Distribution of the cadherin-catenin complex in normal human thyroid epithelium and a thyroid carcinoma cell line

E-cadherin is the major cell-cell adhesion molecule expressed by epithelial cells. Cadherins form a complex with three cytoplasmic proteins, α-, β-, and γ-catenin, and the interaction between them is crucial for anchoring the actin cytoskeleton to the intercellular adherens junctions. The invasive behavior of cancer cells has been attributed to a dysfunction of these molecules. In this study, we examined the distribution of the cadherin-catenin complex in a Chinese human thyroid cancer cell line, CGTH W-2, compared with that in normal human thyroid epithelial cells. In the normal cells, using immunofluorescence staining, E-cadherin and α-, β-, and γ-catenin were found to be localized at the intercellular junction and appeared as 135, 102, 90, and 80 kD proteins on Western blots. In CGTH W-2 cells, no E-cadherin and γ-catenin immunoreactivity was detected by immunofluorescence or Western blotting; α- and β-catenin were detected as 102 and 90 kD proteins on blots but gave a diffuse cytoplasmic immunofluorescence staining pattern in most cells, while β-catenin was also distributed throughout the cytoplasm in most cells but was found at the cell junction in some, where it colocalized with α-actinin. The present data indicate that the loss of cell adhesiveness in these cancer cells may be due to incomplete assembly of the cadherin-catenin complex at the cell junction. However, this defect did not affect the linkage of actin bundles to vinculin-enriched intercellular junctions. J. Cell. Biochem. 70:330–337, 1998. © 1998 Wiley-Liss, Inc.     

Oligomerization of peptides analogous to the cytoplasmic domains of coatomer receptors revealed by mass spectrometry

Members of the p24 family of type I transmembrane proteins are involved in budding of coat protein type I (COPI)-coated vesicles. They serve as coat protein receptors, binding via their cytoplasmic domains to coatomer, a stable cytosolic protein complex that represents the major coat component of these vesicles. Experimental evidence suggest that p23, a member of the p24 family, binds to coatomer in an oligomeric state and that this binding triggers polymerization of the coat protein. Toward an understanding of this process at the molecular level, formation of noncovalent complexes and their relative stabilities were analyzed by Fourier transform ion cyclotron resonance mass spectrometry using nanoelectrospray ionization. Specificity and stability of oligomers formed were established to depend on characteristic peptide sequence motifs and were confirmed by mass spectrometric competition experiments with control peptides. Mutations in the peptide sequence caused decreased interaction and destabilization of the noncovalent complexes. The formation and relative stabilities of dimeric and tetrameric complexes were assessed to be formed by cytoplasmic tails of coatomer receptors. The direct molecular identification provided by mass spectrometry correlates well with biochemical results. Thus, electrospray ionization mass spectrometry proves to be a powerful tool to investigate physiologically relevant peptide complexes.     

KCTD11 inhibits progression of lung cancer by binding to β-catenin to regulate the activity of the Wnt and Hippo pathways

KCTD11 has been reported to be a potential tumour suppressor in several tumour types. However, the expression of KCTD11 and its role has not been reported in human non-small cell lung cancer (NSCLC). Whether its potential molecular mechanism is related to its BTB domain is also unknown. The expression of KCTD11 in 139 NSCLC tissue samples was detected by immunohistochemistry, and its correlation with clinicopathological factors was analysed. The effect of KCTD11 on the biological behaviour of lung cancer cells was verified in vitro and in vivo. Its effect on the epithelial-mesenchymal transition(EMT)process and the Wnt/β-catenin and Hippo/YAP pathways were observed by Western blot, dual-luciferase assay, RT-qPCR, immunofluorescence and immunoprecipitation. KCTD11 is under-expressed in lung cancer tissues and cells and was negatively correlated with the degree of differentiation, tumour-node-metastasis (TNM) stage and lymph node metastasis. Low KCTD11 expression was associated with poor prognosis. KCTD11 overexpression inhibited the proliferation and migration of lung cancer cells. Further studies indicated that KCTD11 inhibited the Wnt pathway, activated the Hippo pathway and inhibited EMT processes by inhibiting the nuclear translocation of β-catenin and YAP. KCTD11 lost its stimulatory effect on the Hippo pathway after knock down of β-catenin. These findings confirm that KCTD11 inhibits β-catenin and YAP nuclear translocation as well as the malignant phenotype of lung cancer cells by interacting with β-catenin. This provides an important experimental basis for the interaction between KCTD11, β-catenin and YAP, further revealing the link between the Wnt and Hippo pathways.     

KHC-4 inhibits β-catenin expression in prostate cancer cells

ABSTRACT

KHC-4 is a 2-phenyl-4-quinolone analogue that exhibits anticancer activity. Aberrant activation of β-catenin signaling contributes to prostate cancer development and progression. Therefore, targeting β-catenin expression could be a useful approach to treating prostate cancer. We found that KHC-4 can inhibit β-catenin expression and its signaling pathway in DU145 prostate cancer cells. Treatment with KHC-4 decreased total β-catenin expression and concomitantly decreased β-catenin levels in both the cytoplasm and nucleus of cells. KHC-4 treatment also inhibited β-catenin expression and that of its target proteins, PI3K, AKT, GSK3β and TBX3. We monitored the stability of β-catenin with the proteasomal inhibitor, MG132, in DU145 cells and found that MG132 reversed KHC-4-induced proteasomal β-catenin degradation. We verified CDK1/β-catenin expression in KHC-4 treated DU145 cells. We found that roscovitine treatment reversed cell proliferation by arresting the cell cycle at the G2/M phase and β-catenin expression caused by KHC-4 treatment. We suggest that KHC-4 inhibits β-catenin signaling in DU145 prostate cancer cells.     

AlphaScreen selectivity assay for β-catenin/B-cell lymphoma 9 inhibitors

The aberrant formation of the β-catenin/B-cell lymphoma 9 (BCL9) protein–protein complex is the driving force for many diseases, including cancer. Crystallographic analyses demonstrate that the surface area in β-catenin for interacting with BCL9 is overlapped with that for the β-catenin/E-cadherin interaction. In this study, a robust AlphaScreen selectivity assay was developed to quantify inhibitor potency for the β-catenin/BCL9 interaction and selectivity for β-catenin/BCL9 over β-catenin/E-cadherin interactions. A pilot screen was performed to demonstrat the feasibility of this assay. This selectivity assay is highly sensitive and suitable for adaptation to high-throughput screening. The establishment of this assay lays the foundation for the discovery of selective inhibitors specific for β-catenin/BCL9 interactions.     

Oncogenic function of DACT1 in colon cancer through the regulation of β-catenin

The Wnt/β-catenin signaling pathway plays important roles in the progression of colon cancer. DACT1 has been identified as a modulator of Wnt signaling through its interaction with Dishevelled (Dvl), a central mediator of both the canonical and noncanonical Wnt pathways. However, the functions of DACT1 in the WNT/β-catenin signaling pathway remain unclear. Here, we present evidence that DACT1 is an important positive regulator in colon cancer through regulating the stability and sublocation of β-catenin. We have shown that DACT1 promotes cancer cell proliferation in vitro and tumor growth in vivo and enhances the migratory and invasive potential of colon cancer cells. Furthermore, the higher expression of DACT1 not only increases the nuclear and cytoplasmic fractions of β-catenin, but also increases its membrane-associated fraction. The overexpression of DACT1 leads to the increased accumulation of nonphosphorylated β-catenin in the cytoplasm and particularly in the nuclei. We have demonstrated that DACT1 interacts with GSK-3β and β-catenin. DACT1 stabilizes β-catenin via DACT1-induced effects on GSK-3β and directly interacts with β-catenin proteins. The level of phosphorylated GSK-3β at Ser9 is significantly increased following the elevated expression of DACT1. DACT1 mediates the subcellular localization of β-catenin via increasing the level of phosphorylated GSK-3β at Ser9 to inhibit the activity of GSK-3β. Taken together, our study identifies DACT1 as an important positive regulator in colon cancer and suggests a potential strategy for the therapeutic control of the β-catenin-dependent pathway.     

Structure-based discovery of a novel inhibitor targeting the β-catenin/Tcf4 interaction

Overactivation or overexpression of β-catenin in the Wnt (wingless) signaling pathway plays an important role in tumorigenesis. Interaction of β-catenin with T-cell factor (Tcf) DNA binding proteins is a key step in the activation of the proliferative genes in response to upstream signals of this Wnt/β-catenin pathway. Recently, we identified a new small molecule inhibitor, named BC21 (C(32)H(36)Cl(2)Cu(2)N(2)O(2)), which effectively inhibits the binding of β-catenin with Tcf4-derived peptide and suppresses β-catenin/Tcf4 driven reporter gene activity. This inhibitor decreases the viability of β-catenin overexpressing HCT116 colon cancer cells that harbor the β-catenin mutation, and more significantly, it inhibits the clonogenic activity of these cells. Down-regulation of c-Myc and cyclin D1 expression, the two important effectors of the Wnt/β-catenin signaling, is confirmed by treating HCT116 cells with BC21. This compound represents a new and modifiable potential anticancer candidate that targets β-catenin/Tcf-4 interaction.     

Mitochondrial proteomic approach reveals galectin-7 as a novel BCL-2 binding protein in human cells

Although the anti-apoptotic activity of Bcl-2 has been extensively studied, its mode of action remains incompletely understood. Deciphering the network of Bcl-2 interacting factors is necessary to better understand the key function of Bcl-2 in apoptosis initiation. To identify novel Bcl-2 mitochondrial partners, we have combined a Bcl-2 immunocapture with a mass spectrometry analysis using highly pure mitochondrial fractions isolated from human cancer cells. We identified at high confidence 127 potential Bcl-2-interacting proteins. Gene ontology mining reveals enrichment for mitochondrial proteins, endoplasmic reticulum-associated proteins, and cytoskeleton-associated proteins. Importantly, we report the identification of galectin-7 (Gal7), a member of a family of β-galactoside-binding lectins that was already known to exhibit a pro-apoptotic function, as a new mitochondrial Bcl-2 interacting partner. Our data further show that endogenous Bcl-2 coimmunoprecipitates with Gal7 and that recombinant Gal7 directly interacts with recombinant Bcl-2. A fraction of Gal7 is constitutively localized at mitochondria in a Bcl-2-dependent manner and sensitizes the mitochondria to the apoptotic signal. In addition, we show that the Bcl-2/Gal7 interaction is abolished following genotoxic stress. Taken together, our findings suggest that the binding of Gal7 to Bcl-2 may constitute a new target for enhancing the intrinsic apoptosis pathway.     

Interaction of Coatomer with Aminoglycoside Antibiotics: Evidence That Coatomer Has at Least Two Dilysine Binding Sites

Coatomer is the soluble precursor of the COPI coat (coat protein I) involved in traffic among membranes of the endoplasmic reticulum and the Golgi apparatus. We report herein that neomycin precipitates coatomer from cell extracts and from purified coatomer preparations. Precipitation first increased and then decreased as the neomycin concentration increased, analogous to the precipitation of a polyvalent antigen by divalent antibodies. This suggested that neomycin cross-linked coatomer into large aggregates and implies that coatomer has two or more binding sites for neomycin. A variety of other aminoglycoside antibiotics precipitated coatomer, or if they did not precipitate, they interfered with the ability of neomycin to precipitate. Coatomer is known to interact with a motif (KKXX) containing adjacent lysine residues at the carboxyl terminus of the cytoplasmic domains of some membrane proteins resident in the endoplasmic reticulum. All of the antibiotics that interacted with coatomer contain at least two close amino groups, suggesting that the antibiotics might be interacting with the di-lysine binding site of coatomer. Consistent with this idea, di-lysine itself blocked the interaction of antibiotics with coatomer. Moreover, di-lysine and antibiotics each blocked the coating of Golgi membranes by coatomer. These data suggest that certain aminoglycoside antibiotics interact with di-lysine binding sites on coatomer and that coatomer contains at least two of these di-lysine binding sites.     

Quantitative proteomics identifies a beta-catenin network as an element of the signaling response to Frizzled-8 protein-related antiproliferative factor

Antiproliferative factor (APF), a Frizzled-8 protein-related sialoglycopeptide involved in the pathogenesis of interstitial cystitis, potently inhibits proliferation of normal urothelial cells as well as certain cancer cells. To elucidate the molecular mechanisms of the growth-inhibitory effect of APF, we performed stable isotope labeling by amino acids in cell culture analysis of T24 bladder cancer cells treated with and without APF. Among over 2000 proteins identified, 54 were significantly up-regulated and 48 were down-regulated by APF treatment. Bioinformatic analysis revealed that a protein network involved in cell adhesion was substantially altered by APF and that β-catenin was a prominent node in this network. Functional assays demonstrated that APF down-regulated β-catenin, at least in part, via proteasomal and lysosomal degradation. Moreover, silencing of β-catenin mimicked the antiproliferative effect of APF whereas ectopic expression of nondegradable β-catenin rescued growth inhibition in response to APF, confirming that β-catenin is a key mediator of APF signaling. Notably, the key role of β-catenin in APF signaling is not restricted to T24 cells, but was also observed in an hTERT-immortalized human bladder epithelial cell line, TRT-HU1. In addition, the network model suggested that β-catenin is linked to cyclooxygenase-2 (COX-2), implying a potential connection between APF and inflammation. Functional assays verified that APF increased the production of prostaglandin E(2) and that down-modulation of β-catenin elevated COX-2 expression, whereas forced expression of nondegradable β-catenin inhibited APF-induced up-regulation of COX-2. Furthermore, we confirmed that β-catenin was down-regulated whereas COX-2 was up-regulated in epithelial cells explanted from IC bladder biopsies compared with control tissues. In summary, our quantitative proteomics study describes the first provisional APF-regulated protein network, within which β-catenin is a key node, and provides new insight that targeting the β-catenin signaling pathway may be a rational approach toward treating interstitial cystitis.