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

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 H-3-thymidine incorporation. Expression of Akt, glycogen synthase kinase 3 beta (GSK3 beta), beta-catenin, p53, and matrix metalloproteinase 2 (MMP2) mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Akt, GSK-3 beta, MMP2, beta-catenin, and p53 protein expression, plus Akt, GSK-3 beta, and beta-catenin phosphorylation, were determined by Western blot. Results: T24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3 beta tyr216 phosphorylation, and beta-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and beta-catenin ser33,37/thr41 phosphorylation, were increased by APF treatment in non-electroporated and nontarget siRNA-transfected cells. Neither mRNA nor total protein expression of Akt, GSK3 beta, or beta-catenin changed in response to APF in these cells. In addition, the changes in cell proliferation, MMP2/p53 mRNA and protein expression, and Akt/GSK3 beta/beta-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, GSK3 beta, and beta-catenin) plus mRNA and protein expression of p53 and MMP2.