Quantitative Proteomics Identifies a β-Catenin Network as an Element of the Signaling Response to Frizzled-8 Protein-Related Antiproliferative Factor

Abstract

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 ␤-catenin was a prominent node in this network. Functional assays demonstrated that APF down-regulated ␤-catenin, at least in part, via proteasomal and lysosomal degradation. Moreover, silencing of ␤-catenin mimicked the antiproliferative effect of APF whereas ectopic expression of nondegradable ␤-catenin rescued growth inhibition in response to APF, confirming that ␤-catenin is a key mediator of APF signaling. Notably, the key role of ␤-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 ␤-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 ␤-catenin elevated COX-2 expression, whereas forced expression of nondegradable ␤-catenin inhibited APF-induced up-regulation of COX-2. Furthermore, we confirmed that ␤-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 ␤-catenin is a key node, and provides new insight that targeting the ␤-catenin signaling pathway may be a rational approach toward treating interstitial cystitis. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.007492, 1-11, 2011. Antiproliferative factor (APF)1 , a nine-residue sialoglycopeptide whose peptide chain is 100% homologous to the putative sixth transmembrane domain of , is secreted by bladder epithelial cells from patients with interstitial cystitis (IC) (2, 3), a prevalent and debilitating pelvic disorder (4, 5). Studies suggest that APF is a potent negative growth factor, which markedly inhibits the proliferation of not only normal bladder epithelial cells but also T24 bladder carcinoma cells and HeLa cervical carcinoma cells (1, 6, 7). Studies have been conducted to investigate the molecular mechanisms underlying the antiproliferative effect of APF using the hypothesis-driven approach; these led to the discoveries that (a) cytoskeleton-associated protein 4 (CKAP4), also known as CLIMP63, is a high-affinity receptor for APF (6); (b) palmitoylation of CKAP4 by ...

The ubiquitin-specific protease USP2a enhances tumor progression by targeting cyclin A1 in bladder cancer

“…Three days after transfection, cell proliferation rate was determined by crystal violet staining. 59 Absorbance was measured at 570 nm.…”

Reviewing Interstitial Cystitis Models and Treatments: A Focus on the Urothelium

“…It was demonstrated that siRNA-mediated knockdown of CKAP4 specifically abrogates APF's effects on cell proliferation, MMP2/p53 protein expression, and Akt/GSK3β/β-catenin phosphorylation on T24 bladder carcinoma cells (75). The Akt/GSK3β/β-catenin signaling importance for APF signal transduction was not only demonstrated in T24 cells (HTB-4), but was also confirmed in an hTERT immortalized human bladder epithelial cell line, TRT-HU1 (75).…”

“…The APF cell inhibitory mechanism is the result of this small molecule binding to the membrane resident palmitoylated CKAP4 receptors causing inhibition of cell proliferation and alteration of the expression of genes relevant to cell-to-cell permeability (E-cadherin, vimentin, and tight junction protein, ZO-1) (74). It was demonstrated that siRNA-mediated knockdown of CKAP4 specifically abrogates APF's effects on cell proliferation, MMP2/p53 protein expression, and Akt/GSK3β/β-catenin phosphorylation on T24 bladder carcinoma cells (75). The Akt/GSK3β/β-catenin signaling importance for APF signal transduction was not only demonstrated in T24 cells (HTB-4), but was also confirmed in an hTERT immortalized human bladder epithelial cell line, TRT-HU1 (75).…”

Quantitative proteomics reveals the downregulation of GRB2 as a prominent node of F806-targeted cell proliferation network

“…Yue et al provided evidences that the proteins influenced in cancer cells by a purified G. lucidum component were searched globally using a proteomic technique and 21 proteins might be target-related proteins of ganoderic acid D [7]. In addition, it has been reported that beta-catenin was a key node of APF-regulated protein network in human bladder cells by a stable isotope labeling with amino acids in a cell culture (SILAC)-based quantitative proteomic approach combined with bioinformatic analyses [8].…”