777 CRUCIAL ROLE OF DICKKOPF-3 IN PROSTATE MORPHOGENESIS IN VITRO AND IN VIVO

Abstract

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2012777 CRUCIAL ROLE OF DICKKOPF-3 IN PROSTATE MORPHOGENESIS IN VITRO AND IN VIVO Yoshiaki Kawano, Diana Romero, Nora Bengoa, Nicole Maltry, Marjorie Walker, Masatoshi Eto, Jonathan Waxman, Christof Niehrs, and Robert Kypta Yoshiaki KawanoYoshiaki Kawano Kumamoto, Japan More articles by this author , Diana RomeroDiana Romero London, United Kingdom More articles by this author , Nora BengoaNora Bengoa Bilbao, Spain More articles by this author , Nicole MaltryNicole Maltry Heidelberg, Germany More articles by this author , Marjorie WalkerMarjorie Walker London, United Kingdom More articles by this author , Masatoshi EtoMasatoshi Eto Kumamoto, Japan More articles by this author , Jonathan WaxmanJonathan Waxman London, United Kingdom More articles by this author , Christof NiehrsChristof Niehrs Heidelberg, Germany More articles by this author , and Robert KyptaRobert Kypta London, United Kingdom More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.865AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To investigate how the tumour suppressor Dickkopf-3 contributes to prostate cancer formation in vitro and in vivo. METHODS RWPE-1 cells were stably transfected with either Dkk-3-targeting pSM2-shRNAmir (RWPE-1/D3-sh cells) or non-targeting vector (RWPE-1/NS-sh) . For 3D acinar morphogenesis assays, either RWPE-1/NS-sh or RWPE-1/D3-sh cells were plated on a thin-layered bed of Matrigel (BD Biosciences) on 8-chamber glass slides and cultured with Keratinocyte SFM (Invitrogen) containing 2% Matrigel. For gene reporter assays, cells were transfected with luciferase and bet-galactosidase-based reporters. Gene expression levels were measured using quantitative real-time PCR. For analysis of Dkk-3 knockout mice and their wild-type littermates, prostates were dissected from 6-8 week-old mice, fixed with 4% paraformaldehyde and embedded in paraffin. Sections were analysed by H&E and immunofluorescent staining. RESULTS RWPE-1/D3-sh cells showed more than a 90% reduction in Dkk-3 mRNA and protein expression, compared to both parental and RWPE-1/NS-sh cells. There were no apparent morphological differences between the cell lines when cultured in 2D. However, in 3D assays, while RWPE-1/NS-sh cells underwent normal acinar morphogenesis with spherically arranged polarisation, RWPE-1/D3-sh cells formed disorganised cell aggregates. Immunofluorescent staining for phosphotylated histone H3 revealed increased cell division in Dkk-3 depleted acini. Simultaneous staining for β4-integrin and β-catenin suggested that partitioning of basal and lateral membranes in Dkk-3 depleted acini was not affected. However, gene reporter assays and analysis of target gene expression indicated changes in Wnt and TGFβ signalling in RWPE-1/D3-sh cells. These results indicate that Dkk-3 knockdown results in changes in key cell signalling pathways, abnormal cell division and disorder of apical membrane integrity, leading to the disruption of acini. Analysis of prostates from mice showed an increased Ki-67 index in all lobes of Dkk-3 knockout mice, compared to wild type, consistent with the results in the in vitro 3D morphogenesis assay. H&E and immunofluorescent staining for ZO-1 and E-Cadherin revealed subtle changes in cellular structure during prostate development. CONCLUSIONS These results suggest that Dkk-3 controls cell proliferation and polarisation to secure organised acinar structure of prostate epithelial cells, and suggest that loss of Dkk-3 expression contributes to prostate cancer development due to aberrant cell proliferation and impaired cellular structure. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e317-e318 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yoshiaki Kawano Kumamoto, Japan More articles by this author Diana Romero London, United Kingdom More articles by this author Nora Bengoa Bilbao, Spain More articles by this author Nicole Maltry Heidelberg, Germany More articles by this author Marjorie Walker London, United Kingdom More articles by this author Masatoshi Eto Kumamoto, Japan More articles by this author Jonathan Waxman London, United Kingdom More articles by this author Christof Niehrs Heidelberg, Germany More articles by this author Robert Kypta London, United Kingdom More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...