Activation of MMPs by EZH2 Figure 3. Expression of TIMP3 is downregulated by EZH2 in prostate cancer cells. (A) qRT–PCR analysis of expression of TIMP3 in the prostate cancer cells (LNCaP, DU145, and PC3) after an infection with EZH2-specific shRNA (sh1) or control shRNA lentivirus (NT). * P,0.05, ** p,0.005 (as compared with control (NT)). (B) Western blot analysis of endogenous levels of EZH2 and TIMP3 protein in prostate cancer cells (LNCaP, DU145, and PC3) infected with EZH2-specific shRNA (sh1) or control shRNA virus (NT). Actin was used as an internal control. (C) Western blot (top) and qRT– PCR (bottom) analyses of expression of EZH2 and TIMP3 in the benign prostate RWPE-1 cells after an infection with EZH2 (WT)-GFP, EZH2 (H689A)- GFP, or control (GFP vector) lentivirus. Actin was used as an internal control. * P,0.05, ** p,0.005 (as compared with control (vector)). The solid arrow indicates EZH2-GFP expressed from the CMV promoter, the dotted arrow indicates endogenous EZH2. (D) Immunostaining of EZH2 and TIMP3 expressed in DU145 cells infected with EZH2-specific shRNA (sh1) or control (NT) lentivirus, using anti-EZH2 antibody (green) and anti-TIMP3 antibody (red). Nuclei were stained with DAPI (blue). The merged images were also shown (EZH2/TIMP3 and DAPI/TIMP3). doi:10.1371/journal.pone.0030393.g003 ECM. EZH2 does this by downregulating expression of TIMP genes (TIMP2 and TIMP3). TIMP2 and TIMP3, compared with TIMP1 and TIMP4, inhibit a broad spectrum of MMPs and several of disintegrin-metalloproteinases, ADAMs and ADAMTSs [12]. Furthermore, TIMP3 is the only member of the TIMP family that tightly binds to ECM and thus is implicated in disease pathogenesis [9,10]. TIMP3 also has an inhibitory effect on angiogenesis by blockage of VEGF binding to VEGF receptor-2 [46] and an ability to promote apoptosis [47–49]. Therefore, the transcriptional repression of TIMP2 and TIMP3 by EZH2 likely enhances ECM degradation and angiogenesis but reduces apoptotic activity, favoring cancer cell invasion and metastasis. The presence or elevated expression of some of MMPs is positively associated with cancer progression [3,50]. Our metas- tasis PCR array analysis shows that some MMP genes, such as MMP7 and MMP13, in PC3 cells were markedly downregulated by knockdown of EZH2 expression (Figure S1). Thus, EZH2 seems to act as an activator, rather than a repressor, of the PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e30393