Phytochemicals Attenuating Aberrant Activation of ��-Catenin in Cancer Cells

Phytochemicals are a rich source of chemoprevention agents but their effects on modulating the Wnt/β-catenin signaling pathway have remained largely uninvestigated. Aberrantly activated Wnt signaling can result in the abnormal stabilization of β-catenin, a key causative step in a broad spectrum of cancers. Here we report the modulation of lithium chloride-activated canonical Wnt/β-catenin signaling by phytochemicals that have antioxidant, anti-inflammatory or chemopreventive properties. The compounds were first screened with a cervical cancer-derived stable Wnt signaling reporter HeLa cell line. Positive hits were subsequently evaluated for β-catenin degradation, suppression of β-catenin nuclear localization and down-regulation of downstream oncogenic targets of Wnt/β-catenin pathway. Our study shows a novel degradation path of β-catenin protein in HeLa cells by Avenanthramide 2p (a polyphenol) and Triptolide (a diterpene triepoxide), respectively from oats and a Chinese medicinal plant. The findings present Avenanthramide 2p as a potential chemopreventive dietary compound that merits further study using in vivo models of cancers; they also provide a new perspective on the mechanism of action of Triptolide.     

Interactions of Plakoglobin and ��-Catenin with Desmosomal Cadherins: BASIS OF SELECTIVE EXCLUSION OF ��- AND ��-CATENIN FROM DESMOSOMES*

Plakoglobin and β-catenin are homologous armadillo repeat proteins found in adherens junctions, where they interact with the cytoplasmic domain of classical cadherins and with α-catenin. Plakoglobin, but normally not β-catenin, is also a structural constituent of desmosomes, where it binds to the cytoplasmic domains of the desmosomal cadherins, desmogleins and desmocollins. Here, we report structural, biophysical, and biochemical studies aimed at understanding the molecular basis of selective exclusion of β-catenin and α-catenin from desmosomes. The crystal structure of the plakoglobin armadillo domain bound to phosphorylated E-cadherin shows virtually identical interactions to those observed between β-catenin and E-cadherin. Trypsin sensitivity experiments indicate that the plakoglobin arm domain by itself is more flexible than that of β-catenin. Binding of plakoglobin and β-catenin to the intracellular regions of E-cadherin, desmoglein1, and desmocollin1 was measured by isothermal titration calorimetry. Plakoglobin and β-catenin bind strongly and with similar thermodynamic parameters to E-cadherin. In contrast, β-catenin binds to desmoglein-1 more weakly than does plakoglobin. β-Catenin and plakoglobin bind with similar weak affinities to desmocollin-1. Full affinity binding of desmoglein-1 requires sequences C-terminal to the region homologous to the catenin-binding domain of classical cadherins. Although pulldown assays suggest that the presence of N- and C-terminal β-catenin “tails” that flank the armadillo repeat region reduces the affinity for desmosomal cadherins, calorimetric measurements show no significant effects of the tails on binding to the cadherins. Using purified proteins, we show that desmosomal cadherins and α-catenin compete directly for binding to plakoglobin, consistent with the absence of α-catenin in desmosomes.     

Functional Cross-talk between ��-Catenin and NF��B Signaling Pathways in Colonic Crypts of Mice in Response to Progastrin

We recently reported a critical role of NFκB in mediating hyperproliferative and anti-apoptotic effects of progastrin on proximal colonic crypts of transgenic mice overexpressing progastrin (Fabp-PG mice). We now report activation of β-catenin in colonic crypts of mice in response to chronic (Fabp-PG mice) and acute (wild type FVB/N mice) progastrin stimulation. Significant increases were measured in relative levels of cellular and nuclear β-catenin and pβ-cat⁴⁵ in proximal colonic crypts of Fabp-PG mice compared with that in wild type littermates. Distal colonic crypts were less responsive. Interestingly, β-catenin activation was downstream of IKKα,β/NFκB, because treatment of Fabp-PG mice with the NFκB essential modulator (NEMO) peptide (inhibitor of IKKα,β/NFκB activation) significantly blocked increases in cellular/nuclear levels of total β-catenin/pβ-cat⁴⁵/and pβ-cat⁵⁵² in proximal colons. Cellular levels of pβ-cat^33,37,41, however, increased in proximal colons in response to NEMO, probably because of a significant increase in pGSK-3β^Tyr216, facilitating degradation of β-catenin. NEMO peptide significantly blocked increases in cyclin D1 expression, thereby, abrogating hyperplasia of proximal crypts. Goblet cell hyperplasia in colonic crypts of Fabp-PG mice was abrogated by NEMO treatment, suggesting a cross-talk between the NFκB/β-catenin and Notch pathways. Cellular proliferation and crypt lengths increased significantly in proximal but not distal crypts of FVB/N mice injected with 1 nm progastrin associated with a significant increase in cellular/nuclear levels of total β-catenin and cyclin D1. Thus, intracellular signals, activated in response to acute and chronic stimulation with progastrin, were similar and specific to proximal colons. Our studies suggest a novel possibility that activation of β-catenin, downstream to the IKKα,β/NFκB pathway, may be integral to the hyperproliferative effects of progastrin on proximal colonic crypts.Accumulating evidence suggests that gastrins play an important role in proliferation and carcinogenesis of gastrointestinal and pancreatic cancers (1, 2). Progastrin and glycine-extended gastrin (G-Gly)³ are predominant forms of gastrins found in many tumors, including colon (3–5). Progastrin exerts potent proliferative and anti-apoptotic effects in vitro and in vivo on intestinal mucosal cells (6–10) and on pancreatic cancer cells (11). Transgenic mice overexpressing progastrin from either the liver (hGAS) or intestinal epithelial cells (Fabp-PG) are at a higher risk for developing pre-neoplastic and neoplastic lesions in colons in response to azoxymethane (12–15). Treatment with G-Gly similarly increased the risk for developing pre-neoplastic lesions in rats (16). Thus progastrin and G-Gly exert co-carcinogenic effects in vivo (12–16).Under physiological conditions, only processed forms of gastrins (G17, G34) are present in the circulation (17). In certain disease states, elevated levels of circulating progastrin (0.1 to >1.0 nm) are measured (1). Because co-carcinogenic effects of progastrin are measured in Fabp-PG mice, which express pathophysiological concentrations of hProgastrin (<1–5 nm) (12), elevated levels of circulating progastrin measured in certain disease states in humans may play a role in colon carcinogenesis. A curious finding was that pre-neoplastic and neoplastic lesions were significantly increased in proximal, but not distal, colons of Fabp-PG mice, in response to azoxymethane (12, 14), which may reflect an increase in proliferation and a decrease in azoxymethane-induced apoptosis in proximal colons of Fabp-PG mice (18). We reported a critical role of NFκB activation in mediating proliferation and the anti-apoptotic effect of progastrin on pancreatic cancer cells (in vitro) and on proximal colonic crypts of Fabp-PG mice (in vivo) (11, 18). Whereas the Wnt/β-catenin pathway is known to play a role in the proliferation of colonic crypts (19), its role in mediating biological effects of progastrin remains unknown.β-Catenin is regulated by canonical (GSK-3β phosphorylation-dependent) and non-canonical (GSK-3β phosphorylation-independent) pathways. In the canonical pathway, inhibition of GSK-3β protects β-catenin against degradation by protein complexes, consisting of GSK-3β, axin, and adenomatous polyposis coli (20). In a resting cell, β-catenin is not present in the cytoplasm or nucleus because of proteasomal degradation of β-catenin that is not bound to E-cadherin (20). Following inactivation of GSK-3β, β-catenin stabilizes in the cytoplasm and translocates to the nucleus where it cooperates with Tcf/Lef for activation of target genes (20). In the current studies, we examined whether β-catenin is activated in proximal versus distal colonic crypts of Fabp-PG mice. Relative levels of β-catenin and its target gene product, cyclin D1, were significantly increased in proximal versus distal colonic crypts of Fabp-PG mice. We next examined a possible cross-talk between NFκB and β-catenin activation and the role of GSK-3β. Our results suggest the novel possibility that β-catenin activation in response to progastrin is downstream to IKKα,β/NFκB p65 activation, and that phosphorylation of GSK-3β at Tyr²¹⁶ may be critically involved.To examine whether differences measured in the response of proximal versus distal colons in Fabp-PG mice were not an artifact of chronic stimulation, we additionally injected WT FVB/N mice with progastrin, as an acute model of stimulation. Our results confirmed that differences we had measured in Fabp-PG mice are not an artifact of chronic stimulation but represent inherent differences in the response of proximal versus distal colonic crypts to circulating progastrins.We and others (18, 21) have previously demonstrated goblet cell hyperplasia in colonic crypts of transgenic mice overexpressing progastrin. In the current studies, we confirmed a significant increase in goblet cell hyperplasia/metaplasia (?) in proximal colonic crypts of Fabp-PG mice. Importantly, goblet cell hyperplasia was reversed to wild type levels by attenuating NFκB activation (and hence β-catenin activation) in NEMO-treated mice. The results of the current studies thus further suggest that pathways which dictate goblet cell lineage may be modulated by progastrin and may be downstream of NFκB/β-catenin activation. This represents a novel paradigm, which needs to be further examined.     

SPARC Suppresses Apoptosis of Idiopathic Pulmonary Fibrosis Fibroblasts through Constitutive Activation of ��-Catenin

Idiopathic pulmonary fibrosis (IPF) is a poorly understood progressive disease characterized by the accumulation of scar tissue in the lung interstitium. A hallmark of the disease is areas of injury to type II alveolar epithelial cells with attendant accumulation of fibroblasts in areas called fibroblastic foci. In an effort to better characterize the lung fibroblast phenotype in IPF patients, we isolated fibroblasts from patients with IPF and looked for activation of signaling proteins, which could help explain the exaggerated fibrogenic response in IPF. We found that IPF fibroblasts constitutively expressed increased basal levels of SPARC, plasminogen activator inhibitor-1 (PAI-1), and active β-catenin compared with control cells. Control of basal PAI-1 expression in IPF fibroblasts was regulated by SPARC-mediated activation of Akt, leading to inhibition of glycogen synthase kinase-3β and activation of β-catenin. Additionally, IPF fibroblasts (but not control fibroblasts) were resistant to plasminogen-induced apoptosis and were sensitized to plasminogen-mediated apoptosis by inhibition of SPARC or β-catenin. These findings uncover a newly discovered regulatory pathway in IPF fibroblasts that is characterized by elevated SPARC, giving rise to activated β-catenin, which regulates expression of downstream genes, such as PAI-1, and confers an apoptosis-resistant phenotype. Disruption of this pathway may represent a novel therapeutic target in IPF.     

Loss of ��-Catenin Decreases the Strength of Single E-cadherin Bonds between Human Cancer Cells

The progression of several human cancers correlates with the loss of cytoplasmic protein α-catenin from E-cadherin-rich intercellular junctions and loss of adhesion. However, the potential role of α-catenin in directly modulating the adhesive function of individual E-cadherin molecules in human cancer is unknown. Here we use single-molecule force spectroscopy to probe the tensile strength, unstressed bond lifetime, and interaction energy between E-cadherins expressed on the surface of live human parental breast cancer cells lacking α-catenin and these cells where α-catenin is re-expressed. We find that the tensile strength and the lifetime of single E-cadherin/E-cadherin bonds between parental cells are significantly lower over a wide range of loading rates. Statistical analysis of the force displacement spectra reveals that single cadherin bonds between cancer cells feature an exceedingly low energy barrier against tensile forces and low molecular stiffness. Disassembly of filamentous actin using latrunculin B has no significant effect on the strength of single intercellular E-cadherin bonds. The absence of α-catenin causes a dominant negative effect on both global cell-cell adhesion and single E-cadherin bond strength. These results suggest that the loss of α-catenin alone drastically reduces the adhesive force between individual cadherin pairs on adjoining cells, explain the global loss of cell adhesion in human breast cancer cells, and show that the forced expression of α-catenin in cancer cells can restore both higher intercellular avidity and intercellular E-cadherin bond strength.The reduction of intercellular adhesion in a solid tumor is a critical step in the progression of tumor cells to metastasis (1). How normal cells lose their ability to form strong adhesions within a tissue is not well understood (2, 3). The loss of adhesion between adjoining epithelial cells and the ensuing onset of metastasis occur through an epithelial-to-mesenchymal transition that often correlates with the loss of cytoplasmic protein α-catenin and a poor prognosis in a wide range of cancers, including breast (4), esophageal (5), gastric (6, 7), cervical (8), and colorectal cancer (9). In normal epithelial tissues, α-catenin localizes to junctions that organize at the interface between adjacent epithelial cells through clustering of cell surface adhesion transmembrane molecule cadherin and its association to the cytoskeleton (10, 11). On the extracellular side, structural studies suggest that cadherin molecules form molecular pairs that interact with cadherin pairs on an adjacent cell through their distal Ca²⁺-binding domains (12). On the intracellular side, cadherin pairs are connected to the cytoskeleton network through specific linker proteins. Until recently it was believed that one critical linker protein between the cytoplasmic domain of cadherin and the actin cytoskeleton was α-catenin, because it can both bind filamentous actin (F-actin) and E-cadherin through β-catenin (13, 14). However, a recent study indicates that α-catenin can either bind the E-cadherin-β-catenin complex as monomer or cross-link actin filaments as homodimer but cannot bind both E-cadherin-β-catenin and F-actin simultaneously (15). Therefore, whether the loss of α-catenin plays a direct role in the loss of adhesion in human cancer cells is unclear.Our recent data using engineered Chinese hamster ovarian cells suggest that α-catenin mediates the rapid strengthening of individual intercellular E-cadherin/E-cadherin bonds following initial molecular recognition between cells bearing E-cadherin molecules (16). Furthermore, α-catenin mediates the formation of additional E-cadherin/E-cadherin bonds once a first bond is formed between adjoining cells to form a nascent intercellular junction (16). Here we hypothesize that the loss of cytoplasmic protein α-catenin in human cancer cells greatly affects the ability of E-cadherin molecules on the surface of these cells to form firm adhesion by reducing the strength of individual intercellular E-cadherin/E-cadherin bonds.Our strategy is to compare parental breast cancer cells that lack α-catenin (MDA-MB-468 cells; denoted here MDA468) with these cells when α-catenin is introduced and exploit high resolution live cell single-molecule force spectroscopy (17) to probe the strength of individual E-cadherin/E-cadherin bonds between adjacent cells (18). The cells are juxtaposed for a controlled time of contact, the probability of successful interactions is subsequently measured, and the mechanical properties (tensile strength, molecular stiffness, and reactive compliance) and biochemical properties (interaction energy, dissociation rate, and bond lifetime) of single intercellular E-cadherin/E-cadherin bonds are analyzed.Our main hypothesis cannot be readily tested using purified proteins. Our ability to measure molecular interactions between live cells (17) rather than recombinant proteins ensures that the proper orientation of cadherin on the cell surfaces and its post-translational modifications are physiological. Moreover, using living cells ensures that the cytoplasmic domain of transmembrane receptors (here human E-cadherin) can interact with cytoplasmic proteins (in particular β-catenin and α-catenin), thereby allowing cell signaling pathways that can influence cell adhesion to function normally.     

GSK-3 Phosphorylates ��-Catenin and Negatively Regulates Its Stability via Ubiquitination/Proteosome-mediated Proteolysis

δ-Catenin was first identified because of its interaction with presenilin-1, and its aberrant expression has been reported in various human tumors and in patients with Cri-du-Chat syndrome, a form of mental retardation. However, the mechanism whereby δ-catenin is regulated in cells has not been fully elucidated. We investigated the possibility that glycogen-synthase kinase-3 (GSK-3) phosphorylates δ-catenin and thus affects its stability. Initially, we found that the level of δ-catenin was greater and the half-life of δ-catenin was longer in GSK-3β^−/− fibroblasts than those in GSK-3β^+/+ fibroblasts. Furthermore, four different approaches designed to specifically inhibit GSK-3 activity, i.e. GSK-3-specific chemical inhibitors, Wnt-3a conditioned media, small interfering RNAs, and GSK-3α and -3β kinase dead constructs, consistently showed that the levels of endogenous δ-catenin in CWR22Rv-1 prostate carcinoma cells and primary cortical neurons were increased by inhibiting GSK-3 activity. In addition, it was found that both GSK-3α and -3β interact with and phosphorylate δ-catenin. The phosphorylation of ΔC207-δ-catenin (lacking 207 C-terminal residues) and T1078A δ-catenin by GSK-3 was noticeably reduced compared with that of wild type δ-catenin, and the data from liquid chromatography-tandem mass spectrometry analyses suggest that the Thr¹⁰⁷⁸ residue of δ-catenin is one of the GSK-3 phosphorylation sites. Treatment with MG132 or ALLN, specific inhibitors of proteosome-dependent proteolysis, increased δ-catenin levels and caused an accumulation of ubiquitinated δ-catenin. It was also found that GSK-3 triggers the ubiquitination of δ-catenin. These results suggest that GSK-3 interacts with and phosphorylates δ-catenin and thereby negatively affects its stability by enabling its ubiquitination/proteosome-mediated proteolysis.δ-Catenin was first identified as a molecule that interacts with presenilin-1 (PS-1)² by yeast two-hybrid assay (1) and was found to belong to the p120-catenin subfamily of armadillo proteins, which characteristically contain 10 Arm repeats (2). In addition to its interaction with PS-1 and its abundant expression in brain (3, 4), several lines of evidence indicate that δ-catenin may play a pivotal role in cognitive function. First, the hemizygous loss of δ-catenin is known to be closely correlated with Cri-du-Chat syndrome, a severe form of mental retardation in humans (5). Second, severe learning deficits and abnormal synaptic plasticity were found in δ-catenin-deficient mice (6). Moreover, in δ-catenin^−/− mice, paired pulse facilitation (a form of short term plasticity) was found to be reduced, and long term potentiation, which is related to the forming and storage mechanisms of memory, was deficient (7, 8). Third, δ-catenin interacting molecules, such as PSs (1, 9), cadherins (10), S-SCAM (2), and PSD-95 (11), have been shown to play important roles in modulating synaptic plasticity. However, even though the maintenance of an adequate δ-catenin level is known to be critical for normal brain function, few studies have been undertaken to identify the factors that regulate δ-catenin stability in cells. We have previously demonstrated that PS-1 inhibits δ-catenin-induced cellular branching and promotes δ-catenin processing and turnover (12).Because of structural similarities among β-catenin, p120-catenin, and δ-catenin and to their shared binding partners (i.e. PS-1 (1, 9) and cadherins (10)), glycogen-synthase kinase-3 (GSK-3) drew our attention as a potential candidate effector of δ-catenin stability in cells. GSK-3 is a serine/threonine kinase and has two highly homologous forms, GSK-3α and GSK-3β, in mammals (13). Although GSK-3α and GSK-3β have similar structures, they differ in mass (GSK-3α (51 kDa) and GSK-3β (47 kDa) (13)) and to some extent in function (14). GSK-3 is a well established inhibitor of Wnt signaling. Moreover, it is known to phosphorylate β-catenin, which results in its degradation via ubiquitination/proteosome-dependent proteolysis (15). GSK-3 is ubiquitously distributed in the human body, but it is particularly abundant in brain (13), and it is interesting that δ-catenin is also abundant in the nervous system (4) and that GSK-3 participates in the progression of Alzheimer disease (16). The majority of GSK-3 substrates have the consensus sequence (Ser/Thr)-Xaa-Xaa-Xaa-(Ser/Thr) (17). Interestingly, we found that δ-catenin has several putative phosphorylation sites targeted by GSK-3, which suggests that δ-catenin can be regulated by GSK-3 in the same way as β-catenin.In this report, we demonstrate that both GSK-3α and -3β interact with and phosphorylate δ-catenin and that this leads to its subsequent ubiquitination and degradation via proteosome-dependent proteolysis. Our results strongly suggest that GSK-3 is a key regulator of δ-catenin stability in cells.