Pharmacologic unmasking of epigenetically silenced genes in breast cancer.
OBJECTIVE
Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of various cancers including breast cancer. Many epigenetically inactivated genes involved in breast cancer development remain to be identified. Therefore, in this study we used a pharmacologic unmasking approach in breast cancer cell lines with 5-aza-2'-deoxycytidine (5-aza-dC) followed by microarray expression analysis to identify epigenetically inactivated genes in breast cancer.


METHODS
Breast cancer cell lines were treated with 5-aza-dC followed by microarray analysis to identify epigenetically inactivated genes in breast cancer. We then used bisulfite DNA sequencing, conventional methylation-specific PCR, and quantitative fluorogenic real-time methylation-specific PCR to confirm cancer-specific methylation in novel genes.


RESULTS
Forty-nine genes were up-regulated in breast cancer cells lines after 5-aza-dC treatment, as determined by microarray analysis. Five genes (MAL, FKBP4, VGF, OGDHL, and KIF1A) showed cancer-specific methylation in breast tissues. Methylation of at least two was found at high frequency only in breast cancers (40 of 40) as compared with normal breast tissue (0 of 10; P<0.0001, Fisher's exact test).


CONCLUSIONS
This study identified new cancer-specific methylated genes to help elucidate the biology of breast cancer and as candidate diagnostic markers for the disease.
Ostrow
Kimberly Laskie
KL
Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins School of Medicine, Baltimore, MD 21231, USA.
Park
Hannah Lui
HL
Hoque
Mohammad Obaidul
MO
Kim
Myoung Sook
MS
Liu
Junwei
J
Argani
Pedram
P
Westra
William
W
Van Criekinge
Wim
W
Sidransky
David
D
D000328:Adult; D000368:Aged; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D020868:Gene Silencing; D006801:Humans; D016547:Kinesins / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics; D008875:Middle Aged; D009185:Myelin Proteins / Q000235:genetics; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D009414:Nerve Growth Factors / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D011510:Proteolipids / Q000235:genetics; D022021:Tacrolimus Binding Proteins / Q000235:genetics
C094065:KIF1A protein, human; C569026:MAL protein, human; D026901:Membrane Transport Proteins; D009185:Myelin Proteins; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D009414:Nerve Growth Factors; D011510:Proteolipids; C510637:VGF protein, human; D016547:Kinesins; D022021:Tacrolimus Binding Proteins; C403485:tacrolimus binding protein 4