WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells.
Epigenetic silencing of secreted wingless-type (Wnt) antagonists through hypermethylation is associated with tobacco smoking and with invasive bladder cancer. The secreted Wnt inhibitory factor-1 (WIF1) has shown consistent growth-inhibitory effect on various cancer cell lines. Therefore, we assessed the mechanisms of action of WIF1 by either restoring WIF1 expression in invasive bladder cancer cell lines (T24 and TSU-PR1) or using a recombinant protein containing functional WIF1 domain. Both ectopic expression of WIF1 and treatment with WIF1 domain protein resulted in cell growth inhibition via G(1) arrest. The G(1) arrest induced by WIF1 is associated with down-regulation of SKP2 and c-myc and up-regulation of p21/WAF1 and p27/Kip1. Conversely, reexpression of SKP2 in WIF1-overexpressing TSU-PR1 cells attenuated the WIF1-induced G(1) arrest. Furthermore, inhibition of nuclear Wnt signaling by either dominant-negative LEF1 or short hairpin RNA of TCF4 also reduced SKP2 expression. The human SKP2 gene contains two TCF/LEF1 consensus binding sites within the promoter. Chromatin immunoprecipitation/real-time PCR analysis revealed that both WIF1 and dominant-negative LEF1 expression decreased the in vivo binding of TCF4 and beta-catenin to the SKP2 promoter. Together, our results suggest that mechanisms of WIF1-induced G(1) arrest include (a) SKP2 down-regulation leading to p27/Kip1 accumulation and (b) c-myc down-regulation releasing p21/WAF1 transcription. Additionally, we show that WIF1 inhibits in vivo bladder tumor growth in nude mice. These observations suggest a mechanism for transformation of bladder epithelium on loss of WIF1 function and provide new targets such as SKP2 for intervention in WIF1-deficient bladder cancer.
Tang
Yaxiong
Y
Department of Urology, University of California at Irvine, Irvine, CA 92868, USA.
Simoneau
Anne R
AR
Liao
Wu-xiang
WX
Yi
Guo
G
Hope
Christopher
C
Liu
Feng
F
Li
Shunqiang
S
Xie
Jun
J
Holcombe
Randall F
RF
Jurnak
Frances A
FA
Mercola
Dan
D
Hoang
Bang H
BH
Zi
Xiaolin
X
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D016193:G1 Phase; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D051821:Lymphoid Enhancer-Binding Factor 1 / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009361:Neoplasm Invasiveness; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D012097:Repressor Proteins / Q000378:metabolism*; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D051820:TCF Transcription Factors / Q000378:metabolism; D058118:Transcription Factor 7-Like 2 Protein; D014158:Transcription, Genetic / Q000187:drug effects; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000473:pathology*; D023041:Xenograft Model Antitumor Assays; D051176:beta Catenin / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C497573:LEF1 protein, human; D051821:Lymphoid Enhancer-Binding Factor 1; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D012097:Repressor Proteins; D044786:S-Phase Kinase-Associated Proteins; D051820:TCF Transcription Factors; C544894:TCF7L2 protein, human; C544895:Tcf7l2 protein, mouse; D058118:Transcription Factor 7-Like 2 Protein; C485569:WIF1 protein, human; D051176:beta Catenin