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Cancer Research 68, 9964, December 1, 2008. doi: 10.1158/0008-5472.CAN-08-1134
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Activation of the Enhancer of Zeste Homologue 2 Gene by the Human Papillomavirus E7 Oncoprotein

Daniela Holland1, Karin Hoppe-Seyler1, Bettina Schuller1, Claudia Lohrey1, Julia Maroldt2, Matthias Dürst2 and Felix Hoppe-Seyler1

1 Molecular Therapy of Virus-Associated Cancers (F065), German Cancer Research Center, Heidelberg, Germany and 2 Gynäkologische Molekularbiologie, Frauenklinik der FSU Jena, Jena, Germany

Requests for reprints: Felix Hoppe-Seyler, Deutsches Krebsforschungszentrum, Molekulare Therapie Virus-assoziierter Tumore (F065), Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany. Phone: 49-6221-424872; Fax: 49-6221-424852; E-mail: hoppe-seyler{at}dkfz.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The malignant phenotype of human papillomavirus (HPV)-positive cancer cells is maintained by the activity of the viral E6 and E7 genes. Here, we identified the polycomb group gene enhancer of zeste homologue 2 (EZH2) as a novel downstream target for the viral oncogenes in HPV-transformed cells. EZH2 expression was activated by HPV16 E7 at the transcriptional level via E7-mediated release of E2F from pocket proteins. RNA interference analyses showed that continuous EZH2 expression is required for the proliferation of HPV-positive tumor cells by stimulating cell cycle progression at the G1-S boundary. In addition to its growth-promoting activity, EZH2 also contributed to the apoptotic resistance of cervical cancer cells. Furthermore, we found that HPV-positive dysplastic and tumorigenic cervical lesions were characterized by high levels of EZH2 protein in vivo. We conclude that the E7 target gene EZH2 is a major determinant for the proliferation of HPV-positive cancer cells and contributes to their apoptotic resistance. Moreover, EZH2 may serve as a novel therapeutic target for the treatment of cervical cancer. [Cancer Res 2008;68(23):9964–72]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Specific types of human papillomaviruses (HPV) cause cervical cancer in humans. The transforming potential of oncogenic HPVs is closely linked to the expression of the viral E6 and E7 genes. Both the E6 and E7 proteins target important antitumor defense pathways in human cells for functional inactivation. For example, E7 interferes with the growth-inhibitory pRb pathway (1) and can act as a mitotic mutator (2), whereas E6 can block p53-mediated apoptosis (3) and induces telomerase expression (4). To elucidate novel potential downstream cellular targets for the HPV E6/E7 genes, we recently performed a whole genome microarray analysis (~26,000 genes) in HeLa cervical cancer cells, in which endogenous E6/E7 expression was silenced by RNA interference (RNAi). A total of 360 cellular genes were found to be down-regulated, thereby representing potential targets for being activated by E6/E7 (5). They included the enhancer of zeste homologue 2 (EZH2) gene.

EZH2 encodes a polycomb group protein, which acts as a histone methyltransferase (6–8). EZH2 is involved in several key regulatory mechanisms within eukaryotic cells, such as control of embryonal development or of cell proliferation (9). More recently, there is evidence linking EZH2 to human tumorigenesis. For example, for both prostate cancer and breast cancer, EZH2 expression is often observed in proliferative and more aggressive tumor subgroups and has diagnostic and/or prognostic value (10, 11). Notably, EZH2 is not only a potential proliferation marker but seems to directly contribute to the deregulation of cell growth as a bona fide oncogene. Overexpression of EZH2 conferred cellular growth advantage in vitro, promoted invasion, and exhibited oncogenic properties in nude mice (11–14). Vice versa, inhibition of EZH2 expression by RNAi can result in growth inhibition of cancer cells (10, 15, 16). Here, we identify EZH2 as a novel target gene being activated by oncogenic HPVs.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cells and transfections. HPV18-positive HeLa cervical carcinoma cells, HPV16-positive CaSki and SiHa cervical carcinoma cells, HPV-negative U2OS osteosarcoma, and MCF-7 breast cancer cells were all maintained in DMEM (pH 7.2) supplemented with 10% FCS, 50 units/mL penicillin, and 50 µg/mL streptomycin sulfate. Primary human cervical keratinocytes were grown in keratinocyte growth medium 2 with supplements (Promocell). Plasmids were transfected by calcium phosphate coprecipitation (17) into cell lines or with Fugene HD (Roche Diagnostics) into primary human keratinocytes. Synthetic small interfering RNAs (siRNA) were transfected with Oligofectamine (Invitrogen). For long-term treatment with synthetic siRNAs (>72 h), cells were transfected a second time at 72 h. For transfections, cells were plated on 6-cm dishes at 30% to 50% confluency. Oligofectamine (8 µL) and siRNAs at final concentrations between 50 and 200 nmol were both diluted in Opti-MEM I reduced serum medium (Invitrogen) and mixed in a final volume of 400 µL transfection solution. Complete medium was exchanged against 1.6 mL Opti-MEM I, which was supplemented 4 h later with 1 mL DMEM containing 30% FCS.

Plasmids and synthetic siRNAs. siRNAs either were expressed from vector pSuper or were chemically synthesized (Dharmacon Research). siRNAs were generated against the following target sequences, as previously described (5, 18): si16E6/E7, 5'-CCGGACAGAGCCCAUUACA-3' (HPV16 nucleotides 700–718); si18E6/E7, 5'-CCACAACGUCACACAAUGU-3' (HPV18 nucleotides 755–773); si16E6, 5'-ACCGUUGUGUGAUUUGUUA-3' (HPV16 nucleotides 385–403); and si18E6, 5'-CUAACACUGGGUUAUACAA-3' (HPV18 nucleotides 385–403). EZH2 targeting siRNA siEZH2-1 (13) and vector pSuper-p53 (19) have been characterized in detail before. siEZH2-2 (5'-GAAUGGAAACAGCGAAGGA-3'; EZH2 nucleotides 341–359; NM_004456) is provided as a predesigned siRNA (Dharmacon Research). The siRNA "siControl" (5'-UAGCGACUAAACACAUCAA-3'; Dharmacon Research) contains at least four mismatches to all known human genes. Luciferase reporter plasmids EZH2wt (pGL3bprhEZH2; –1095/+48), EZH2{Delta}E2F (pGL3bprhEZH2; –151/+48; ref. 13), and p21Luc (17) have been previously described. E7 expression plasmid pCMV16E7HA-Flag (20) was kindly provided by Dr. Karl Münger (Brigham and Women's Hospital, Boston, MA). Corresponding plasmids pCMV18E7HA-Flag, pCMV11E7HA-Flag, pCMV6bE7HA-Flag, and pCMV16E7({Delta}DLYC)HA-Flag were created accordingly in the same vector backbone using inserts from the previously described pOZ-CE7 vector system (21).

Luciferase assays. Cells were plated on 6-cm dishes and transfected by calcium phosphate coprecipitation with 2 µg of the respective reporter gene constructs and either 0.5 µg of E6 or E7 expression plasmids or the corresponding basic vector. To correct for possible variations in transfection efficiencies, 0.5 µg of the internal standards CMV-Gal or 1.5 µg actin-Gal was included (17). To study the effects of p53 depletion on EZH2 promoter activity, 2 µg of reporter gene construct and 4 µg of pSuper-p53 or basic vector pSuper, respectively, were cotransfected with 0.25 µg CMV-Gal. At 48 to 72 h after transfection, cells were harvested and luciferase activities were determined, as further detailed elsewhere (17). Each value represents the mean of at least two independent experiments, each performed in duplicate, and SDs are indicated.

Protein and RNA analyses. Protein extracts were prepared 16 to 96 h after transfection, as indicated and described previously (17). For Western blot analyses, 30 µg of protein extract were separated by 12.5% SDS-PAGE, transferred to an Immobilon-P membrane (Millipore), and analyzed by enhanced chemiluminescence (Amersham Biosciences). The following antibodies were used: anti-18E7 antibody 18E7C (20), anti-16E7 (NM2) antibody (kind gift of Dr. Martin Müller, German Cancer Research Center, Heidelberg, Germany), anti-tubulin antibody CP06 (Calbiochem), anti-EZH2 antibody AC22 (Cell Signaling), anti-p21WAF-1 antibody OP64 (Calbiochem), anti-p53 antibody DO-1 (BD Pharmingen), anti-cyclin D1 antibody DSC6 (Cell Signaling), anti-cyclin E antibody HE-12 (Santa Cruz Biotechnology), rabbit anti-Flag antibody (Sigma), and anti-β-actin antibody AC-74 (Sigma). Northern blot analyses were performed as described (22). To account for loading variations between individual lanes, filters were reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Colony formation assays. Cells were grown on 6-cm dishes and transfected with 6 µg of individual pSuper vectors, as indicated, together with 0.2 µg of pSV2Neo to allow for selection of transfected cells. After 8 to 12 d of selection for G418 (Invitrogen) resistance, colonies were fixed with formaldehyde and stained with crystal violet.

Cell proliferation assays. 5-Bromo-2'-deoxyuridine (BrdUrd) incorporation was measured using the Cell Proliferation ELISA, BrdUrd (Roche Diagnostics). Briefly, cells were treated for 48 or 72 h with either 96 µmol/L A771726 (Axxora) or 1 mmol/L hydroxyurea (Calbiochem). Subsequently, cells were labeled with BrdUrd for 2 h and further processed as described in the supplier's manual.

Cell cycle analyses. Cells were trypsinized, pelleted, and resuspended in citrate buffer. Subsequently, PBS containing 1 mg/mL RNase A (Roche Diagnostics), 20 µg/mL propidium iodide (Sigma), 0.5% NP40, and 50 mmol/L EDTA was added. Cell cycle analyses were performed using a FACSCalibur (BD Biosciences) with CellQuest Pro software provided by the manufacturer. Apoptotic cells were excluded and quantitation of the percentage of cells in individual cell cycle phases was performed using FlowJo software (Tree Star, Inc.), applying the Dean-Jett-Fox model (23). Statistical significance of differences in measured variables (G1 and S phase populations) between controls and treated groups was determined by unpaired t test. Differences were considered significant at P < 0.05.

Senescence assays and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling analyses. For analysis of senescence, cells were grown as described for colony formation assays. Staining for senescence-associated β-galactosidase (SA-β-Gal) activity was performed 10 d after transfection, following previously described protocols (24). Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) analyses for detection of apoptosis were performed by using the in situ cell death detection kit (Roche Diagnostics) 72 h after transfection with synthetic siRNAs. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Roche Diagnostics). Apoptotic strand breaks and total DNA were visualized by epifluorescence microscopy (Olympus Vanox-T).

Immunohistochemical analysis. Serial cryosections (5 µm) of normal cervical tissue, high-grade cervical intraepithelial neoplasia (CIN; CIN2+), and cervical carcinomas were mounted on 3-aminopropyl-triethocysilane–coated slides and fixed in 4% paraformaldehyde in TBS for 4 min at 4°C. Endogenous peroxidase activity was blocked by 0.6% hydrogen peroxidase in TBS for 30 min at room temperature. Anti-EZH2 (0.8 µg/mL; BD Pharmingen) or anti-Ki-67 (rabbit polyclonal, 1:60; USBiological) antibodies were incubated overnight at 4°C. Subsequent thorough washing in TBS-1% Tween 20 was performed. Specifically bound primary antibodies were detected by the use of the EnVison technology (DAKO) in accordance with the manufacturer's instructions. Finally, all sections were counterstained with hematoxylin and mounted in glycerol gelatin.

RNA-RNA in situ hybridization. Digoxigenin (DIG)-UTP–labeled sense and antisense RNA were generated from linearized HPV16 E6*I-E7 (98–670 bp) plasmid DNA by in vitro transcription using T3 RNA polymerase (DIG RNA labeling mix, Roche Diagnostics) followed by partial hydrolysis. This cDNA was cloned from a HPV16-positive cervical carcinoma (25) and detects all mRNA encoding spliced/unspliced E6 and E7. For RNA-RNA in situ hybridization, serial cryosections (7 µm) of normal cervical tissue, high-grade CIN (CIN2+), and cervical carcinomas were mounted on silane-coated slides, fixed in 4% paraformaldehyde in 2x saline-sodium phosphate-EDTA [SSPE; 0.3 mol/L NaCl, 23 mmol/L NaH2PO4, 2 mmol/L EDTA (pH 7.4)], digested with proteinase K (0.5 µg/mL), and prehybridized at 42°C for 3 h. Each section was covered with 20 µL of hybridization solution [50% formamide, 2x SSPE, 10% dextran sulfate, 10 mmol/L Tris-HCl (pH 7.5), 1x Denhardt's solution, 500 µg/mL tRNA, 100 µg/mL herring sperm DNA, 0.1% SDS] containing 150 ng/µL DIG-RNA probe and incubated at 42°C overnight. After hybridization, slides were washed once in 50% formamide, 2x SSPE, 0.1% SDS for 30 min at 50°C; treated with RNase A (50 µg/mL in 2x SSC, 0.1% SDS); and washed again in 50% formamide, 0.5x SSPE, 0.1% SDS for 30 min at 37°C. For detection, the tyramide signal amplification system (Perkin-Elmer) was used. In brief, slides were rinsed in wash buffer (TNT) and endogenous peroxidase activity was blocked by 0.6% hydrogen peroxidase in TNT at room temperature followed by 30-min incubation with antibody conjugate [anti-DIG-POD, 1:500, in blocking buffer (2-nitro-5-thiobenzoate); Roche Diagnostics] at 37°C. Unspecific bound antibodies were removed by washing thrice for 5 min each in TNT. Sections were incubated in the dark with biotin-labeled tyramide (1:50 in amplification diluent) for 10 min. After three washes of 5 min each in TNT, streptavidin-horseradish peroxidase (HRP; diluted 1:1,000 in blocking buffer) was applied to the sections for 30 min. Finally, sections were rinsed thrice for 5 min each in wash buffer followed by 5-min incubation with the standard HRP chromogenic substrate 3,3'-diaminobenzidine at room temperature in the dark. For counterstaining, 2-min incubation in hematoxylin was performed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Silencing of E6/E7 expression blocks EZH2 expression. Previous microarray studies of HeLa cells raised the possibility that EZH2 may represent a novel target gene, which is activated by the HPV E6/E7 oncogenes (5). To validate this observation, we analyzed EZH2 expression on silencing of E6 expression alone, or on combined silencing of E6/E7 expression, by Western and Northern blot analysis of HPV-positive cancer cells. As shown in Fig. 1A , we observed a strong down-regulation of EZH2 protein levels after blocking HPV18 E6/E7 expression in HeLa cells. In contrast, a siRNA targeting E6 alone did not affect EZH2 protein concentrations but led to an increase of p53 and p21 protein levels, as expected for interfering with E6 expression (17). Strong repression of EZH2 expression on E6/E7 silencing was also observed at the RNA level (Fig. 1A). To investigate whether these effects are a peculiarity of HeLa cells and whether they can be also observed for other oncogenic HPV types, we analyzed HPV16-positive SiHa and CaSki cervical cancer cells. Again, inhibition of E6/E7 expression, but not of E6 expression alone, resulted in a strong reduction of EZH2 protein levels (Fig. 1B). To further control the specificity of these effects, we analyzed HPV-negative U2OS osteosarcoma and MCF-7 breast cancer cells. As shown in Fig. 1C, none of the HPV-targeting siRNAs affected EZH2 expression in these cells, corroborating that the RNAi-mediated repression of EZH2 expression is HPV dependent. Time course experiments revealed that a significant reduction of E7 levels was detectable at 18 to 24 hours following siRNA treatment and preceded the reduction of EZH2 levels, which started to be visible at 48 hours (Fig. 1D). These observations would be consistent with the idea that down-modulation of EZH2 expression is a consequence of silencing viral E6/E7 expression. Taken together, these results show that EZH2 expression is strongly reduced in HPV-positive cervical cancer cells on silencing of endogenous E6/E7 expression, thereby defining EZH2 as a novel target gene for activation by the HPV oncogenes.


Figure 1
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  		Figure 1. Silencing of E6/E7 expression blocks EZH2 expression. A, left, Western blot analysis of HPV18-positive HeLa cells 96 h after transfection with siRNAs against HPV18 E6/E7 (si18E6/E7), E6 alone (si18E6), or EZH2 (siEZH2-1 and EZH2-2). siControl, control siRNA. Tub, tubulin protein to monitor comparable loading between lanes. Right, Northern blot analysis of EZH2 mRNA 96 h after transfection of HeLa cells with si18E6/E7 or siEZH2-1. GAPDH, loading control. B, Western blot analysis of HPV16-positive CaSki and SiHa cells 96 h after transfection with siRNAs against HPV16 E6/E7 (si16E6/E7) or E6 alone (si16E6). C, Western blot analysis of U2OS osteosarcoma and MCF-7 breast cancer cells 96 h after transfection with si18E6/E7, si18E6, or siEZH2-1. D, time course experiments analyzing EZH2 and HPV18 E7 protein levels following si18E6/E7 treatment of HeLa cells for the indicated time periods. All experiments were independently repeated at least thrice, with consistent results.

 
Transcriptional stimulation of the EZH2 promoter by E7. To investigate whether E6/E7-mediated regulation of EZH2 expression occurs at the transcriptional level, we performed reporter gene assays using a construct in which the luciferase gene is under transcriptional control of the wild-type EZH2 promoter (13). On ectopic expression in p53 wild-type MCF-7 cells, HPV16 E6 did not affect the EZH2 promoter and HPV18 E6 very marginally stimulated its activity (Fig. 2A ). Under the same experimental conditions, both HPV16 and HPV18 E6 strongly reduced the activity of the p21 promoter (Fig. 2A), which is a transcriptional target for p53 (26), showing that they are expressed at functionally relevant levels. The slight activation of the EZH2 promoter by HPV18 E6 in the reporter gene analyses may be mediated via E6-induced degradation of p53 because p53 has been found to act as a potential inhibitor of EZH2 expression (15). Indeed, we found a weak activation of the EZH2 promoter on RNAi-mediated inhibition of p53 expression at conditions where p21 promoter activity is down-regulated (Fig. 2A).


Figure 2
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  		Figure 2. HPV E7 regulates EZH2 expression. A, luciferase reporter gene assays of EZH2 (black columns) and p21 (dashed columns) promoter activities on coexpression of HPV16 E6 (16E6) or HPV18 E6 (18E6) in MCF-7 cells. Indicated are relative luciferase activities (RLA) above the activities of the individual reporter constructs in the presence of cotransfected vector control (arbitrarily set at 1.0). Bars, SD. Right, EZH2 (black columns) and p21 (dashed columns) promoter activities on cotransfection of the p53-targeting siRNA vector pSuper-p53 in MCF-7 cells. Indicated are relative luciferase activities above the activities of the individual reporter constructs in the presence of the cotransfected vector control pSuper (arbitrarily set at 1.0). B, MCF-7 cells were transfected with luciferase reporter plasmids containing either the wild-type EZH2 (black columns) or an EZH2 promoter construct from which the putative E2F-binding sites have been deleted (EZH2{Delta}E2F; white columns). Luciferase activities on cotransfection of expression vectors for HA-Flag–tagged HPV18 E7, HPV16 E7, HPV16 E7{Delta}DLYC, HPV11 E7, and HPV6b E7 proteins are indicated relative to the activities of the reporter constructs in the presence of cotransfected vector control (arbitrarily set at 1.0). E7 expression levels in MCF-7 cells are monitored by Western blot (right) using an anti-Flag antibody. C, analysis of EZH2 promoter activities in primary human cervical keratinocytes on cotransfection of expression vectors for HPV18 E7, HPV16 E7, HPV16 E7{Delta}DLYC, HPV11 E7, and HPV6b E7. Luciferase activities are indicated relative to the activities of the reporter construct in the presence of the cotransfected vector control (arbitrarily set at 1.0). E7 expression levels in primary cervical keratinocytes are monitored by Western blot (right) using an anti-Flag antibody. D, HPV18-positive HeLa, HPV16-positive SiHa cells, and primary human cervical keratinocytes (CxK) were treated with the antiproliferative agents A771726 and hydroxyurea (HU). Growth inhibition was measured by BrdUrd assay (percent inhibition of BrdUrd incorporation). EZH2 and HPV E7 expression (HeLa: HPV18 E7; SiHa: HPV16 E7) was monitored by Western blot. Actin, loading control. Repression of EZH2 expression in cervical keratinocytes was approximately 80% (A771726) or 60% (hydroxyurea), as assessed by densitometry. Experiments were independently repeated at least thrice, with consistent results.

 
In contrast to the, at most, marginal effects of E6 on EZH2 promoter activity, we observed that ectopic expression of both high-risk (HPV16 or HPV18) and low-risk (HPV6b and HPV11) E7 reproducibly led to an activation of the wild-type EZH2 promoter in MCF-7 cells (Fig. 2B). Because EZH2 is a putative E2F target, we investigated the effects of the HPV E7 proteins on a truncated EZH2 promoter from which the putative E2F-binding sites have been deleted (13). We found that the mutant promoter has almost completely lost its responsiveness toward E7 (Fig. 2B). This indicates that E7-induced stimulation of the EZH2 promoter is mediated by E2F factors.

Because E7 can activate E2F-responsive promoters via release of E2F from pRb and from other pocket proteins, such as p107 and p130 (1), we investigated the dependence of E7-mediated EZH2 activation on the interaction with pocket proteins. As shown in Fig. 2B, a mutant E7 protein (16E7{Delta}DLYC), which has lost the ability to bind to pocket proteins (21), was no longer able to transactivate the EZH2 promoter, albeit expressed at similar levels as wild-type HPV16 E7.

These investigations were extended to primary human cervical keratinocytes, the natural host cells for HPVs. Again, HPV16, HPV11, and HPV6b E7 were able to transactivate the EZH2 promoter (Fig. 2C), in line with the ability of these factors to induce E2F-dependent promoters (27, 28). This transactivation potential was virtually completely lost in the 16E7{Delta}DLYC mutant. Interestingly, we observed that HPV18 E7, in contrast to the other investigated E7 proteins, did not detectably activate the EZH2 promoter in primary human cervical (Fig. 2C) or in primary human foreskin keratinocytes (data not shown).

Because EZH2 expression is strongly cell growth regulated and elevated in growing cells (13), we investigated whether EZH2 induction may be a secondary effect of E7-induced cell proliferation. To this end, HPV18-positive HeLa cells, HPV16-positive SiHa cells, and human primary cervical keratinocytes were treated with the antiproliferative antimetabolites A771726 (29) and hydroxyurea (30). Both agents led to efficient growth inhibition, as shown by a strong reduction of BrdUrd incooperation (Fig. 2D). In line with being growth regulated, EZH2 concentrations were substantially reduced in growth-inhibited primary keratinocytes (Fig. 2D). In contrast, however, no reduction was observed in both HPV16- and HPV18-positive cells, which maintained endogenous E7 expression under growth inhibition.

EZH2 is required for the proliferation of HPV-positive cancer cells. Because EZH2 can possess growth-stimulatory activities (10, 12–15), we investigated the consequences of RNAi-mediated silencing of EZH2 expression in HPV-positive cancer cells. Cell cycle analyses in HeLa cells revealed that interfering with EZH2 expression resulted in a significant (P < 0.05) cell cycle arrest in G1 (Fig. 3A ). Thus, EZH2 activity is required for G1-S cell cycle progression in HPV-positive cancer cells.


Figure 3
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  		Figure 3. EZH2 is required for the proliferation of HPV-positive cervical cancer cells. A, cell cycle analyses of HeLa cells treated with siControl, siEZH2-1, siEZH2-2, or si18E6/E7, respectively. Columns, percentages of cells in the G1, S, or G2 phases of the cell cycle; bars, SD. B, Western blot analysis of cyclin D1 and cyclin E levels 60 h after RNAi-mediated EZH2 depletion of HeLa cells, synchronized by serum starvation. C, colony formation assays of HPV18-positive HeLa cells and HPV16-positive CaSki and SiHa cells. HPV-negative U2OS cells are growth inhibited on EZH2 depletion (13) and served as positive control. Cells were stably transfected with pSuper-EZH2-1, pSuper-16E6/E7 (CaSki and SiHa), or pSuper-18E6/E7 (HeLa and U2OS), respectively. Control, pSuper or pSuper-control transfectants. Experiments were independently repeated at least thrice, with consistent results.

 
EZH2 has been implicated in stimulating expression of a series of proliferative genes, including several cyclin genes, such as cyclin D1 and cyclin E (13). Expression of both proteins was strongly reduced on RNAi-mediated repression of EZH2 expression (Fig. 3B), indicating that the G1-S arrest on EZH2 depletion is linked to reduced expression of growth-promoting cyclins.

Next, we analyzed the phenotypic effects of long-term inhibition of EZH2 expression in HPV-positive cancer cells. We performed colony formation assays following stable transfection of pSuper constructs, which block EZH2 expression by RNAi. As observed for HPV-negative U2OS control cells, the proliferation of which depends on EZH2 (13), we found that interference with EZH2 expression led to a clear decrease in the colony formation capacity of HPV18-positive HeLa as well as of HPV16-positive SiHa and CaSki cells. Similar effects were observed on RNAi-mediated silencing of E6/E7 expression in all HPV-positive cell lines examined (Fig. 3C).

At late time points (10 days of siRNA treatment), silencing of E6/E7 expression resulted in efficient induction of senescence of HeLa cells (Fig. 4A ). This was indicated by the appearance of flattened and enlarged cells with long cytoplasmic projections and by induction of the SA-β-Gal gene (24), a well-characterized marker for HeLa cell senescence (31). On the contrary, long-term inhibition of EZH2 expression resulted in only very few residual cells, which did not exhibit SA-β-Gal staining (Fig. 4A).


Figure 4
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  		Figure 4. Silencing of E6/E7, but not of EZH2 expression, leads to senescence. A, cells were stably transfected with either pSuper-EZH2-1, pSuper-18E6/E7, or pSuper control. Ten days after transfection, cells were stained for expression of the senescence marker SA-β-Gal (blue). Top, bright-field microscopy; bottom, phase-contrast microscopy. B, Western blot analysis of p53 and p21 levels in HeLa cells, following silencing of EZH2 or E6/E7 expression, respectively. Tub, tubulin as loading control. All experiments were independently repeated at least thrice, with consistent results.

 
Activation of the p21 gene has been implicated to play a major role for the induction of senescence on silencing of HPV E6/E7 oncogene expression (31). We therefore investigated whether the phenotypic differences between siEZH2-treated and siE6/E7-treated cells may be linked to a differential p21 response. As shown in Fig. 4B, RNAi-mediated interference with E6/E7 expression led to strong increases of both p53 and p21 protein levels. On the contrary, silencing of EZH2 expression led to only a marginal increase of p53 and p21 concentrations. Thus, induction of senescence correlated with the differential induction of p21 on silencing E6/E7 or EZH2 expression, respectively.

EZH2 contributes to the apoptotic resistance of HPV-positive cancer cells. The strong reduction of cell numbers following long-term inhibition of EZH2 expression raised the question whether EZH2 may be involved in apoptosis regulation of HPV-positive cancer cells. Therefore, we repressed EZH2 or E6/E7 expression in HeLa cells by RNAi and subsequently performed TUNEL analyses to detect apoptotic cells. Silencing of E6/E7 expression by RNAi did not affect apoptosis rate (Fig. 5 ), as has been previously reported (32). In contrast, inhibition of EZH2 expression was linked to increased apoptosis, as indicated by condensed chromatin and positive TUNEL signals (Fig. 5). Thus, in addition to its proliferative activity, EZH2 also contributes to the apoptosis resistance of HeLa cells.


Figure 5
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  		Figure 5. Silencing of EZH2, but not of E6/E7 expression, induces apoptosis in HeLa cells. A, exemplary TUNEL analyses of HeLa cells treated for 72 h with siEZH2-1, si18E6/E7, or siControl, respectively. Nuclei were stained with DAPI. B, percentage of TUNEL-positive cells on treatment with siEZH2-1, siEZH2-2, or si18E6E7. Experiments were performed at least thrice, and three independent microscopic fields were examined for each sample. Bars, SD.

 
HPV-positive lesions express high levels of EZH2 in vivo. The identification of EZH2 as a target gene for the HPV oncogenes in vitro raised the question about the relation between HPV E6/E7 and EZH2 expression in vivo. To this end, we analyzed biopsies from normal cervical tissue, HPV16-positive high-grade lesions (CIN2+), and HPV16-positive cervical carcinomas. Ten biopsies of each entity were investigated. We regularly found that HPV-positive areas expressing E6/E7 mRNA (Fig. 6 ), or which stained positive for the surrogate HPV marker p16 (data not shown), also exhibited elevated levels of EZH2 protein. Typical examples are provided in Fig. 6, showing a clear spatial overlap between (a) the signals for E6/E7 mRNA, (b) expression of EZH2 protein, and (c) expression of the proliferation marker Ki-67 in a squamous cell carcinoma of the cervix and in a CIN2/3 lesion. These findings are consistent with our in vitro evidence showing stimulation of EZH2 expression through the activity of the viral E7 oncogene. Notably, we also observed increased expression of EZH2 distinctly in the proliferating parabasal layers of normal cervical tissue, as indicated by the colocalization of Ki-67 and EZH2 signals (Fig. 6). These data indicate that activation of EZH2 expression may also play a role for the proliferation of parabasal keratinocytes in normal cervical epithelium.


Figure 6
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  		Figure 6. HPV-positive lesions express high levels of EZH2 in vivo. Analysis of a squamous cell carcinoma of the cervix, a CIN2/3 lesion adjacent to normal epithelium (boundaries between normal tissue to the left and dysplastic tissue to the right are indicated by arrows), and normal cervical epithelium for expression of E6/E7 mRNA by in situ hybridization and for EZH2 or Ki-67 protein expression by immunohistochemistry. HE, hematoxylin staining. Bar, 200 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
The present work defines EZH2 as a novel target gene that is activated by the HPV E7 oncogene in cervical cancer cells. Moreover, RNAi analyses revealed that continuous expression of EZH2 is required for the proliferation of HPV-positive cancer cells in vitro and contributes to their apoptotic resistance.

HPV-mediated stimulation of EZH2 expression occurs, at least in large parts, through transcriptional activation of the EZH2 promoter via E7-mediated release of E2F factors from inhibitory pocket proteins. This notion is supported by the findings that (a) E7 can activate the EZH2 promoter in luciferase reporter gene assays, (b) an EZH2 promoter devoid of E2F-binding sites is no longer inducible, and (c) a HPV16 E7 mutant defective for binding to pocket proteins lost the ability to transactivate the EZH2 promoter. Furthermore, the observation that growth-inhibited HPV-positive cancer cells did not exhibit a reduction of EZH2 expression levels indicates that activation of EZH2 expression is not just a secondary effect in response to E7-induced cell proliferation.

The EZH2 promoter was also stimulated by the E7 proteins of low-risk HPV types HPV6b and HPV11, in line with the ability of these proteins to activate E2F-regulated promoters (27, 28). Notably, we observed that HPV18 E7, in contrast to all the other investigated E7 proteins, did not detectably stimulate the EZH2 promoter in primary cervical or foreskin keratinocytes despite clearly activating the EZH2 promoter in MCF-7 cells. This suggests a cell type–dependent regulation of HPV18 E7 activity in keratinocytes. In this context, it should be noted that HPV18 is preferentially associated with the development of adenocarcinomas and relatively rarely found in the much more common squamous cell carcinomas of the cervix (33). One may speculate that differential activities of individual E7 proteins in a specific cellular context could be related to the predominance of the respective HPV types in carcinomas of different histopathology.

Alike an inhibition of E6/E7 expression, silencing of EZH2 had a profound antiproliferative effect in HPV-positive cancer cell lines. The mechanisms by which the EZH2 protein can augment cellular proliferation are still poorly defined but may include the stimulation of cyclin expression (13). Other potential pathways associated with growth stimulation by EZH2 include interference with retinoic acid receptor signaling (34), stimulation of Wnt signaling (35), and repression of tumor suppressor genes, such as p16 (36, 37). We did not obtain experimental evidence for a direct interaction between HPV16 and HPV18 E7 with EZH2, as assessed by mammalian two-hybrid analyses (data not shown).

Growth inhibition on interference with EZH2 expression has been linked to G1 arrest in some tumor cell lines (13, 35), whereas G2-M arrest has been observed in others, either in combination with G1 arrest (13) or alone (10, 15). In HeLa cells, we detected a clear increase in G1 and a decrease in S-phase populations on intracellular EZH2 depletion, without evidence for an increase in G2 populations. Thus, our data indicate that the EZH2 protein stimulates cell cycle progression in HPV-positive cancer cell lines primarily at the G1-S boundary.

Both long-term silencing of EZH2 or E6/E7 expression led to efficient inhibition of HPV-positive cancer cells in colony formation assays. However, the underlying mechanisms are different. Inhibition of E6/E7 resulted in induction of senescence. This was associated with p53 reactivation, as expected from blocking E6 expression, and strong reconstitution of p21 expression. In contrast, interference with EZH2 expression led to only a marginal induction of p53 and p21. This lack of efficient p21 induction could be explained by the retention of endogenous E6 expression, which leads to continuous p53 degradation (38).

On the other hand, silencing of EZH2, but not of E6/E7 expression, was associated with the induction of apoptosis. There are, thus far, only very limited and partly contradictory data available about possible effects of EZH2 on apoptosis regulation. No obvious signs for apoptosis induction were observed in a study of primary human embryonic lung fibroblasts, following RNAi-mediated silencing of EZH2 expression (15). Similarly, siRNA treatment of nontumorigenic breast epithelial MCF-10A cells did not induce apoptosis; however, it led to increased apoptosis in MCF-7 breast cancer cells (39). We also observed a modest induction of apoptosis on interference with EZH2 expression in certain kidney cancer cell lines, indicating that the significance of EZH2 for apoptosis regulation may be influenced in a cell type–dependent manner (40). In line, siRNA-mediated inhibition of EZH2 expression did not affect the viability of adherently growing prostate carcinoma precursor cells but rather led to anoikis (41). Our finding that interference with EZH2 expression resulted in increased apoptosis in HeLa cells shows that EZH2 has a role in apoptosis regulation in adherently growing cervical cancer cells. Induction of apoptosis was relatively modest (affecting 10–20% of treated cells) and comparable with that observed on repression of EZH2 expression in kidney cancer cells (40). In view of the profound inhibitory effects observed in colony formation assays, it is likely that growth inhibition is the primary effect of EZH2 repression.

If E7-mediated activation of EZH2 expression is relevant under in vivo conditions, one would expect that HPV-positive lesions contain high levels of EZH2 protein. This is indeed the case, as we found in an analysis of a series of HPV-positive lesions where we invariably observed a strict correlation between HPV E6/E7 oncogene expression and EZH2 protein levels. However, there was no obvious correlation between EZH2 protein levels and the degree of malignancy of the lesions. Whereas the regular detection of high EZH2 protein levels in HPV-positive lesions is fully compatible with EZH2 being a target for oncogenic HPVs, it is notable that we also clearly detected EZH2 expression in the parabasal cells of the cervical mucosa, which represents the main proliferative cell layer in normal cervix (42). Thus, EZH2 expression is unlikely to serve as a novel tumor marker for cervical cancer, in contrast to the situation reported for breast cancer where EZH2 expression is elevated in precancers and cancers but nearly undetectable in normal breast samples (43). Yet, our functional data about the growth-promoting and antiapoptotic EZH2 activities in cervical cancer–derived cells, as well as the functional studies in other tumor systems cited above, strongly argue against the possibility that EZH2 is only an innocent proliferation marker in cervical cancer. Rather, it seems possible that oncogenic HPVs could conquer similar pathways to stimulate tumor cell growth as they are physiologically used by the parabasal cells of the cervix to activate their proliferation.

The findings that EZH2 contributes to both the proliferation and the apoptotic resistance of cervical cancer cells furthermore indicate that EZH2 may serve as a novel therapeutic target for the treatment of cervical cancer. In line, there is recent preclinical data showing that interference with EZH2 is not only growth inhibitory in vitro but can also exert antitumor effects in mouse models (16, 41, 44). Future strategies to inhibit EZH2 function for therapeutic purposes could include the further development of nucleic acid–based approaches blocking EZH2 expression, such as atelocollagen-mediated delivery of EZH2-targeting siRNAs (44), or the application of 3-deazaneplanocin A, an agent that is able to deplete intracellular EZH2 protein (39).


   Disclosure of Potential Conflicts of Interest
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Landesstiftung Baden-Württemberg (F. Hoppe-Seyler).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Julia Bulkescher, Angela Heilig, and Lars Jansen for expert technical assistance; Drs. Kristian Helin and Adrian Bracken for EZH2 plasmids; Dr. Karl Münger for E7 vectors; Dr. Martin Müller for HPV16 E7 antibodies; and Dr. Hans-Jürgen Stark for help with microscopy.


   Footnotes
 
Note: D. Holland and K. Hoppe-Seyler contributed equally to this work.

Received 3/26/08. Revised 9/11/08. Accepted 9/11/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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Correction: Article on Activation of EZH2 by HPV
Cancer Res., April 15, 2009; 69(8): 3721 - 3721.
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