Molecular Profiling of Prostatic Acinar Morphogenesis Identifies PDCD4 and KLF6 as Tissue Architecture–Specific Prognostic Markers in Prostate Cancer

Prostatic Diseases
DOI: 10.1016/j.ajpath.2012.10.024 Publication Date: 2012-12-04T21:45:17Z
ABSTRACT
Histopathological classification of human prostate cancer (PCA) relies on the morphological assessment tissue specimens but has limited prognostic value. To address this deficiency, we performed comparative transcriptome analysis prostatic acini generated in a three-dimensional basement membrane that recapitulates differentiated characteristics and gene expression profile glandular epithelial tissue. We then applied an acinar morphogenesis–specific to two independent cohorts patients with PCA (total n = 79) found those tumors expressing profile, which designated acini-like tumors, had significantly lower risk postoperative relapse compared correlation (hazard ratio, 0.078; log-rank test P 0.009). Multivariate analyses showed superior prediction performance using system clinical criteria Gleason scores. prioritized genes identified programmed cell death protein 4 (PDCD4) Kruppel-like factor 6 (KLF6) as critical regulators surrogate markers architectures, form signature robustly predicts prognosis remarkable accuracy several large series 161; concordance index, 0.913 0.951). Thus, by exploiting genomic program associated differentiation, related molecular enhance PCA. Prostate is leading cause cancer-related men. For early-stage localized cancer, radical prostatectomy offers best opportunity eradicate disease. However, approximately 15% 30% disease at diagnosis develop recurrence within 5 10 years, most these subsequently show poor therapeutic outcome.1Bill-Axelson A. Holmberg L. Ruutu M. Haggman Andersson S.O. Bratell S. Spangberg Busch C. Nordling Garmo H. Palmgren J. Adami H.O. Norlen B.J. Johansson J.E. 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RWPE-1 (ATCC, Manassas, VA) maintained keratinocyte–serum-free medium (Invitrogen, Carlsbad, CA) supplemented bovine pituitary extract, ng/mL epidermal factor, 0.5% penicillin-streptomycin (Invitrogen).22Bello Webber M.M. Kleinman H.K. Wartinger D.D. Rhim Androgen responsive adult lines immortalized papillomavirus 18.Carcinogenesis. 1997; 18: 1215-1223Crossref (331) LNCaP, DU-145, PC-3 (ATCC) RPMI 1640 (for LNCaP cells) Dulbecco's modified Eagle's DU-145 cells; Invitrogen) 10% fetal penicillin-streptomycin. embedded thick layer (3D) reconstituted (rBM) gel (Matrigel; BD Biosciences, San Jose, CA), previously described.23Weaver V.M. Petersen O.W. Larabell Briand Damsky Reversion phenotype vivo blocking antibodies.J 137: 231-245Crossref (1205) Whole immunofluorescent staining described.24Lee Lee E.H. Three-dimensional models cells.Nat Methods. 4: 359-365Crossref (1000) Confocal imaging Nikon Digital Eclipse C1 confocal microscope (Nikon, Tokyo, Japan). antibodies include rat anti-α6 (clone GoH3; Millipore) mouse anti-GM130 35; Biosciences). nuclei counterstained Hoechst 33342 DAPI (Invitrogen). Total RNA samples extracted cultivated 3D rBM different lengths time (36 48 hours 7 8 days) TRIzol (Invitrogen) purified RNeasy minikit DNase (Qiagen, Valencia, CA). Experiments triplicate. Whole-genome Affymetrix Human Genome U133A 2.0 Plus GeneChip platform, protocol (Affymetrix, Santa Clara, hybridization intensity data processed Operating software (Affymetrix), filtered P/A/M flags retain present all three least one conditions. false-discovery rate <0.025 select differentially expressed comparison group. deposited National Center Biotechnology Information's Omnibus (http://www.ncbi.nlm.nih.gov/geo; accession number GSE30304). (1.0 μg) template cDNA synthesis Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Madison, WI). (100 ng) PCR amplification LightCycler FastStart DNA MASTERPLUS SYBR Green I Kit, quantitative real-time RT-PCR amplified Kit (Roche Diagnostics GmbH, Mannheim, Germany). Oligonucleotide primers designed Primer Bank (http://pga.mgh.harvard.edu/primerbank/index.html, last accessed June 10, 2012). sequences available request. median centered experiments average linkage clustering Cluster version 2.11 TreeView 1.60 (Eisen Lab, University California, Berkeley). clustering, selected uploaded DAVID (http://david.abcc.ncifcrf.gov, March 28, 2012) ranked list annotations executed system. surveyed ontology biological process, < 0.05, top annotations, along genes, diagram output Cytoscape (http://www.cytoscape.org, Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, search enriched networks functions genes. 50 who underwent Brigham Women's Hospital (BWH cohort; Boston, MA) information reported kindly provided Massimo (Dana-Farber Institute, Boston).10Singh 29 Stanford (Stanford, Karolinska Institute (Solna, Sweden), Johns Hopkins (Baltimore, (the SU/KI/JHU cohort) downloaded GSE3933).16Lapointe measure resemblance specimen organoid, mapped probe sets microarray platforms same Entrez-gene IDs normalized mean levels microarrays) fluorescence ratios each probes across architectures. tumor, calculated Pearson's coefficient organoid probes. identify, set optimally predicted described supervised modifications.25Wang Y. Klijn Zhang Sieuwerts Look M.P. Yang Talantov Timmermans Meijer-van Gelder M.E. Yu Jatkoe Berns E.M. Atkins Foekens J.A. Gene-expression distant metastasis lymph-node-negative primary cancer.Lancet. 365: 671-679Abstract (1991) Briefly, gene, univariate Cox regression level (on log2 scale) length relapse-free BWH cohort. 1000 bootstrap cohort samples. determined estimated standardized calculating values samples, respectively. ensure consistency model, expressional changes during cystic expected positive cellular aggregates) negative up-regulated cysts) relapse, coefficient. rank ordered values, multiple repeatedly adding descendingly list, starting top-ranked (Equation 1) patient set:Recurrencescore=∑i=3kbixi(1) where k set, bi ith probe, xi probe. index (C-index) evaluate predictive analysis, 1.0 perfect discrimination.26Pencina D'Agostino Overall C discrimination analysis: specific population confidence interval estimation.Stat 2109-2123Crossref (1149) Sustained knockdown achieved retrovirus lentivirus-mediated interference (RNAi) oligonucleotide described27Narla DiFeo Yao Banno Hod Reeves Qiao R.F. Camacho-Vanegas O. Levine Kirschenbaum Friedman S.L. Martignetti Targeted inhibition KLF6 splice variant, SV1, suppresses spread.Cancer 65: 5761-5768Crossref (137) pSUPER.retro.neo (OligoEngine, Seattle, WA) short hairpin oligonucleotides (MISSION shRNA lentiviruses). Amphotropic produced Phoenix ampho (a gift Garry Nolan, University), packaging vectors pCgp pVSV-G boost viral titer. Formalin-fixed, paraffin-embedded (FFPE) tissues 61 Chimei Foundational Medical CFMC Mackay Memorial acquired conformity Institutional Review Board–approved protocols (Table 1). biochemical prostate-specific antigen (PSA) 0.4 consecutive 0.2 increasing.28Stephenson Kattan M.W. Eastham Dotan Z.A. Bianco Jr., F.J. Lilja Scardino P.T. Defining prostatectomy: proposal definition.J Oncol. 24: 3973-3978Crossref (409) Tissue sections deparaffinized, hydrated, immersed citrate buffer pH 6.0 epitope retrieval microwave. Endogenous peroxidase activity quenched 3% hydrogen 15 minutes, slides incubated horse block non-specific immunoreactivity. antibody detected DAKO EnVision kit (Dako, Carpinteria, anti-PDCD4 (1:4000, clone EPR3431), anti-KLF6 (1:50, polyclonal), anti–ATP-binding cassette transporter (ABCG1; 1:500, EP1366Y) (all Epitomics, Burlingame, All immunohistochemical (IHC) evaluated expert pathologists (C.-F.Li C.-K. Chen), quantified histological (H-score).29Budwit-Novotny D.A. McCarty K.S. E.B. Soper Mutch Creasman W.T. Flowers Immunohistochemical estrogen receptor endometrial adenocarcinoma monoclonal antibody.Cancer 1986; 46: 5419-5425PubMed ScholarTable 1Patient CharacteristicsClinical cohortBWH group (n 50)SU/KI/JHU 29)CFMC 61)MMH 50)Mean (SD) age (years)59.4 (6.8)59.0 (6.2)65.1 (5.6)67.4 (5.4)Gleason 4-746 (92.0)22 (75.9)29 (47.5)20 (40.0) 8-94 (8.0)7 (24.1)32 (52.5)30 (60.0)Mean baseline (ng/mL)8.3 (5.8)NA18.8 (30.8)15.2 (3.6)Tumor stage I-II30 (60.0)19 (65.5)53 (86.9)42 (84.0) ≥III20 (40.0)10 (34.5)8 (13.1)8 (16.0)Recurrence9 (18.0)7 (24.1)11 (18.0)12 (24.0)Medium (months)42.911.587.970.2MMH, Hospital; NA, available. Open table tab MMH, statistical programming language R 2.14.0 (http://cran.r-project.org) SPSS 10.0 (SPSS, Chicago, IL), conduct data. two-tailed Student's t-test simple significance testing, tests analysis. C-statistics conducted survcomp package. cutoff discriminates groups respect maximal Youden's index.30Pepe Statistical Evaluation Tests Classification Prediction. 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