Cancer Letters 336 (2013) 53–60

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Cancer Letters

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EZH2 blockade by RNA interference inhibits growth of ovarian cancer byfacilitating re-expression of p21waf1/cip1 and by inhibiting mutant p53

0304-3835/$ - see front matter Published by Elsevier Ireland Ltd.http://dx.doi.org/10.1016/j.canlet.2013.04.012

⇑ Corresponding author. Address: Department of Surgery, Wayne State Univer-sity, John D. Dingell VA Medical Center, Lab. 4242 4646 John R, Detroit, MI 48201,United States. Tel.: +1 313 576 3002; fax: +1 313 576 1112.

E-mail address: rbatchu@med.wayne.edu (R.B. Batchu).1 These authors contributed equally to this work.

Shelly Seward a,b,d,1, Assaad Semaan a,c,d,1, Aamer M. Qazi a,c,d,1, Oksana V. Gruzdyn a,c,d,Sreedhar Chamala a,d, Christopher C. Bryant f, Sanjeev Kumar g, David Cameron a,d,Seema Sethi d,e, Rouba Ali-Fehmi d,e, Robert Morris b,d, David L. Bouwman a,d, Adnan R. Munkarah b,h,Donald W. Weaver a,d, Scott A. Gruber a,c, Ramesh B. Batchu a,c,d,⇑a Laboratory of Surgical Oncology & Developmental Therapeutics, Department of Surgery, Wayne State University, Detroit, MI 48201, United Statesb Department of Ob/Gyn, Wayne State University, Detroit, MI 48201, United Statesc John D. Dingell VA Medical Center, Detroit, MI 48201, United Statesd Karmanos Cancer Institute, Detroit, MI 48201, United Statese Department Pathology, Wayne State University, Detroit, MI 48201, United Statesf MD Anderson Cancer Center Orlando, Gynecologic Oncology, Orlando, FL 32806, United Statesg Division of Gynecologic Surgery, Mayo Clinic Cancer Center, Rochester, MN 55905, United Statesh Department of Women’s Health Services, Henry Ford Health System, Detroit, MI 48202, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 February 2013Received in revised form 29 March 2013Accepted 10 April 2013

Keywords:Enhancer of zeste homolog 2 (EZH2)Epithelial ovarian cancer (EOC)p21waf1/cip1

RNA interference (RNAi)m p53

The enhancer of zeste homolog 2 (EZH2) methyltransferase is a transcriptional repressor. EZH2 is abnor-mally elevated in epithelial ovarian cancer (EOC). We demonstrated that EZH2 knockdown inhibited cellgrowth, activated apoptosis, and enhanced chemosensitivity. Further, silencing of EZH2 resulted in re-expression of p21waf1/cip1 and down-regulation of mutant p53. Finally, EZH2 knockdown contributed toattenuated EOC growth in SCID mice.

Published by Elsevier Ireland Ltd.

1. Introduction

In 2012, there were an estimated 22,280 new cases of epithelialovarian cancer (EOC) and 15,500 deaths in the US, with 5-year sur-vival approaching less than 40% [1,2]. The combination of carbo-platin and paclitaxel, in addition to optimal surgical tumordebulking, represents the standard of care in ovarian cancer treat-ment. Although standard care achieves an initial complete clinicalresponse in many cases, recurrence rates are high and new thera-peutic strategies are needed [3].

The polycomb group complex proteins that form the polycombrepressive complexes (PRC 1 and 2) are chromatin modifiers thatplay an important role in the transcriptional control of variousgenes involved in cell growth and proliferation [4]. Enhancer of

zeste homolog 2 (EZH2) is the catalytic subunit of the PRC2 com-plex which trimethylates histone H3 at lysine residue 27(H3K27me3), often at the sites of tumor suppressor genes [5]. Thisepigenetic modification leads to dense packaging of chromatinmaking it less accessible to the transcriptional machinery, eventu-ally resulting in silencing of these genes.

Although EZH2 is not over-expressed in normal adult differen-tiated tissues, it is often over-expressed in human cancers [6]. Highlevels of EZH2 have been associated with aggressive forms of pros-tate, breast, hepatocellular, gastric cancer, as well as melanomamaking it a potential marker of solid tumors [7]. RNA interference(RNAi)-mediated reduction in EZH2 expression significantly sup-pressed tumor growth and proliferation in multiple cancers [8].Moreover, pharmacologic interference in EZH2 function inducedselective apoptosis in cancer cells but not in normal cells [6].

Although EZH2 over-expression in patients with EOC has beenassociated with tumor progression [9,10] and accumulation ofstem cell-like populations with acquired drug resistance [11], themechanism by which EZH2 achieves its effects remains elusive.

54 S. Seward et al. / Cancer Letters 336 (2013) 53–60

Inhibition of tumor suppressor genes is a key event in carcinogen-esis [12]. Recently, an inverse correlation between the expressionof EZH2 and that of tumor suppressor genes such as E-cadherinin gastric cancer [13], p27kip (CDKN1B) in pancreatic cancer [14],p57kip2 (CDKI) in breast cancer [15] and RAS association domainfamily 1 (RASSF1) in ovarian cancer has been described [16]. Theloss of p21waf1/cip1, a G1 phase cell cycle inhibitor, is frequently ob-served in ovarian cancers and is known to confer a growth advan-tage [17]. In fact, p21waf1/cip1 acts downstream of p53, which allowscells to repair damaged DNA and eventually inhibits carcinogenesis[18]. p53 regulates the expression of p21waf1/cip1 [19] and is oftenmutated in ovarian cancer [20]. Moreover, it was suggested thatwild type p53 actively inhibits the expression of EZH2 contributingto cell cycle arrest [21].

The purpose of this study was to examine the effect of EZH2knockdown by RNAi on the expression of p21waf1/cip1. We demon-strate for the first time in EOC that EZH2 knockdown impairs cellproliferation in vitro and growth of tumor xenografts in vivo byfacilitating the re-expression of tumor-suppressor p21waf1/cip1

and by inhibiting p53 mutant expression, thus providing a novelmolecular mechanism for tumor growth inhibition.

2. Materials and methods

2.1. Reagents, antibodies and cell culture

All reagents, unless specified, were from Sigma Chemical Co, St. Louis. EZH2 andp21waf1/cip1 antibodies were purchased from Millipore (Danvers, MA). Cyclins, cy-clin-dependent kinases (CDKs), poly (ADP-ribose) polymerase (PARP) and b-actinand other apoptosis-related antibodies were purchased from Santa Cruz Biotech-nology (Santa Cruz, CA). Ab6 antibody recognizing amino acids 21–25 that detectsboth mutant and wild-type p53, and Ab3 antibody recognizing amino acids 213–217 that detects only mutant p53 were both obtained from Oncogene ResearchProducts (Cambridge, MA). Advanced stage serous EOC cell lines were obtainedfrom American Type Culture Collection (SKOV-3 ATCC # CRL-HTB-77 and MDAH-2774 ATCC # CRL-10303). Cell lines were propagated in McCoy’s 5A medium sup-plemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin,and 100 mg/ml streptomycin (Thermo Fisher Scientific, Pittsburgh, PA). Cells werecultured in a humidified atmosphere with 5% CO2 at 37 �C. Trypsin (0.05%)/EDTAsolution was used to detach the cells from the culture flask for propagation pur-poses. Non-target negative control shRNA and EZH2 shRNA vector pLKO.1 were ob-tained from Sigma–Aldrich (St. Louis, MO). The non-target control vector sequenceis: 50 CCG GCA ACA AGA TGA AGA GCA CCA ACT CGA GTT GGT GCT CTT CAT CTT GTTGTT TTT 30 . The EZH2-shRNA vector sequence is: 50 CCG GCC CAA CAT AGA TGG ACCAAA TCT CGA GAT TTG GTC CAT CTA TGT TGG GTT TTT G 30 .

2.2. Patients

Using departmental databases, we identified control ovarian tissue and patientswith EOC treated at Wayne State University between 1985 and 2004. Patients withEOC who underwent primary surgery with no prior chemotherapy or radiationtherapy were included in the study (n = 178). Both serous type EOC (n = 131) andnon-serous type EOC (n = 47) were included in this study.

This protocol was approved by the Wayne State University Institutional ReviewBoard and received a waiver of consent for review of archived material and medicalrecords. A retrospective chart review was performed to retrieve demographic, clin-ical, surgical, and pathological data. Survival data were retrieved using the Surveil-lance Epidemiology and End Results (SEERs) database and the institutionalcomputerized Clinical Information System. Surgical staging was determined basedon International Federation of Gynecologic Oncology (FIGO) criteria. Histologic typewas independently determined by two pathologists using previously-describedWorld Health Organization criteria [22]. Tumors were classified into serous, clearcell, endometrioid, and mucinous types, and designated as low and high grade usingM.D. Anderson criteria [23].

2.3. Immunohistochemical (IHC) staining

Tissue microarray sections were stained with antibodies to EZH2. Four- to 5-lm-thick sections were deparaffinized, antigen retrieved by steam treatment in acitrate buffer, quenched for 10 min with 3% hydrogen peroxide, and pre-incubatedwith blocking serum at 1:20 in 2% bovine serum albumin/phosphate-buffered sal-ine (PBS) solution for 15 min at room temperature. After incubation with primaryantibodies, slides were rinsed with PBS, and the secondary antibody was appliedat 1:500 in PBS for 30 min at room temperature. After rinses with PBS for 30 s, slideswere incubated with streptavidin/peroxidase at 1:500 in PBS for 30 min at room

temperature and then rinsed with PBS and incubated for 15 min with 0.06% diam-inobenzidine and counterstained with Harris modified hematoxylin (Fisher Health-care, Hanover Park, IL). The slides were then examined under a transmission lightmicroscope. Staining intensity was scored as low or high.

2.4. Western blotting

Proteins were separated using 4–20% SDS Tris–glycine gels and western transferof proteins to nitrocellulose papers was performed using the iBlot dry blotting de-vice (InVitrogen Corp, Carlsbad, CA). The blocking agent used was 3% nonfat milkpowder, and secondary antibodies conjugated to HRPO (Santa Cruz Biotechnology,Santa Cruz, CA) were used. Antibody reactions were then visualized using the en-hanced chemiluminescence Western blotting detection reagent (Amersham Phar-macia, Uppsala, Sweden).

2.5. Cell proliferation assays

Standard prototype growth curves and the number of viable cells were deter-mined for each cell line (treated and control groups) in triplicate experimentsaccording to the CCK-8 (Dojindo, Gaithersburg, MD) manufacturer’s instructions.Growth curves were plotted as a percentage of the value of non-target scramblednegative control pLKO.1 vector treated minus the value of untreated cells on day0. Day 3 values were considered for determination of the 50% cell proliferation inhi-bition (IC50) for a given treatment. In some cases, a parallel manual count was alsoperformed with trypan blue and counting by the exclusion method using a hemo-cytometer was undertaken to confirm the CCK-8 assay results.

2.6. Colony formation assay

Cells were transfected with 4 lg EZH2-shRNA or control shRNA using Effectene(Qiagen, Valencia, CA). Transfected cells were trypsinized after 48 h and seeded at aconcentration of 1000 cells per well in 6-well plates containing McCoy’s 5A supple-mented with 10% FBS. After incubation for 12 days, the cells were washed and fixedin absolute methanol for 15 min at �20 �C and stained with crystal violet in 25%methanol. The colonies were then counted, photographed, and quantified using Im-ageJ software, a publically-available, Java-based image processing program devel-oped at the National Institutes of Health.

2.7. Cell invasion and wound-healing assays

The cell invasion assay was performed with a modified Boyden chamber usingfilters with a pore size of 8 lm. Briefly, EZH2-shRNA transfected cells (104/650 ll)were added to the upper chamber with the lower chamber filled with mediumand incubated for 18 h at 37 �C in a 5% CO2 humidified chamber. Cells on the uppersurface of the membrane were then removed using wet cotton swabs. The under-side of the chamber was washed twice with PBS, and stained with 5 lM CalceinAM stain (InVitrogen, Carlsbad, CA). The migrated cells on the matrigel coatedmembrane side facing the lower chamber were counted under a fluorescent micro-scope in six random fields and photographed. For wound healing assays, EZH2-shRNA and scrambled vector transfected cells were plated at an equal densityand grown to 80% confluency. Monolayer cells were disrupted mechanically witha pipette tip to generate a wound ridge free of cells and the extent of cell migrationwas measured by their ability to close the artificially-created gap and was docu-mented under a light microscope.

2.8. Apoptosis assay

The apoptosis assay was performed using the Annexin V-FITC apoptosis detec-tion Kit I (Calbiochem, Gibbstown, NJ) according to the manufacturer’s instructions.Briefly, MDAH-2774 cells were transfected with EZH2-shRNA or a control vectorand allowed to grow for 48 h. Both floating and non-floating cells were collectedby trypsinization, washed twice in PBS, and re-suspended in Annexin-V bindingbuffer at a concentration of 106 cells/ml. An aliquot of 100 ll of this suspensionwas stained with 5 ll of Annexin-V-FITC and 5 ll of propidium iodide, and incu-bated for 15 min at room temperature in the dark. Subsequently, the DNA contentof the sub G1 cell population, representing apoptotic cells, was suspended in 400 llof binding buffer and subjected to fluorescence-activated cell sorting (FACS)analysis.

2.9. Chemosensitivity assay

Paclitaxel is a widely-used chemotherapeutic agent against ovarian cancer,which at 1 lM is effective in arresting ovarian cancer cells at the G2/M phase ofthe cell cycle [24]. Untreated, control- transfected, and EZH2-shRNA-transfectedcells were exposed to the mitotic inhibitor paclitaxel at 1 lM concentration. Cellproliferation analysis was then conducted as stated above.

S. Seward et al. / Cancer Letters 336 (2013) 53–60 55

2.10. Chromatin immunoprecipitation (ChIP) assay

ChIP was performed using the Chromatin Immunoprecipitation Assay kit (EZ-Magna ChIPTM A, Upstate, Temecula, CA) according to the manufacturer’s instruc-tions. Briefly, 1 � 107 MDAH-2774 cells, both control and transfected with EZH2-shRNA for 48 h, were cross-linked with 1% formaldehyde for 10 min at 25 �C. Cellswere harvested, lysed, and sonicated (8 � 15 s with 50 s cooling). Soluble chromatinwas immunoprecipitated with Anti-EZH2 (Cat # 07-689, Millipore, Billerica, MA).DNA–protein immune complexes were eluted and reverse cross-linked, and DNAwas extracted using spin filter columns (provided). Standard end-point PCR wasperformed using JumpStart REDTaq PCR mix (Sigma, St. Louis, MO). The presenceof the p21waf1/cip1 promoter domain in immunoprecipitated DNA was identifiedusing the following primers: Forward primer 50-GGT GTC TAG GTG CTC CAG GT-30; Reverse primer 50-GCA CTC TCC AGG AGG ACA CA-30 . The optimal reaction con-ditions were determined for PCR primers. Parameters were denaturation at 94 �Cfor 1 min and annealing at 59 �C for 1 min, followed by elongation at 68 �C for2 min. The amplified p21waf1/cip1 promoter region targeting the �93 region fromthe transcriptional start site was analyzed after 30 cycles on 1% agarose/ethidiumbromide gel electrophoresis showing a 255 bp band. In control samples, the pri-mary antibodies were replaced with non-immune rabbit IgG.

2.11. DNA microarray

DNA microarray analysis was performed using the Human Whole Genome One-Array v5 (Phalanx Biotech, Palo Alto, CA). Total RNA was extracted from control andEZH2-shRNA-transfected samples using RNeasy Mini Kit (Qiagen, Valencia, CA). RNAquality and integrity were determined utilizing an Agilent 2100 Bioanalyzer (PaloAlto, CA) and absorbance at A260/A280. Raw intensity signals for each microarraywere captured using a Molecular Dynamics™ Axon 4100A scanner, measured usingGenePixPro™ Software.

2.12. Mice xenograft studies

Six week old CB17/Cr-SCID mice were obtained from Taconic Farms (German-town, NY) and maintained under pathogen-free conditions (n = 24). MDAH-2774cells (4 � 106) transfected with EZH2-shRNA or control vector were suspended in100 ll of PBS. These two sets of cells were injected subcutaneously into oppositeflanks of each mouse. Once the tumors grew to a palpable size beginning 2 weeksafter injection, tumor diameters were measured every 3 days using digital calipersfollowing a cutaneous shave of the tumor area. Tumor volume in mm3 was calculatedusing the following formula: (L �W2) where L is the maximum length and W is themaximum width of the tumor. Statistical analysis was performed using Student’s un-paired t-test. All animal experiments included in this study were done in compliancewith Institutional Animal Care and Use Committee/Wayne State Universityguidelines.

2.13. Statistical analysis

Statistical analyses were performed using the SPSS for Windows software (ver-sion 13.0; SPSS Inc., Chicago, IL). The correlation between EZH2 expression andother prognostic variables was assessed using the Fisher’s exact test. All data pointswere expressed as the mean ± SD from at least three independent experiments. Allp-values were two-sided and a value <0.05 was considered statistically significant.

3. Results

3.1. EZH2 expression is associated with poor prognosis and itsknockdown leads to inhibition of cell proliferation

The clinical significance of EZH2 in a cohort of ovarian cancerpatients has been studied previously, but not analyzed accordingto histologic subtypes [25]. The tumors of 178 patients were eval-uated for EZH2 expression using immunohistochemical stainingand correlations were made to clinical features. There was a signif-icant positive correlation between high EZH2 expression and indi-cators of poor prognosis in serous histology (serous 76% vs. non-serous 45%, p = 0.0001), advanced FIGO stage (stage III/IV 74% vs.stage I/II 51%, p = 0.008), and high-grade tumors (high 78% vs.low 53%, p = 0.009). Fig. 1A shows staining of control and represen-tative stages in the development of EOC. This correlation promptedus to explore its significance in tumor progression by performingRNAi-mediated knockdown studies.

We observed a significant inhibition in cell proliferation in bothcell lines treated with EZH2-shRNA compared with control-treated

cells. Fast-growing MDAH-2774 cell lines had a 73.6% reduction inthe number of live cells at day 3 compared with the baselinegrowth of control cells, whereas the slow-growing cell lineSKOV-3 had a 44.5% reduction in the number of live cells comparedwith controls by day 3 (Fig. 1B). Western blot analysis confirmedthat transfection with EZH2-shRNA resulted in a substantial de-crease in EZH2 protein levels by 48 h in the transfected cell lines(Fig. 1C). We further analyzed the long-term effect of EZH2 knock-down on growth by performing clonogenic assays on cells at day12. Both cell lines had a decrease in the number and size of colo-nies formed after transfection with EZH2-shRNA (Fig. 1D).

3.2. EZH2 knockdown inhibits cell migration and induces apoptosis

Since EZH2 over-expression is associated with a more aggres-sive tumor phenotype as observed by IHC, we performed woundhealing and trans-well invasion assays to assess whether EZH2silencing can inhibit cell migration and invasion. The MDAH-2774 cell line was selected for the remaining studies because itis faster growing and had the highest rate of EZH2 knockdown.In MDAH-2774 cells transfected with the control vector, we ob-served 96.8% wound healing within 20 h. On the other hand, inMDAH-2774 cells transfected with EZH2-shRNA, we only observeda 35.2% wound closure in the same time period (Fig. 2A). Further,EZH2 knockdown inhibited cell migration by 75% compared withcontrol cells as measured by the Boyden chamber assay (Fig. 2B).

To determine whether EZH2-shRNA growth inhibition was dueto enhanced apoptosis, MDAH-2774-transfected cells were as-sessed using the apoptosis detection kit and counted by FACS.The percentage of late apoptotic cells increased from 3% in the con-trol-transfected cells to 17.5% in the cells transfected with EZH2-shRNA (Fig. 2C). Further, using Western blot analysis, we observedan increase in the expression of pro-apoptotic bak, inhibition of theexpression of anti-apoptotic bcl-2, and increased cleavage of PARPwith EZH2 knockdown in MDAH-2774 cells compared with con-trols by 48 h, indicating activation of the intrinsic apoptotic path-way (Fig. 2D). EZH2 knockdown did not alter b actin expressionlevels indicating the absence of any nonspecific protein down-regulation.

3.3. EZH2 silencing down-modulates mutant p53 resulting in theincrease of chemosensitivity

We then assessed the effect of EZH2 inhibition on the suscepti-bility of MDAH-2774 cells to paclitaxel chemotherapy. Either pac-litaxel exposure or EZH2-shRNA transfection alone resulted in a70% decrease in the number of live cells, while the combined treat-ment resulted in a 95% decrease in the number of live cells by day 5(Fig. 3A). Since we have shown a decrease in metastatic potentialwith EZH2-shRNA inhibition as well as increased sensitivity to che-motherapy, we evaluated the expression of wild-type and mutantp53 after EZH2-shRNA transfection. Western blot analysis withmutant-specific p53 antibody (p53213–217) showed a band of signif-icantly lesser intensity with EZH2 knockdown when comparedwith pantropic p53 antibody that recognizes both wild type andmutant variants (p5321–25) (Fig. 3B).

3.4. EZH2 silencing enhances expression of p21waf1/cip1

We explored the possibility of EZH2 as a modulator of not onlytumor suppressor gene regulation, but of CDK regulation via theCDK inhibitor p21waf1/cip1. ChIP assays using anti-EZH2 antibodiesrevealed the presence of an interaction between EZH2 and thep21waf1/cip1 promoter. This interaction was significantly inhibitedby EZH2-shRNA transfection, indicating a possible epigenetic tran-scriptional re-activation of p21waf1/cip1 expression (Fig. 4A). Deple-

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Fig. 1. Immuno-histochemical staining of EZH2 and effects of its knockdown on cell growth and colony formation. (A) (i) Normal ovarian section-EZH2 negative (40�); (ii–iv)EOC tissues FIGO stages II, III and IV respectively (40�). Six patient samples were stained for each disease stage. (B) Cell growth inhibition by EZH2-shRNA: (i) MDAH-2774and (ii) SKOV-3 cells were transfected and relative cell viability was measured by CCK-8 colorimetric assay at the indicated times as described in Section 2. (iii). Bar graphdepicts the mean percentage of proliferation on day 5 normalized to control from independent experiments performed in triplicate (�p < 0.05). (C) The knockdown of EZH2 in(i). MDAH-2774 and (ii). SKOV-3 cells was evaluated by western blot analysis at indicated time intervals. (D) Inhibition of colony-forming ability of the cells by EZH2depletion: (i) MDAH-2774 and (ii) SKOV-3. (iii) Bar graph depicts the number of colonies expressed as relative intensity (measured by Image j program) normalized to controlfrom independent experiments performed in triplicate (�p < 0.05).

56 S. Seward et al. / Cancer Letters 336 (2013) 53–60

tion of EZH2 showed an up-regulation of p21waf1/cip1 protein levelsalong with down-regulation of global H3k27me3 as assessed byWestern blot analysis corroborating the ChIP assay results (Fig. 4B).

3.5. Inhibition of in vivo tumor growth by EZH2-shRNA

MDAH-2774 control cells and EZH2-shRNA transfected cellswere subcutaneously injected into the dorsal flanks of SCID mice.Tumor xenografts formed by cells transfected with EZH2-shRNAwere smaller than control tumors throughout the study period of50 days (Fig. 5A). The mean tumor volume was significantly lessin the transfected group compared with the control group through-out the study period (Fig. 5B).

4. Discussion

Many studies have demonstrated high levels of expression ofEZH2 in several epithelial cancers, linking its over-expression tomalignant transformation.[7] EZH2’s ability to promote tumor pro-gression in breast and prostate cancers has attracted attention to itas a potential target for anticancer therapies. Based on these obser-vations, our aim was to evaluate the potential of EZH2 gene silenc-ing in preventing the progression of EOC and to establish theassociated signaling mechanisms.

Extensive IHC analysis of 178 patients clearly indicated a signif-icant positive correlation between high EZH2 expression with ser-ous tumor histology, advanced FIGO stage, and high tumor grade.

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Fig. 2. Analysis of cell migration, invasion, and apoptosis with EZH2 knockdown. (A) Cell migration by wound healing assay in MDAH-2774 cells: The extent of closure wasmonitored under phase-contrast microscopy and photographs were taken at 20 h (Upper panel). The p value was compared to the control vector (�p < 0.05), and results werethe mean of independent experiments in triplicate (Lower panel). (B) Trans-well migration assay in Boyden chamber using EZH2-shRNA- and control vector- transfectedMDAH-2774 cells: migrated cells at the lower surface of the trans-well filter were stained and counted (Upper panel). The bar graph represents the average migrated cellnumber from independent experiments performed in triplicate (�p < 0.05) (Lower panel). (C) Control vector- and EZH2-shRNA- transfected MDAH-2774 cells (1 � 105 cells)were fixed, stained both with Annexin V FITC and propidium iodide (20 lg/ml), then processed for flow cytometric analysis. (D) Western blot analysis of PARP protein, bak,bcl-2, and control b actin in MDAH-2774 cells transfected with control vector or EZH2-shRNA. Native and cleaved PARP are indicated by arrows.

S. Seward et al. / Cancer Letters 336 (2013) 53–60 57

This positive correlation between high EZH2 expression andaggressive tumor pathologic features further emphasizes its poten-tial as a target for therapeutic intervention in ovarian cancer.

RNAi-based therapies are especially appealing as they can tar-get tumor-specific proteins that are difficult to target by traditionalpharmacological approaches and with much less toxicity [26]. Ourability to significantly knockdown EZH2 protein expression in mul-tiple ovarian cancer cell lines using shRNA suggests that RNAi is a

feasible modality to decrease expression of EZH2 in ovarian cancer.In addition, we demonstrated that this decrease in expression actu-ally translates into a functional decrease in in vitro cancer cell pro-liferation and colony formation. Halting growth capacity at thecellular level might translate into a potential decrease in clinicaltumor burden.

Metastasis is regarded as a multi-step process and cellular inva-sion constitutes a critical part of this process. Although the vast

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Fig. 3. Transfection of EZH2-shRNA enhances chemosensitivity and inhibits mutant p53 expression in MDAH-2774 cells. (A) Paclitaxel-mediated cytotoxicity with or withoutEZH2-shRNA transfection in MDAH-2774 cells. Con: Control; ES: EZH2-shRNA; P: Paclitaxel. (B) Expression of mutant and wild-type p53 after transfection of EZH2-shRNA.p5321–25 and P5346–55 recognizes both wild type and mutant while p53213–217 antibody detects only mutant p53 under non-denaturing conditions.

58 S. Seward et al. / Cancer Letters 336 (2013) 53–60

majority of work on EZH2 has focused on its activity as a methyltransferase, primarily involved in chromatin restructuring, EZH2has also been detected in the cytoplasm and has been recentlyimplicated in migration and invasion of cancer cells by influencingactin polymerization [27]. In fact, Varambally et al. [28] demon-strated that over-expression of EZH2 was sufficient to increasethe invasive potential of prostate cancer cells. Furthermore, severalstudies documented a correlation between high EZH2 levels andtumor relapse [6], distant metastasis, and invasiveness [29]. In linewith these observations, our study revealed significant inhibitionof cell motility and membrane transduction after EZH2-shRNA-mediated knockdown.

Earlier studies have shown that EZH2 knockdown inhibits cellcycle progression [30]. We observed that inhibition of ovarian tu-mor cell growth with EZH2 knockdown was associated with an in-crease in cell death via apoptosis. This apoptotic effect was alsodemonstrated by the decrease in expression of anti-apoptotic bcl-2 and an increase in expression of the pro-apoptotic bak, eventuallyleading to increased cleavage of PARP, indicating activation of thecaspase 3 mediated intrinsic apoptotic pathway. Further studiesto investigate the exact mechanism EZH2 plays in the apoptoticpathway are underway.

Drug resistance remains a significant impediment to efficaciousclinical cancer treatment. The correlation between EZH2 over-expression and chemo-resistance has been previously described[31]. Since elevated expression of EZH2 is associated with a drugresistant phenotype, therapy inhibiting EZH2 expression would

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Fig. 4. EZH2 interaction with p21waf1/cip1 by ChIP assay and Western blot analysis of mep21waf1/cip1 promoter region. The top panel shows results using Anti-EZH2 antibody and2) transfected MDAH-2774 cells. A 255 bp band is shown targeting �93 region of p21waf1

In the bottom panel, GADPH primers were used as a positive control using anti-acytyl Hiused with normal rabbit IgG antibody as a negative control (lane 2). In both panels, thindicated proteins 48 h after transfection of MDAH-2774 cells with EZH2-shRNA or con

be expected to decrease drug resistance and enhance cancer celldeath. Inhibiting EZH2 expression with siRNA in a known cis-platin-resistant EOC cell line reversed the cells resistance and in-creased cell death [9]. We showed that EZH2 depletion withRNAi in ovarian cancer cells made the cells more sensitive to thecytotoxic effects of a different chemotherapeutic agent, paclitaxel.In the future, the use of non-toxic RNAi therapy may allow the useof lower doses of chemotherapy, decreasing the toxic side-effectsof these agents while increasing overall efficacy.p21waf1/cip1 playsan important role as a G1 to S phase check point to induce cell cy-cle arrest [32]. Loss of p21waf1/cip1 expression is frequently ob-served in EOC and correlates with a more aggressive form of thedisease [17]. ChIP analyses revealed that EZH2 protein in cancercells is closely associated with p21waf1/cip1, pointing to EZH2 asbeing involved with p21waf1/cip1 regulation. Furthermore, knock-down of overexpressed EZH2 by RNAi led to recovery of normalp21waf1/cip1 expression as seen by Western blot analysis. Addition-ally, Western blot confirmed the decreased EZH2 protein expres-sion also correlated with a decrease in tri-methylated H3K27,confirming its functional inhibition. This may be an additionalpathway conducive to EZH2 RNAi therapy.

Mutations in p53 are known to be associated with decreasedsensitivity to chemotherapy [33] and increased tumor invasivenessand metastatic potential. Further, p53 is known to up-regulate theexpression of p21waf1/cip1 [19] but is mutated in EOC [34]. Mutatedp53 enhances metastatic potential, tissue invasiveness, and che-moresistance [35]. Our results indicate that while total expression

e3

/cip1

2 4

RI

0

2 4 6

RI

0

2 4 6

RI

0

ctin

thylated H3k27 and p21waf1/cip1. (A) ChIP assay shows direct binding of EZH2 to thep21waf1/cip1 primers performed with control vector- (lane 1) and EZH2-shRNA- (lane/cip1 promoter after performing a standard PCR reaction as described in the Section 2.stone H3 as the immunoprecipitating antibody (lane 1) and the same primers weree input lanes indicate equal loading of the ChIP samples. (B) Expression levels of

trol vector.

0

20

40

60

80

100

0 10 20 30 40 50 60Tum

or V

olum

e (m

m3)

Control

EZH2 shRNA

Days

Control EZH2-shRNA A B

Fig. 5. Xenograft studies with EZH2-shRNA-transfected MDAH-2774 cell line. (A) In contrast to control vector-transfected cells, the EZH2-shRNA-transfected cells have areduced ability to form tumors in SCID mice. (B) Tumor volume was monitored over time and depicted in a box plot.

EZH2-shRNA mutant p53

p21waf1/cip1

In vivo tumor regression Cell proliferation

Apoptosis

Cell Migration

Cell Invasiveness

Chemosensitivity H3k27me3

Fig. 6. Schematic representation of the effects of EZH2 knockdown leading to tumor regression in EOC.

S. Seward et al. / Cancer Letters 336 (2013) 53–60 59

levels of p53 are unaltered with EZH2 knockdown, there is signif-icantly less expression of mutant p53.

EZH2 knockdown has also been recently linked with the re-expression of the tumor suppressor gene RASSF1 in ovarian cancer[16], corroborating our finding that silencing tumor suppressorgenes is one of the mechanisms by which EZH2 promotes carcino-genesis in EOC. We observed a reduction in the levels of mutantbut not wild type p53 in response to EZH2 knockdown. It is wellestablished that activation of p21waf1/cip1 is an important primaryfunction of p53 in eliciting growth arrest [18]. Thus, in additionto interacting directly with the p21waf1/cip1 promoter to cause itsinactivation, EZH2 might be causing a down-regulation inp21waf1/cip1 expression by increasing the levels of mutant p53, anupstream regulator of p21waf1/cip1 expression. A recent report iden-tified over 60 direct and indirect targets of EZH2 in EOC that in-clude a putative stem cell marker known as ALDH1A1[36].Together, these data support a key role for EZH2 in the mainte-nance of a drug-resistant, tumor-sustaining subpopulation of cellsin ovarian cancers undergoing chemotherapy [11].

In addition to the in vitro tumor suppression seen with EZH2knockdown, our in vivo experiments indicate that EZH2-depletedcells have reduced ability to form tumors in the SCID mouse xeno-graft model. From a translational standpoint, this finding confirmsthe exciting possibility of a novel target in EOC treatment whichcan potentially supplement currently used more toxic chemother-apeutic agents.

The mechanism behind EZH2 over-expression is not entirelyclear, but changes in the levels of specific microRNAs can influenceits expression. Along these lines, our laboratory previously re-ported miR-101-mediated down-regulation of EZH2 in EOC [37],corroborating its negative regulation of EZH2 expression in EOCsimilar to results demonstrated for other cancers [38].

Our observations demonstrate for the first time that knockdownof EZH2 leads to an increase in EOC cell apoptosis, enhanced sensi-tivity to paclitaxel via down-regulation of mutant p53, re-expres-sion of p21waf1/cip1, and in vivo reduction of xenograft tumorgrowth (Fig. 6). Our findings suggest that EZH2-shRNA holds prom-

ise as a potential therapeutic modality for EOC and warrants fur-ther testing for possible clinical application.

Conflict of interest statement

The authors have no financial gains to disclose.

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