overexpressing endogenous Rad6B were isolated on the basis of Rad6B promoter activity, and the consequences of Wnt disruption on β-catenin levels, transcriptional activity and Rad6B expression were determined by expressing a dominant negative mutant of Wnt coreceptor, LRP6. Disruption of Wnt signaling inhibited Rad6B expression and β-catenin accumulation. Both Wnt signaling disruption and Rad6B silencing separately enhanced localization of β-catenin on the cell membranes and decreased intracellular β-catenin accumulation. In summary, our data suggest that the Rad6B overexpression frequently observed in breast carcinomas contribute to β-catenin stabilization and transcriptional activation by inhibiting the CK1-mediated initiating phosphorylation and by ubiquitinating K394. However, the Rad6B induced β-catenin stabilization requires intact Wnt signaling. Supported by DOD W81XWH07-1-0562. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1000.

Molecular association between β-catenin degradation complex and Rac guanine exchange factor DOCK4 is essential for Wnt/β-catenin signaling

The canonical Wnt/beta-catenin pathway is a highly conserved signaling cascade that is involved in development and stem cell renewal. The deregulation of this pathway is often associated with increased cell growth and neoplasia. The small GTPase Rac has been shown to influence canonical Wnt signaling by regulating beta-catenin stability through an unknown mechanism. We report that DOCK4, a guanine nucleotide exchange factor (GEF) for Rac and a member of the CDM family of unconventional GEFs, mediates Wnt-induced Rac activation in the canonical Wnt/beta-catenin pathway. DOCK4 expression regulates cellular beta-catenin levels in response to the Wnt signal, in vitro. Biochemical studies demonstrate that DOCK4 interacts with the beta-catenin degradation complex, consisting of the proteins adenomatosis polyposis coli, Axin and glycogen synthase kinase 3beta (GSK3beta). This molecular interaction enhances beta-catenin stability and Axin degradation. Furthermore, we observe that DOCK4 is phosphorylated by GSK3beta, which enhances Wnt-induced Rac activation. Using a T-cell factor reporter zebrafish we confirm that DOCK4 is required for Wnt/beta-catenin activity, in vivo. These results elucidate a novel intracellular signaling mechanism in which a Rac GEF, DOCK4 acts as a scaffold protein in the Wnt/beta-catenin pathway.

Presenilin 1 Independently Regulates β-Catenin Stability and Transcriptional Activity

Presenilin 1 (PS1) regulates beta-catenin stability; however, published data regarding the direction of the effect are contradictory. We examined the effects of wild-type and mutant forms of PS1 on the membrane, cytoplasmic, nuclear, and signaling pools of endogenous and exogenous beta-catenin by immunofluorescence microscopy, subcellular fractionation, and in a transcription assay. We found that PS1 destabilizes the cytoplasmic and nuclear pools of beta-catenin when stabilized by Wnt or Dvl but not when stabilized at lower levels of the Wnt pathway. The PS1 mutants examined were less able to reduce the stability of beta-catenin. PS1 also inhibited the transcriptional activity of endogenous beta-catenin, and the PS1 mutants were again less inhibitory at the level of Dvl but showed a different pattern of inhibition toward transcription below Dvl. The transcriptional activity of exogenously expressed wild-type beta-catenin and two mutants, DeltaN89beta-catenin and DeltaSTbeta-catenin, were also inhibited by wild-type and mutant PS1. We conclude that PS1 negatively regulates the stability and transcriptional activity of beta-catenin at different levels in the Wnt pathway, that the effect on transcriptional activity appears to be independent of the GSK-3beta mediated degradation of beta-catenin, and that mutations in PS1 differentially affect the stability and transcriptional activity of beta-catenin.

The Ubiquitin-Proteasome Pathway and Serine Kinase Activity Modulate Adenomatous Polyposis Coli Protein-mediated Regulation of β-Catenin-Lymphocyte Enhancer-binding Factor Signaling

The tumor suppressor function of the adenomatous polyposis coli protein (APC) depends, in part, on its ability to bind and regulate the multifunctional protein, beta-catenin. beta-Catenin binds the high mobility group box transcription factors, lymphocyte enhancer-binding factor (LEF) and T-cell factor, to directly regulate gene transcription. Using LEF reporter assays we find that APC-mediated down-regulation of beta-catenin-LEF signaling is reversed by proteasomal inhibitors in a dose-dependent manner. APC down-regulates signaling induced by wild type beta-catenin but not by the non-ubiquitinatable S37A mutant, beta-catenin. Bisindoylmaleimide-type protein kinase C inhibitors, which prevent beta-catenin ubiquitination, decrease the ability of APC to down-regulate beta-catenin-LEF signaling. All these effects on LEF signaling are paralleled by changes in beta-catenin protein levels. Lithium, an inhibitor of glycogen synthase kinase-3beta, does not alter the ability of APC to down-regulate beta-catenin protein and beta-catenin-LEF signaling in the colon cancer cells that were tested. These results point to a role for beta-catenin ubiquitination, proteasomal degradation, and potentially a serine kinase other than glycogen synthase kinase-3beta in the tumor-suppressive actions of APC.