Polycomb repressor complex-2 is a novel target for mesothelioma therapy.
OBJECTIVE
Polycomb group (PcG) proteins are critical epigenetic mediators of stem cell pluripotency, which have been implicated in the pathogenesis of human cancers. This study was undertaken to examine the frequency and clinical relevance of PcG protein expression in malignant pleural mesotheliomas (MPM).


METHODS
Microarray, quantitative reverse transcriptase PCR (qRT-PCR), immunoblot, and immunohistochemistry techniques were used to examine PcG protein expression in cultured MPM, mesothelioma specimens, and normal mesothelial cells. Lentiviral short hairpin RNA techniques were used to inhibit EZH2 and EED expression in MPM cells. Proliferation, migration, clonogenicity, and tumorigenicity of MPM cells either exhibiting knockdown of EZH2 or EED, or exposed to 3-deazaneplanocin A (DZNep), and respective controls were assessed by cell count, scratch and soft agar assays, and murine xenograft experiments. Microarray and qRT-PCR techniques were used to examine gene expression profiles mediated by knockdown of EZH2 or EED, or DZNep.


RESULTS
EZH2 and EED, which encode components of polycomb repressor complex-2 (PRC-2), were overexpressed in MPM lines relative to normal mesothelial cells. EZH2 was overexpressed in approximately 85% of MPMs compared with normal pleura, correlating with diminished patient survival. Overexpression of EZH2 coincided with decreased levels of miR-101 and miR-26a. Knockdown of EZH2 orEED, or DZNep treatment, decreased global H3K27Me3 levels, and significantly inhibited proliferation, migration, clonogenicity, and tumorigenicity of MPM cells. Common as well as differential gene expression profiles were observed following knockdown of PRC-2 members or DZNep treatment.


CONCLUSIONS
Pharmacologic inhibition of PRC-2 expression/activity is a novel strategy for mesothelioma therapy.
Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Clinton D
CD
Kemp
Mahadev
M
Rao
Sichuan
S
Xi
Suzanne
S
Inchauste
Haresh
H
Mani
Patricia
P
Fetsch
Armando
A
Filie
Mary
M
Zhang
Julie A
JA
Hong
Robert L
RL
Walker
Yuelin J
YJ
Zhu
R Taylor
RT
Ripley
Aarti
A
Mathur
Fang
F
Liu
Maocheng
M
Yang
Paul A
PA
Meltzer
Victor E
VE
Marquez
Assunta
A
De Rienzo
Raphael
R
Bueno
David S
DS
Schrump
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000328:Adult; D000368:Aged; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071221:Enhancer of Zeste Homolog 2 Protein; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D007124:Immunoenzyme Techniques; D008654:Mesothelioma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010997:Pleural Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism*; D063151:Polycomb Repressive Complex 2; D063146:Polycomb-Group Proteins; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000241:Adenosine; D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; D000071221:Enhancer of Zeste Homolog 2 Protein; D035683:MicroRNAs; D063151:Polycomb Repressive Complex 2; D063146:Polycomb-Group Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D014157:Transcription Factors; C491488:EED protein, human; C534072:MIRN101 microRNA, human; C528163:MIRN26 microRNA, human; C048460:3-deazaneplanocin; C498409:EZH2 protein, human