Polycomb group protein enhancer of zeste 2 is an oncogene that promotes the neoplastic transformation of a benign prostatic epithelial cell line

Abstract

Polycomb group protein enhancer of zeste 2 (EZH2) is a master regulatory protein that plays a critical role in development as part of the polycomb repressive complex 2. Polycomb repressive complex 2 controls numerous cell cycle and regulatory genes through trimethylation of histone 3, which results in chromatin condensation and transcriptional silencing. EZH2 overexpression has been correlated with high incidence of more aggressive, metastatic prostate cancers. Although this correlation means EZH2 could prove valuable as a biomarker in clinical settings, the question remains whether EZH2 is actually responsible for the initiation of these more aggressive tumor types. In this study, EZH2-mediated neoplastic transformation of the normal prostate epithelial cell line benign prostate hyperplasia 1 (BPH1) was confirmed by in vivo tumor growth and in vitro colony formation. Furthermore, EZH2 transformation resulted in increased invasive behavior of BPH1 cells, indicating that EZH2 may be responsible for aggressive behavior in prostate cancers. BPH1 was also transformed with the classic oncogenes myristoylated Akt and activated Ras(V12) to allow phenotype comparisons with the EZH2-transformed cells. This study marks the first demonstration of neoplastic transformation in prostate cells mediated by EZH2 and establishes that EZH2 possesses stronger transforming activity than Akt but weaker activity than activated Ras.

Figure 1. Lentiviral schematics and EZH2 overexpression in BPH1

A. Schematic diagram of EZH2 overexpression and GFP control lentiviral vectors. LTR: Long Terminal Repeat; cppt: central polypurine tract; CMV: human cytomegalovirus promoter; IRES: internal ribosomal entry site; EGFP: enhanced green fluorescent protein; wpre: woodchuck post-transcriptional regulatory element. B. qRT-PCR analysis of EZH2 and EED levels in transduced BPH1 populations. EZH2 overexpression achieved in the BPH1-EZH2 population was 40-fold the endogenous levels. C. Western Blot analysis of overexpression of EZH2 in BPH1-EZH2 cells. α-EZH2: antibody specific for endogenous and overexpressed EZH2; α-HA: antibody specific for HA-tagged EZH2; α-GFP: antibody specific for GFP protein expression; α-Actin: antibody specific for β-Actin, used as a loading control. In α-HA panel, the top band represents full size HA-tagged EZH2. Lower bands represent degradation of HA-tagged EZH2. Blots shown are cropped for space considerations. Full blots can be seen in Supplemental Figure 1.

Figure 2. Immunocytochemistry confirmed increase in Histone 3 Lysine 27 Tri-Methylation in EZH2-overexpressing BPH1

BPH1-EZH2 and BPH1-GFP were grown and fixed on coverslips, then stained with an anti-Histone 3 Lysine 27 Tri-Methylation antibody (α-H3K27Me3). GFP panel shows EGFP expression in transduced cells. α-H3K27Me3 panel shows staining for tri-methylation on Histone 3 at Lysine 27. Overlay panel allows direct comparison of GFP and H3K27Me3 panels. DAPI panel shows all cells within the field of view. Scale bar is 25 microns. Nuclear staining intensity was quantified in GFP positive and GFP negative cells in each field. Fold change was calculated by average nuclear intensity of GFP positive cells divided by average nuclear intensity of GFP negative cells.

Figure 3. EZH2 overexpression was sufficient to transform BPH1 cells

BPH1 cells were marked with GFP, EZH2, Akt, or Ras(V12) lentivirus. A. Soft Agar Colony growth assay with all BPH1 sublines. EZH2 overexpression by lentiviral transduction was sufficient to cause transformation of BPH1 cells. BPH1 cells infected with the classical oncogenes Akt and Ras served as positive controls for colony formation. B. Soft Agar growth results plotted by colony number alone. BPH1-GFP control grew very few colonies. BPH1-Akt (p=0.02) showed mild colony formation, BPH1-Ras (p=0.00019) showed stronger colony formation, and BPH1-EZH2 (p=0.00081) fell directly between the two. C. Soft Agar growth results plotted by colony size. BPH1-Akt p=0.0247, BPH1-Ras p=0.004, and BPH1-EZH2 p=0.0087. * p< 0.05, ** p<0.01, *** p<0.001 as calculated by Student's T-Test relative to BPH1-GFP control. Results of four independent experiments are shown.

Figure 4. Overexpression of EZH2 increased invasive behavior of BPH1 cells

BPH1 sublines were assayed for invasive behavior towards either A. 10% FBS containing media or B. 3T3-conditioned serum free media. Cells were allowed to invade for 48 hours through a matrigel-coated 8 micron membrane then were fixed and stained with DAPI. Assays were performed in duplicate. Scale bar is 100 Microns. C. Quantification of cells invaded towards 10% FBS containing media by total membrane area covered. D. Quantification of cells invaded towards 3T3 containing media by total membrane area covered. * p< 0.05, ** p<0.01. AU: Arbitrary Units. All statistical analyses performed using Student's T-Test against BPH1-GFP control values.

Figure 5. EZH2-mediated transformation of BPH1 cell line resulted in tumor growth in SCID mice

BPH1 sublines were implanted in the flanks of SCID mice. A. Equivalent numbers of cells, confirmed by comparable optical signal, were implanted for each group on Day 0. Differences in optical signal at study endpoint demonstrated differential tumor growth rates among the groups. B. Physical caliper measurements demonstrated rapid growth by Ras tumors and slower growth by Akt tumors. BPH1-EZH2 (EZH2) tumors grew very slowly due to limited vasculature and nutrient delivery, but still grew relative to uninfected BPH1 (UI), BPH1-GFP (GFP), and BPH1-GFP/VEGFA (GFP/VEGFA) controls. When angiogenesis was stimulated by VEGF-A expression, BPH1-EZH2/VEGF-A (EZH2/VEGFA) tumors grew faster than BPH1-Akt (Akt), but slower than BPH1-Ras (Ras). C. Time required for each BPH1 tumor type to reach 1cm radius. BPH1-Ras tumors were largest at excision and BPH1-Akt tumors were smaller. Poorly vascularized BPH1-EZH2 tumors were smaller than BPH1-Akt, but well vascularized BPH1-EZH2/VEGF-A tumors were much larger than BPH1-Akt. D. Tumor mass at removal confirmed expansion of BPH1-Ras (p=0.0002), BPH1-Akt (p=0.001), BPH1-EZH2 (p=0.03) and BPH1-EZH2/VEGF-A (p=0.002), but not BPH1-GFP and BPH1-GFP/VEGFA, cells in vivo. qRT-PCR on extracted tumors for (E) HoxA9, (F) ADRB2, and (G) VEGF-A. E. Changes in HoxA9 expression corresponded with increased (BPH1-EZH2, 0.5-fold of BPH1-GFP, p=0.0006) or decreased (BPH1-Akt, 5.4-fold of BPH1-GFP, p=1.8×10⁻⁷) EZH2 activity. F. Changes in ADBR2 expression corresponded with increased (BPH1-EZH2, 0.7-fold of BPH1-GFP, p=0.0003) or decreased (BPH1-Akt, 1.2-fold of BPH1-GFP, p=0.46) EZH2 activity. G. Differences in observed tumor vasculature at excision were corroborated by differences in VEGF-A expression (BPH1-EZH2, p=0.01; BPH1-Akt, p=1.0×10⁻⁷; BPH1-Ras, p=0.0002). * p< 0.05, ** p<0.01, *** p<0.001 as calculated by Student's T-Test relative to GFP control. H. BPH1-EZH2 and BPH1-EZH2/VEGFA tumors stained for exogenous EZH2 expression (HA), GFP, and vasculature (CD31). GFP expression is indicative of cells transduced with either EZH2 or VEGFA virus (in BPH1-EZH2/VEGFA sample) and overlays with HA expression. CD31 expression on vasculature is greatly increased in the BPH1-EZH2/VEGFA tumor compared to the BPH1-EZH2 tumor. Additional panels are shown in Supplemental Figure 4. Scale bar is 200 microns.