Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells.

ABSTRACT

BACKGROUND: Urinary bladder cancer is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. Antiproliferative factor (APF) is a potent glycopeptide inhibitor of epithelial cell proliferation that was discovered in the urine of patients with interstitial cystitis, a disorder with bladder epithelial thinning and ulceration. APF mediates its antiproliferative activity in primary normal bladder epithelial cells via cytoskeletal associated protein 4 (CKAP4). Because synthetic asialo-APF (as-APF) has also been shown to inhibit T24 bladder cancer cell proliferation at nanomolar concentrations in vitro, and because the peptide segment of APF is 100% homologous to part of frizzled 8, we determined whether CKAP4 mediates as-APF inhibition of proliferation and/or downstream Wnt/frizzled signaling events in T24 cells. METHODS: T24 cells were transfected with double-stranded siRNAs against CKAP4 and treated with synthetic as-APF or inactive control peptide; cells that did not undergo electroporation and cells transfected with non-target (scrambled) double-stranded siRNA served as negative controls. Cell proliferation was determined by 3H-thymidine incorporation. Expression of Akt, glycogen synthase kinase 3ß (GSK3ß), ß-catenin, p53, and matrix metalloproteinase 2 (MMP2) mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Akt, GSK-3ß, MMP2, ß-catenin, and p53 protein expression, plus Akt, GSK-3ß, and ß-catenin phosphorylation, were determined by Western blot. RESULTS: T24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3ß tyr216 phosphorylation, and ß-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and ß-catenin ser33,37/thr41 phosphorylation, were increased by APF treatment in non-electroporated and non-target siRNA-transfected cells. Neither mRNA nor total protein expression of Akt, GSK3ß, or ß-catenin changed in response to APF in these cells. In addition, the changes in cell proliferation, MMP2/p53 mRNA and protein expression, and Akt/GSK3ß/ß-catenin phosphorylation in response to APF treatment were all specifically abrogated following CKAP4 siRNA knockdown. CONCLUSIONS: Synthetic as-APF inhibits cell proliferation in T24 bladder carcinoma cells via the CKAP4 receptor. The mechanism for this inhibition involves regulating phosphorylation of specific cell signaling molecules (Akt, GSK3ß, and ß-catenin) plus mRNA and protein expression of p53 and MMP2.