Activation of MMPs by EZH2 Figure 2. Identification of the downstream target genes of EZH2 associated with metastasis of prostate cancer. (A) RT2 profiler PCR array for Human Tumor Metastasis genes in DU145 cells after an infection with EZH2-specific shRNA or control (NT) lentivirus. The scatter plot of the test vs control samples indicates the validity of the experiment. (B) 12 genes whose expressions are highly affected by EZH2 knockdown are shown (also see Tables S1 and S2). Total RNA was isolated from DU145 cells after an infection with EZH2-specific shRNA or control (NT) lentivirus and processed for PCR array (C) Immunofluorescence images of normal prostate (Normal 1 and Normal 2) and prostate tumor tissue sections (PCa1 and PCa2), using anti-EZH2 antibody (green) and anti-TIMP3 antibody (red). Nuclei were stained with DAPI (blue). doi:10.1371/journal.pone.0030393.g002 EZH2. Our RT-PCR analysis showed that TIMP1 was not regulated by EZH2, whereas TIMP4 was repressed when EZH2 expression is knocked down (Figure 6). In contrast, TIMP2 expression was significantly increased by EZH2 knockdown, but was not increased when the mutant EZH2 protein (EZH2-H689A) is expressed. Our ChIP studies show that TIMP2, like TIMP3, is also subject to EZH2-mediated histone methylation and subse- quent promoter DNA methylation (Figure 7). Down-regulation of the TIMP2 gene in prostate cancer cells was shown to be associated with promoter methylation [13], suggesting that epigenetic regulatory factors, such as HDAC and DNMT, may be involved in regulation of TIMP2. Our results show that the epigenetic modifier EZH2 may be the factor that mediates epigenetic inactivation of TIMP2. Transcriptional repression of TIMP3 by EZH2 promotes invasion of prostate cancer cells To assess the functional link between the downregulation of TIMP3 by EZH2 (Figure 3) and the decreased invasive phenotype of prostate cancer cells by EZH2 knockdown (Figures 1C and 1D), we overexpressed EZH2 and TIMP3 separately or together in RWPE-1 cells (Figure 8A) and examined the invasive activity of the cells using Transwell Boyden chamber assay (Figure 8B). The invasive phenotype of RWPE-1 cells was induced by EZH2 overexpression as observed above (Figure 1D), but it was significantly suppressed by cooverexpression of TIMP3 (Figure 8B), providing direct evidence that the repression of TIMP3 by EZH2 results in increased invasive activity of prostate cancer cells. MMP-2 and MMP-9 are known to be the prominent MMPs responsible for ECM degradation, and thus we tested whether enzymatic activity of these two MMPs is regulated by EZH2 using gelatin zymography assay. MMP-2 activity was found not to be changed significantly by EZH2 knockdown. However, MMP-9 activity was decreased by ,28% in PC3 cells and ,50% in DU145 cells, respectively, treated with EZH2-specific shRNA, compared with control cells (NT) (Figure 8C). Taken together, these results suggest that EZH2-mediated transcriptional repres- sion of TIMP3 directly leads to activation of MMP-9. Discussion The balance between MMPs and TIMPs is a critical parameter for the degradation of ECM and thus a crucial step for cancer invasion and metastasis. In this study, we show that aberrant upregulation of EZH2 expression in prostate cancer cells shift this balance towards MMPs and thereby promote degradation of the PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30393