Autophagy induction impairs Wnt/β-catenin signalling through β-catenin relocalisation in glioblastoma cells

Autophagy is an evolutionary conserved process mediating lysosomal degradation of cytoplasmic material. Its involvement in cancer progression is highly controversial, due to its dual role in both limiting tumoural transformation and in protecting established tumoral cells from unfavorable conditions. Little is known about the cross-talk between autophagy and intracellular signalling pathways, as well as about autophagy impact on signalling molecules turnover.An aberrantly activated Wnt/β-catenin signalling is responsible for tumour proliferation, invasion, and stemness maintenance. Here we show that autophagy negatively regulates Wnt/β-catenin signalling in glioblastoma multiforme (GBM) cells, through Dishevelled degradation. We also provide the first evidence that autophagy promotes β-catenin relocalisation within the cell, by inducing a decrease of the nuclear protein fraction. In particular, upon autophagy induction, β-catenin appears mainly localized in sub-membrane areas where it associates with N-cadherin to form epithelial-like cell-cell adhesion structures.Our data indicate, for the first time, that autophagy induction results in Wnt signalling attenuation and in β-catenin relocalisation within the GBM cell. These findings further support the idea that autophagy modulation could represent a potential therapeutical strategy to contrast GBM progression.     

Cell cycle control of Wnt/β-catenin signalling by conductin/axin2 through CDC20

Wnt/β-catenin signalling regulates cell proliferation by modulating the cell cycle and is negatively regulated by conductin/axin2/axil. We show that conductin levels peak at G2/M followed by a rapid decline during return to G1. In line with this, Wnt/β-catenin target genes are low at G2/M and high at G1/S, and β-catenin phosphorylation oscillates during the cell cycle in a conductin-dependent manner. Conductin is degraded by the anaphase-promoting complex/cyclosome cofactor CDC20. Knockdown of CDC20 blocks Wnt signalling through conductin. CDC20-resistant conductin inhibits Wnt signalling and attenuates colony formation of colorectal cancer cells. We propose that CDC20-mediated degradation of conductin regulates Wnt/β-catenin signalling for maximal activity during G1/S.     

Biphasic and dosage-dependent regulation of osteoclastogenesis by β-catenin

Wnt/β-catenin signaling is a critical regulator of skeletal physiology. However, previous studies have mainly focused on its roles in osteoblasts, while its specific function in osteoclasts is unknown. This is a clinically important question because neutralizing antibodies against Wnt antagonists are promising new drugs for bone diseases. Here, we show that in osteoclastogenesis, β-catenin is induced during the macrophage colony-stimulating factor (M-CSF)-mediated quiescence-to-proliferation switch but suppressed during the RANKL-mediated proliferation-to-differentiation switch. Genetically, β-catenin deletion blocks osteoclast precursor proliferation, while β-catenin constitutive activation sustains proliferation but prevents osteoclast differentiation, both causing osteopetrosis. In contrast, β-catenin heterozygosity enhances osteoclast differentiation, causing osteoporosis. Biochemically, Wnt activation attenuates whereas Wnt inhibition stimulates osteoclastogenesis. Mechanistically, β-catenin activation increases GATA2/Evi1 expression but abolishes RANKL-induced c-Jun phosphorylation. Therefore, β-catenin exerts a pivotal biphasic and dosage-dependent regulation of osteoclastogenesis. Importantly, these findings suggest that Wnt activation is a more effective treatment for skeletal fragility than previously recognized that confers dual anabolic and anti-catabolic benefits.     

RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling

The Wnt/β-catenin signalling pathway plays essential roles in embryonic development and adult tissue homeostasis, and deregulation of this pathway has been linked to cancer. Axin is a concentration-limiting component of the β-catenin destruction complex, and its stability is regulated by tankyrase. However, the molecular mechanism by which tankyrase-dependent poly(ADP-ribosyl)ation (PARsylation) is coupled to ubiquitylation and degradation of axin remains undefined. Here, we identify RNF146, a RING-domain E3 ubiquitin ligase, as a positive regulator of Wnt signalling. RNF146 promotes Wnt signalling by mediating tankyrase-dependent degradation of axin. Mechanistically, RNF146 directly interacts with poly(ADP-ribose) through its WWE domain, and promotes degradation of PARsylated proteins. Using proteomics approaches, we have identified BLZF1 and CASC3 as further substrates targeted by tankyrase and RNF146 for degradation. Thus, identification of RNF146 as a PARsylation-directed E3 ligase establishes a molecular paradigm that links tankyrase-dependent PARsylation to ubiquitylation. RNF146-dependent protein degradation may emerge as a major mechanism by which tankyrase exerts its function.     

AGR3 promotes the stemness of colorectal cancer via modulating Wnt/β-catenin signalling

Cancer cells with stem cell properties have been acknowledged to be responsible for cancer initiation and progression. Wnt/β-catenin signalling is a major signal pathway promoting the stemness of cancer cells. Anterior gradient 3 (AGR3), a member of the protein disulfide isomerase (PDI) family, was found to be overexpressed in several cancers. However, the roles and mechanisms of AGR3 in colorectal cancer (CRC) have not been previously described. In our study, we find that AGR3 is highly expressed in CRC and associated with poor prognosis. Functional studies show that AGR3 promotes the stemness of CRC cells. Mechanically, AGR3 activates Wnt/β-catenin signalling and promotes the nuclear translocation of β-catenin to upregulate stemness related genes. Wnt/β-catenin signalling inhibition counteracts the promoting effect of AGR3 on cancer stemness. Moreover, the effect of AGR3 on Wnt/β-catenin signalling and cancer stemness depends on the presence of frizzled 4 (FZD4). Thus, our study first uncovers the stemness-promoting role and the oncogenic mechanism of AGR3 in CRC, which might provide a novel target for designing anti-CRC strategies.     

Hsp90α/β associates with the GSK3β/axin1/phospho-β-catenin complex in the human MCF-7 epithelial breast cancer model

Hsp90α/β, the signal transduction chaperone, maintains intracellular communication in normal, stem, and cancer cells. The well characterised association of Hsp90α/β with its client kinases form the framework of multiple signalling networks. GSK3β, a known Hsp90α/β client, mediates β-catenin phosphorylation as part of a cytoplasmic destruction complex which targets phospho-β-catenin to the 26S proteasome. The canonical Wnt/β-catenin pathway promotes stem cell self-renewal as well as oncogenesis. The degree of Hsp90α/β involvement in Wnt/β-catenin signalling needs clarification. Here, we describe the association of Hsp90α/β with GSK3β, β-catenin, phospho-β-catenin and the molecular scaffold, axin1, in the human MCF-7 epithelial breast cancer cell model using selective inhibition of Hsp90α/β, confocal laser scanning microscopy and immunoprecipitation. Our findings suggest that Hsp90α/β modulates the phosphorylation of β-catenin by interaction in common complex with GSK3β/axin1/β-catenin.     

LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling

R-spondins are secreted Wnt signalling agonists, which regulate embryonic patterning and stem cell proliferation, but whose mechanism of action is poorly understood. Here we show that R-spondins bind to the orphan G-protein-coupled receptors LGR4 and LGR5 by their Furin domains. Gain- and loss-of-function experiments in mammalian cells and Xenopus embryos indicate that LGR4 and LGR5 promote R-spondin-mediated Wnt/β-catenin and Wnt/PCP signalling. R-spondin-triggered β-catenin signalling requires Clathrin, while Wnt3a-mediated β-catenin signalling requires Caveolin-mediated endocytosis, suggesting that internalization has a mechanistic role in R-spondin signalling.     

Wnt/β-catenin signaling pathway may regulate cell cycle and expression of cyclin A and cyclin E protein in hepatocellular carcinoma cells

Wnt/β-catenin signaling pathway and cell cycle play the key roles during the genesis and development of hepatocellular carcinoma (HCC). The cytoplasmic protein β-catenin is a multifunctional protein and a central molecule in the Wnt signaling pathway. Cell cycle is regulated by a a series of regulatory factors. Current researches indicated that expression of cyclin D1 and c-myc decreased after silencing β-catenin gene in HCC, but it is unclear if other cyclins are affected. To determine the relation, small interference RNA(siRNA) against β-catenin was transfected into HCC cell line HepG2, and cell cycle and cyclin A and cyclin E protein expression were detected. We demonstrated that cell cycle was arrested in G0/G1 at 72 h after the transfection and with the time passing, the cell cycle began to transfer from G0/G1 to G2/M through S and had a trend to revert at 96 h. In addition, β-catenin protein expression was decreased at both 72 and 96 h, although the level was slightly higher at 96 h than that at 72 h. However, cyclin A and cyclin E protein expression increased at 72 h and decreased at 96 h. These findings suggest that silencing β-catenin gene may induce the changes of cell cycle and cyclin A and cyclin E expression. Wnt/β-catenin signaling pathway probably takes part in the genesis and development of HCC through regulating cell cycle and the expression of cyclin A and cyclin E.     

Epidermal growth factor can signal via β-catenin to control proliferation of mesenchymal stem cells independently of canonical Wnt signalling

Bone marrow mesenchymal stem/stromal cells (MSCs) maintain bone homeostasis and repair through the ability to expand in response to mitotic stimuli and differentiate into skeletal lineages. Signalling mechanisms that enable precise control of MSC function remain unclear. Here we report that by initially examining differences in signalling pathway expression profiles of individual MSC clones, we identified a previously unrecognised signalling mechanism regulated by epidermal growth factor (EGF) in primary human MSCs. We demonstrate that EGF is able to activate β-catenin, a key component of the canonical Wnt signalling pathway. EGF is able to induce nuclear translocation of β-catenin in human MSCs but does not drive expression of Wnt target genes or T cell factor (TCF) activity in MSC reporter cell lines. Using an efficient Design of Experiments (DoE) statistical analysis, with different combinations and concentrations of EGF and Wnt ligands, we were able to confirm that EGF does not influence the Wnt/β-catenin pathway in MSCs. We show that the effects of EGF on MSCs are temporally regulated to initiate early “classical” EGF signalling mechanisms (e.g via mitogen activated protein kinase) with delayed activation of β-catenin. By RNA-sequencing, we identified gene sets that were exclusively regulated by the EGF/β-catenin pathway, which were distinct from classical EGF-regulated genes. However, subsets of classical EGF gene targets were significantly influenced by EGF/β-catenin activation. These signalling pathways cooperate to enable EGF-mediated proliferation of MSCs by alleviating the suppression of cell cycle pathways induced by classical EGF signalling.     

Opposing effects of Tcf3 and Tcf1 control Wnt stimulation of embryonic stem cell self-renewal

The co-occupancy of Tcf3 with Oct4, Sox2 and Nanog on embryonic stem cell (ESC) chromatin indicated that Tcf3 has been suggested to play an integral role in a poorly understood mechanism underlying Wnt-dependent stimulation of mouse ESC self-renewal of mouse ESCs. Although the conventional view of Tcf proteins as the β-catenin-binding effectors of Wnt signalling suggested Tcf3-β-catenin activation of target genes would stimulate self-renewal, here we show that an antagonistic relationship between Wnt3a and Tcf3 on gene expression regulates ESC self-renewal. Genetic ablation of Tcf3 replaced the requirement for exogenous Wnt3a or GSK3 inhibition for ESC self-renewal, demonstrating that inhibition of Tcf3 repressor is the necessary downstream effect of Wnt signalling. Interestingly, both Tcf3-β-catenin and Tcf1-β-catenin interactions contributed to Wnt stimulation of self-renewal and gene expression, and the combination of Tcf3 and Tcf1 recruited Wnt-stabilized β-catenin to Oct4 binding sites on ESC chromatin. This work elucidates the molecular link between the effects of Wnt and the regulation of the Oct4/Sox2/Nanog network.     

A genetic study of the role of the Wnt/beta-catenin signalling in Paneth cell differentiation

Wnt/β-catenin signalling plays a key role in the homeostasis of the intestinal epithelium. Whereas its role in the maintenance of the stem cell compartment has been clearly demonstrated, its role in the Paneth cell fate remains unclear. We performed genetic studies to elucidate the functions of the Wnt/β-catenin pathway in Paneth cell differentiation. We analysed mice with inducible gain-of-function mutations in the Wnt/β-catenin pathway and mice with a hypomorphic β-catenin allele that have not been previously described. We demonstrated that acute activation of Wnt/β-catenin signalling induces de novo specification of Paneth cells in both the small intestine and colon and that colon cancers resulting from Apc mutations expressed many genes involved in Paneth cell differentiation. This suggests a key role for the Wnt/β-catenin pathway in Paneth cell differentiation. We also showed that a slight decrease in β-catenin gene dosage induced a major defect in Paneth cell differentiation, but only a modest effect on crypt morphogenesis. Overall, our findings show that a high level of β-catenin activation is required to determine Paneth cell fate and that fine tuning of β-catenin signalling is critical for correct Paneth cell lineage.