@article{812068f2cf2c4f1e8ab3bf838ac5d1e9,
title = "A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer",
abstract = "DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1,505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.",
keywords = "Biomarker, DNA methylation, ERG, Polycomb, Prostate cancer",
author = "Jacob Schwartzman and Solange Mongoue-Tchokote and Angela Gibbs and Lina Gao and Corless, {Christopher L.} and Jennifer Jin and Luai Zarour and Celestia Higano and True, {Lawrence D.} and Vessella, {Robert L.} and Beth Wilmot and Daniel Bottomly and McWeeney, {Shannon K.} and Bova, {Steven G.} and Partin, {Alan W.} and Motomi Mori and Alumkal, {Joshi J.}",
note = "Funding Information: RWPE-1 cells (ATCC, Manassas, VA) were cultured in K-SFM (#10724, Invitrogen, Carlsbad, CA). For RNAi experiments, cells were seeded in six-well plates the day before treatment, then transfected with siRNA oligos at 50 nM (final concentration) using Oligofectamine according to the manufacturer{\textquoteright}s protocol Billerica, MA), or normal rabbit IgG antibody (#12-370, (#12252-011, Invitrogen, Carlsbad, CA). Media was changed at Millipore, Billerica, MA) were added to sheared, formaldehyde 4 h post-transfection and cells were harvested at 96 h post-trans-crosslinked chromatin derived from ~500,000 cells to immu-fection for protein and RNA. siRNA oligonucleotide duplexes noprecipitate DNA overnight at 4°C. Chromatin was removed were purchased from Dharmacon, Inc., (Lafayette, CO): EZH2 prior to immunoprecipitation for input controls. Immune com-(NM_152998) siGENOME SMARTpool #M-004218-03; lucif- plexes were collected with protein A/G (3:1)-magnetic beads erase non-targeted control (NTC): CGU ACG CGG AAU ACU (Dynabeads #100-02D and #100-04D, Invitrogen, Carlsbad, UCG AdT dT. CA). After extensive washing, immune complexes were released, RNA extraction and RTPCR. Total RNA was extracted crosslinks were reversed, and DNA was purified with a MinElute from PC3 or RWPE-1 cell lysates in TriZol reagent (Invitrogen, PCR purification kit (#28004, Qiagen Inc., Valencia, CA) Carlsbad, CA) according to the manufacturer{\textquoteright}s instructions. Total according to the manufacturer{\textquoteright}s instructions and eluted with 60 RNA was extracted from paraffin-embedded prostate cancer ul buffer EB. Quantitative PCR was performed using an ABI samples with a High Pure FFPE RNA Micro kit (#04823125001, 7500 Fast instrument (Applied Biosystems, Carlsbad, CA). Ten Roche Diagnostics Corporation, Indianapolis, IN) accord-microliters real-time reactions containing 2 μl of the immu-ing to the manufacturer{\textquoteright}s instructions. RNA was re-suspended noprecipated or input DNA in 1x SYBR GreenER mastermix in sterile DI H2O and quantified by spectrophotometry with a (Invitrogen, Carlsbad, CA) were performed in triplicate with the Nano-Drop ND-1000 (NanoDrop Products, Wilmington, DE). following thermocycling conditions: 50°C for 2 min, 95°C for One microgram of RNA from each treatment condition was 10 min, then 40 cycles of amplification at 95°C for 15 seconds reverse-transcribed using an Omniscript RT kit (#205113, and 60°C for 1 min. Qiagen Inc., Valencia, CA) according to the manufacturer{\textquoteright}s ERG promoter ChIP Sense GTG CTG GGT TCC TTT instructions. PCR and electrophoresis for ERG mRNA expres-CCT G; ERG promoter ChIP Antisense CAG GTT TGG ACA sion was performed as for MSPCR above, but using 12.5 ng of CTA CGC G. cDNA template per reaction and 40 cycles of 95°C for 30 sec- onds, 60°C for 30 seconds, 72°C for 30 seconds. The presence of Disclosure of Potential Conflict of Interest the TMPRSS2-ERG fusion was determined by quantitative PCR, No potential conflicts of interest were disclosed. which was performe{\"U}d on a LightCycler 480 -instBrumOent (ERochFe T#JPTDJFODF Diagnostics Corporation, Indianapolis, IN) with the following Acknowledgments thermocycling conditions: 95°C fo%r 10 min, then 50 cycles of We thank Mr. and Mrs. Robert and Diana Gerding for their 95°C for 10 seconds, 60°C for 20 seconds aPnd 7O2°C fPor 1 sUecoEnd. JTkind aUSnd gJCenerVous sUupFport of prostate cancer research, Carol GAPDH was used as an endogenous control. Beadling, Ph.D. and Michael Heinrich, MD for sharing their ERG RTPCR Sense CAG CGT CCT CAG TTA GAT CCT; TMPRSS2-ERG primers, and Ms. Chris Koontz for editorial and ERG RTPCR Antisense CGA ACT TGT AGG CGT AGC G; submission assistance. Beta-Actin RTPCR Sense TTC CTT CCT GGG CAT GGA GT; Beta-Actin RTPCR Antisense TCT TCA TTG TGC TGG This publication was made possible with support from the GTG CC; TMPRSS2-ERG Sense CGC GAG CTA AGC AGG Oregon Clinical and Translational Research Institute (OCTRI), AG; TMPRSS2-ERG probe 6FAM-CGC GGC AGG AAG CCT grant number 5 KL2 RR024141-04 from the National Center TAT CAG-TAMRA; TMPRSS2-ERG Antisense CGA CTG for Research Resources (NCRR), a component of the National GTC CTC ACT CAC AA; GAPDH Sense AG CCG CAT CTT Institutes of Health (NIH) and NIH Roadmap for Medical CTT TTG C; GAPDH Antisense GCC CAA TAC GAC CAA Research NIH RR024141. Additional support includes: Flight ATC C. Attendant Medical Research Institute (FAMRI) Young Clinical Chromatin immunoprecipitation (ChIP) and ChIP PCR. Scientist Award; OHSU Knight Cancer Institute Pilot Award; ChIP experiments were performed as described with modifica-NIHP50-CA97186, Pacific Northwest Prostate Cancer SPORE; tions.32 Two to five micrograms of anti-H3K27me3 (#ab6002, NIH P30-CA015704, Cancer Center Support Grant; NIH Abcam, Cambridge, MA), H3K4me2 (#07-030, Millipore, 5UL1RR024140.",
year = "2011",
month = oct,
doi = "10.4161/epi.6.10.17727",
language = "English (US)",
volume = "6",
pages = "1248--1256",
journal = "Epigenetics",
issn = "1559-2294",
publisher = "Landes Bioscience",
number = "10",
}