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

Abstract

BackgroundUrinary 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.MethodsT24 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.ResultsT24 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.ConclusionsSynthetic 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.

Transmembrane domain is crucial to the subcellular localization and function of Myc target 1

“…It was reported that CKAP4 plays important roles in several cellular processes, including endoplasmic reticulum anchoring 18 and morphology determination 19, regulation of Dicer function 20, cell proliferation 21 and tumour migration 22, 23. Knockdown of CKAP4 (Fig.…”

Prognostic significance of cytoskeleton‐associated membrane protein 4 and its palmitoyl acyltransferase DHHC2 in hepatocellular carcinoma

“…To our knowledge to date, there is evidence linking the CKAP4 gene to tumor growth in patients with bladder cancer and cervical carcinoma. 8,9 Our recent work indicates that CKAP4 is a prognostic marker in patients with intrahepatic cholangiocellular carcinoma and is clinicopathologically associated with tumor progression and metastasis. 14 To our knowledge, systematic investigation of the prognostic significance of CKAP4 in HCC has not been reported to date, especially with long-term follow-up and a large number of patients.…”

“…[4][5][6] Recently, it has been identified as a cell surface receptor for tissue plasminogen activator and surfactant protein A, as well as antiproliferative factor. 3,[7][8][9] It has been shown that CKAP4 may inhibit the proliferation of bladder cancer cells by combining its ligand antiproliferative factor, whose mechanism may be related to the Wnt signaling pathway. Recent studies have shown that palmitoylation by DHHC2, a putative tumor suppressor, is required for CKAP4 trafficking from the endoplasmic reticulum to the plasma membrane and for its nuclear localization.…”

Resveratrol induces apoptosis of bladder cancer cells via miR-21 regulation of the Akt/Bcl-2 signaling pathway

“…Inhibition of Akt activity is considered to be an effective strategy for bladder cancer treatment (25). Bcl-2 is also a direct participant in the apoptotic pathway and has tumorigenic potential (18).…”