Research

EZH2 overexpression in natural killer/T-cell lymphoma confers growth advantage independently of histone methyltransferase activity (Blood, May 2013)

The role of enhancer of zeste homolog 2 (EZH2) in cancer is complex and may vary depending on the cellular context. We found that EZH2 is aberrantly overexpressed in the majority of natural killer/T-cell lymphoma (NKTL), an aggressive lymphoid malignancy with very poor prognosis. We show that EZH2 upregulation is mediated by MYC-induced repression of its regulatory micro RNAs and EZH2 exerts oncogenic properties in NKTL. Ectopic expression of EZH2 in both primary NK cells and NKTL cell lines leads to a significant growth advantage. Conversely, knock-down of EZH2 in NKTL cell lines results in cell growth inhibition. Intriguingly, ectopic EZH2 mutant deficient for histone methyltransferase activity is also able to confer growth advantage and rescue growth inhibition on endogenous EZH2 depletion in NKTL cells, indicating an oncogenic role of EZH2 independent of its gene-silencing activity. Mechanistically, we show that EZH2 directly promotes the transcription of cyclin D1 and this effect is independent of its enzymatic activity. Furthermore, depletion of EZH2 using a PRC2 inhibitor 3-deazaneplanocin A significantly inhibits growth of NK tumor cells. Therefore, our study uncovers an oncogenic role of EZH2 independent of its methyltransferase activity in NKTL and suggests that targeting EZH2 may have therapeutic usefulness in this lymphoma.

junli2Figure 3. EZH2 overexpression in NKTL promotes cell growth independent of histone methyltransferase activity. (A) Primary NK cells transduced by EZH2 exhibit a growth advantage. Primary NK cells expressing ectopic EZH2 were monitored by a coexpressed GFP marker. Using our established viral infection protocol, we routinely obtained a transduction efficiency of 4.3% for normal NK cells. If EZH2 infection does not alter the cell growth, the EZH2-infected cell will not gain a growth advantage; thus, the percentage of GFP1 cells should remain at 4.3%. However, the percentage of GFP1 cells increased from approximately 4.3% at day 2 to ;34.4% at day 5, indicating an acquired growth advantage in these EZH2-infected cells. Antibiotic selection of positively infected cells was not done.

(B) Scatterplot representation of the correlation between the percentage of Ki-67–positive cells and the percentage of EZH2- positive cells. Spearman correlation coefficient r for EZH2 vKi67 5 0.73; P , .0001.

(C) MTS proliferation assay showing that ectopic expression of EZH2 promotes cell growth of NKTL cell lines without requiring SET domain activity. Cells were cotransfected with pMAX-GFP and the control empty vector pcDNA4.1 or EZH2 expression plasmids. Cells transfected were subjected to proliferation assays for up to 96 hours. The cell growth (expressed as a percentage of the empty vector control) was determined by MTS assay as described in Materials and Methods. The mean values of triplicate samples are shown, and error bars indicate standard deviations.

(D) Western blot analysis of EZH2, H3K27m3, and H3K27m2 in indicated samples. Expression of EZH2 WT and the SET-domain mutant was detected by the MYCtag antibody. H3 was used as a loading control.

Authors:

Junli Yan,1 Siok-Bian Ng,1-3 Jim Liang-Seah Tay,3 Baohong Lin,4 Tze Loong Koh,4 Joy Tan,3 Viknesvaran Selvarajan,2,3, Shaw-Cheng Liu,1 Chonglei Bi,1 Shi Wang,2 Shoa-Nian Choo,2,3 Norio Shimizu,5 Gaofeng Huang,1 Qiang Yu,6 and Wee-Joo Chng1,3,4

1Cancer Science Institute of Singapore, National University of Singapore, Singapore
2Department of Pathology, National University Health System, Singapore
3Yong Loo Lin School of Medicine, National University of Singapore, Singapore
4Department of Haematology-Oncology, National University Cancer Institute of Singapore, National University Health System, Singapore
5Department of Virology, Tokyo Medical and Dental University, Tokyo, Japan
6Department of Cancer Biology and Pharmacology, Genome Institute of Singapore, Agency for Science, Technology and Research, Biopolis, Singapore