title: 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 3β (GSK3β), β-catenin, p53, and matrix metalloproteinase 2 (MMP2) mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Akt, GSK-3β, MMP2, β-catenin, and p53 protein expression, plus Akt, GSK-3β, and β-catenin phosphorylation, were determined by Western blot.
  
  
  RESULTS
  T24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3β tyr216 phosphorylation, and β-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and β-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, GSK3β, or β-catenin changed in response to APF in these cells. In addition, the changes in cell proliferation, MMP2/p53 mRNA and protein expression, and Akt/GSK3β/β-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, GSK3β, and β-catenin) plus mRNA and protein expression of p53 and MMP2.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.
authors.forename: Hanief M
authors.initials: HM
authors.lastname: Shahjee
authors.forename: Kristopher R
authors.initials: KR
authors.lastname: Koch
authors.forename: Li
authors.initials: L
authors.lastname: Guo
authors.forename: Chen-Ou
authors.initials: CO
authors.lastname: Zhang
authors.forename: Susan K
authors.initials: SK
authors.lastname: Keay
mesh_terms: D015153:Blotting, Western; D002295:Carcinoma, Transitional Cell / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D006023:Glycoproteins / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014162:Transfection; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology
chemical_list: C081876:CKAP4 protein, human; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C490708:antiproliferative factor APF, human; D051057:Proto-Oncogene Proteins c-akt