Regulation of transcription factor E2F3a and its clinical relevance in ovarian cancer

2010;70:4613-4623. Published OnlineFirst May 11, 2010.Cancer Res Daniel Reimer, Michael Hubalek, Svenja Riedle, et al.

Directed Proliferation in Ovarian Cancer−Receptor E2F3a Is Critically Involved in Epidermal Growth Factor

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Tumor and Stem Cell Biology
Cancer
Research

E2F3a Is Critically Involved in Epidermal Growth FactorReceptor–Directed Proliferation in Ovarian Cancer

Daniel Reimer1, Michael Hubalek1, Svenja Riedle3, Sergej Skvortsov2, Martin Erdel4, Nicole Concin1, Heidi Fiegl1,Elisabeth Müller-Holzner1, Christian Marth1, Karl Illmensee5, Peter Altevogt3, and Alain G. Zeimet1

Abstract

Authors' A2Radiothe3D015-TuHeidelberCytogenetAustria; an

Note: SupResearch O

Corresponand GynecInnsbruck,E-mail: alai

doi: 10.115

©2010 Am

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We describe for the first time a new integral molecular pathway, linking transcription factor E2F3a to epi-dermal growth factor receptor (EGFR) activation in ovarian cancer cells. Investigations on the role of E2Ffamily members in EGFR-mediated mitogenic signaling revealed that E2F3a was selectively upregulatedfollowing EGFR activation, whereas all other E2F family members remained unaffected. In contrast, EGFtreatment of healthy ovarian surface epithelial and mesothelial cells yielded a selective upregulation ofproliferation-promoting E2F1 and E2F2 without influencing E2F3a expression. In ovarian cancer cell lines,the extent of EGF-induced proliferative stimulus was closely related to the magnitude of E2F3a increase,and proliferation inhibition by E2F3a knockdown was not overcome by EGF exposure. Furthermore, theEGFR-E2F3a axis was found to be signal transducer and activator of transcription 1/3 dependent and theratio of IFN-regulatory factor (IRF)-1 to IRF-2 was shown to be determinative for E2F3a control. In a pilotstudy on 32 primary ovarian cancer specimens, a highly significant correlation between activated EGFR andE2F3a expression was disclosed. This new integral pathway in the EGFR-driven mitogenic cell response, whichthrough its key player E2F3a was found to be essential in triggering proliferation in ovarian cancer cells,provides new insights into EGFR signaling and could represent the basis for appealing new therapeutic ap-proaches in ovarian cancer. Cancer Res; 70(11); 4613–23. ©2010 AACR.

Introduction

The epidermal growth factor receptor (EGFR) is com-monly expressed in various tumor entities, including ovariancarcinomas, and has been largely implicated in the prog-ression of malignant diseases (1–4). Although its selectivetargeting poses an appealing new approach in cancer treat-ment, the functional complexity of the various EGFR down-stream pathways in oncogenesis and cancer progression isfar from fully elucidated at the molecular level. EGF andother growth factors are known to affect key transitionpoints in the cell cycle in terms of exit from quiescenceand entry into G1-phase or G1-S checkpoint transition (5,6). In this context, the E2F family of transcription factorsand its governor, the retinoblastoma tumor suppressorprotein, are crucially involved in regulating these transition

ffiliations: Departments of 1Obstetrics and Gynecology andrapy, Innsbruck Medical University, Innsbruck, Austria;morimmunology, German Cancer Research Center,g, Germany; 4Laboratory for Molecular Biology andics, Krankenhaus der Barmherzigen Schwestern Linz, Linz,d 5Genesis Fertility Center, Patras, Greece

plementary data for this article are available at Cancernline (http://cancerres.aacrjournals.org/).

ding Author: Alain G. Zeimet, Department of Obstetricsology, Innsbruck Medical University, Anichstrasse 35, A-6020Austria. Phone: 43-512-504-23051; Fax: 43-512-504-23055;[email protected].

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points during the cell cycle (7–10). On the basis of their func-tion, the E2F family can be divided into three subgroups.(a) E2F1 and E2F2 act primarily as “proliferation promoters”and are required for activation of genes involved in G1-S tran-sition and cell cycle progression (11). However, under certaincircumstances, including malignant transformation, E2F1 isalso involved in regulating apoptosis by targeting key genesof the p53 and p73 pathways (12). (b) E2F4 to E2F8 arepredominantly nuclear in G0–early G1 phase and act as“proliferation inhibitors” (11). (c) In the context of cell cycleregulation, E2F3 has recently been shown to occupy a uniqueposition: Using two alternative promoters and two 5′ exons,the E2F3 gene encodes for two proteins that exhibit opposingfunctions. In fact, the E2F3a isoform is mainly expressed inproliferating cells and reaches peak levels in late G1 phase(13). In contrast, the NH2-terminal–truncated E2F3b iso-form is permanently expressed throughout the cell cycle,where it exhibits inhibitory properties (14). However, the roleof the E2F family of transcription factors has not yet beenstudied in the context of EGFR-mediated effects on cell cyclecontrol.We here report for the first time that E2F3a upregula-

tion is essential for EGFR-directed proliferation of ovariancancer cells, whereas the expression of all other E2F familymembers remains unaffected following EGF stimulation. Inaddition, we elucidate the molecular background down-stream from the EGFR and show that the interplay betweenIFN-regulatory factor (IRF)-1 and IRF-2 is crucially involvedin the induction of E2F3a expression. Furthermore, as proof

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of principle, we show a highly significant in vivo correlationbetween E2F3a transcripts and the activated EGFR in a pilotstudy on 32 primary ovarian cancer specimens.

Materials and Methods

Cell culture experimentsEstablished ovarian cancer cell lines and the mesothelioma

cell line CRL-5820 (American Type Culture Collection/LGCPromochem GmbH) were cultured in serum-free medium aspreviously described (15). Ovarian surface epithelial (OSE)and human peritoneal mesothelial cells (HPMC) were obtainedfrom postmenopausal patients during surgery for other thaninflammatory or malignant conditions. All included patientsgave written consent for tissue use in research, and the studywas approved by the local Institutional Ethics Review Board.Cell culture of HPMCandOSEwas performed as published pre-viously (15, 16). For EGF and IFNγ treatment, cells were platedin 25-cm2 culture flasks and allowed to attach overnight. Con-fluent cells (75–80%) were washed with PBS and treated witheither 100 ng/mL EGF (Sigma) or 10 ng/mL IFNγ (BoehringerIngelheim) over various time periods. Alternatively, cellswere preincubated with the EGFR inhibitors cetuximab, erloti-nib, or lapatinib (concentrations are given in figures) for 60minutes and incubated for an additional 12 hours in freshmedium containing 100 ng/mL EGF and the EGFR inhibitor.To inhibit relevant EGF-induced signaling pathways, celllines were preincubated with 2 μmol/L pyridone-6 or 20μmol/L AG490 [Janus-activated kinase (JAK)/signal transduc-er and activator of transcription 1/3 (STAT1/3)], 10 mmol/LGF109203X (protein kinase C), or 20 μmol/L PD98059 [mito-gen-activated protein kinase (MAPK) kinase] for 60 minutesand incubated for an additional 12 hours with 100 ng/mLEGF and the inhibitors. On completion of incubation, cellswere counted with an electronic particle counter (Coulter)and collected for subsequent analyses.

Cell proliferation assaysCell proliferation was determined with the MTT assay (Sigma).

Briefly, cellswere cultured in 96-well plates thedaybefore the exper-iment at a density of 5 × 103 per well and then incubated for 1 to 6days. To eachwell, 200μL freshmediumand 20μLMTT (5mg/mLin PBS) were added, after which the cells were incubated at 37°C for4 hours. After removal of all growthmedium,MTT crystal particleswere dissolved in 100 μL DMSO by shaking for 2 to 3 minutes.Absorbance of formazan dye was then estimated at a test wave-length of 570 nm and a reference wavelength of 630 nm.

Primers and probes for reverse transcription-PCRsSpecific primers and probes for E2F1, E2F2, E2F3a, E2F3b,

IRF-1, IRF-2, and TATA box-binding protein (TBP) were deter-mined with the computer program “Primer Express” (AppliedBiosystems; Supplementary Table S1).

Total RNA extraction and real-time PCR amplificationTotal RNA extraction, DNase treatment, and reverse tran-

scription were done as previously described (16). Reversetranscription-PCR (RT-PCR) was performed using an ABI

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Prism 7900 Detection System (Applied Biosystems) as recentlydescribed (17). PCR efficacies were acquired by amplifying se-rially diluted cDNA from HTB-77. PCR assays were conductedin triplicate. Gene expression levels were determined with thecomparative CT method according to User Bulletin 2 (AppliedBiosystems). Transcript levels were normalized to TBP.

siRNA-based knockdown of E2F3a, IRF-1, and IRF-2Oligonucleotides for siRNAs directed against E2F3a, IRF-1,

and IRF-2 were purchased from Eurofins MWG (Supplemen-tary Table S2). Briefly, cells were plated in a six-well plate at adensity of 1 × 105 per well in growth medium without anti-biotics and allowed to seed overnight. After medium removal,cells were washed three times with Opti-MEM I medium (In-vitrogen). Opti-MEM I medium (800 μL) was added to eachwell. siRNA (2 μL of a 100 μmol/L stock) was diluted in 183μL Opti-MEM I medium. Oligofectamine (4 μL; Invitrogen)was mixed with 11 μL Opti-MEM I medium and incubatedat room temperature for 5 minutes. Diluted siRNA and dilut-ed Oligofectamine were combined, and after incubation for20 to 25 minutes, the transfection mixture was added toeach well. Cells were incubated for 4 hours at 37°C. Growthmedium (500 μL containing 30% FCS) was added to eachwell without removing the transfection complex. In thecase of EGF treatment, transfected cells were incubated with100 ng/mL EGF for 12 hours. For proliferation assays, ovariancancer cells were plated in a 96-well plate at a density of 2.8 ×103 per well in growth medium without antibiotics. After24 hours, growth medium was removed, cells were washed,and 80 μL Opti-MEM I medium was added to each well.siRNA (0.2 μL of a 100 μmol/L stock) was diluted in 16.4 μLOpti-MEM I medium. Oligofectamine (0.4 μL) was mixedwith 3 μL Opti-MEM I medium and incubated at room tem-perature. Diluted siRNA and diluted Oligofectamine werecombined and mixed gently. After incubation at room tem-perature for 20 to 25 minutes, the transfection mixture wasadded. Cells were incubated for 4 hours at 37°C. Growth me-dium (50 μL containing 30% FCS) was added to each wellwithout removing the transfection complex and used forsubsequent MTT assays.

ImmunoblottingWestern blots were performed as described previously

(18). The following antibodies were purchased from SantaCruz Biotechnology: rabbit anti-E2F3a (1:200), rabbit anti–IRF-1 (1:100), rabbit anti–IRF-2 (1:100), and anti–β-actin.The following antibodies were from Cell Signaling Technolo-gy: rabbit anti-EGFR (1:450) and rabbit anti–glyceraldehyde-3-phosphate dehydrogenase (1:10,000). Protein band intensi-ties were defined as the mean of pixels within the area of theband (mean) limited by a preformed rectangular area (area)after subtraction of the background pixels. Quantificationwas performed with Scion Image (Scion Corp.) software.

ImmunohistochemistryImmunohistochemical staining was performed on paraffin-

embedded 4-μm sections (n = 32) using the UltraViewUniversal DAB Detection kit (Ventana) based on a mouse

Cancer Research




EGFR-E2F3a Axis in Ovarian Cancer


anti-rabbit Ventana BenchMark automated slide-stainingsystem. After deparaffinization, antigens were retrieved withcell-conditioning solution 1 (standard); slides were then incu-bated for 1 hour at 37°C with the rabbit anti–phospho-EGFTyr845 (1:50; Cell Signaling Technology). Counterstainingwas performed with Nexes Hematoxylin (1:2), followed byNexes Bluing Reagent. Negative controls were obtained byomitting the primary antibody, and paraffin-embeddedEGF-treated HTB-77 served as positive control. The amountof activated EGFR was independently evaluated by twopathologists. Expression was determined by calculating atotal staining score defined as the product of the percentageof stained cells and the staining intensity (0–3).

Results

EGF induces the expression of E2F3a but does not affectthat of the other E2F family members in ovarian cancercell linesEGF treatment of the ovarian cancer cell lines HOC-7,

HTB-77, and A2780 yielded a transient upregulation ofE2F3a transcript levels (Fig. 1A–C) without affectingexpression of the other E2F family members. In HOC-7 andHTB-77, a significant increase in E2F3a expression wasrevealed after 40 minutes with peak levels after 12 hours,

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followed by a decline toward baseline after 24 hours. Inlow EGFR-expressing A2780 cells, the E2F3a peak was lesspronounced. E2F3b in HTB-77 and A2780 was unaffectedon EGF exposure, but in HOC-7, EGF caused a significant de-cline in E2F3b expression with a nadir of 0.5-fold expressionafter 6 hours. Similar results about E2F3 isoform expressionwere observed in the ovarian cancer cell lines OVCAR-3 andMDAH-2774 (Supplementary Fig. S1A and B).At the protein level, significant upregulation of E2F3a was

observed after 60 minutes and reached a plateau in HOC-7 andHTB-77 after 3 hours of EGF treatment (Fig. 1D). The same wastrue for A2780 cells, where differences were near the detectionlimit. In contrast to the observed E2F3a protein peaks, EGF-induced enhancement of cell growth reached statistical sig-nificance only after 36 hours with a steady-state plateau after72 hours (Fig. 2A). In accordance with E2F3a transcript levels,HOC-7 exhibited highest growth enhancement, followed byHTB-77 and A2780. In contrast, E2F3a inducibility did notcorrelate with the constitutive expression of EGFR (Supple-mentary Fig. S1D) in the ovarian cancer cell lines.Interestingly, 100 ng/mL EGF did not significantly increase

proliferation in EGFR-overexpressing SKOV-6 (Fig. 2A).Because E2F3a expression levels were also not inducibleunder EGF exposure (Fig. 2B), we were tempted to speculatethat E2F3a induction is crucially involved in EGF-mediated

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Figure 1. A to C, HOC-7, HTB-77, and A2780cells under EGF. EGF treatment yielded aselective, time-dependent upregulation ofE2F3a mRNA, whereas E2F3b was notinducible. *, P < 0.05; **, P < 0.01.Columns, mean of three independentexperiments; bars, SD. D, immunoreactivityof EGF-induced 45-kDa E2F3a protein (arrow)increase in HOC-7 and HTB-77 analyzedby Western blot. Shown are representativeblots of three independent experiments.

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ovarian cancer cell proliferation. Furthermore, when EGF wassubstituted by transforming growth factor-α (TGF-α) in allthese experiments, the outcome was essentially the same.It is noteworthy that in healthy OSE, neither E2F3a nor E2F3b

expression was influenced by EGF or TGF-α treatment. How-ever, EGF treatment of these cells yielded an induction of theother proliferation-promoting E2F family members (i.e., E2F1and E2F2; Fig. 2C). In line with this, exposure of healthy meso-thelial cells to 100 ng/mL EGF did not influence the expressionof both E2F3 isoforms but did significantly enhance E2F1 andE2F2 transcript levels (Fig. 2D). In contrast to EGF-responsiveovarian cancer cells, incubation of malignant mesotheliomaCRL-5820 cells neither yield E2F3a transcript level upregula-tion (Fig. 2E) nor enhanced cell proliferation (Fig. 2A).

E2F3a upregulation is reversed by EGFR-inhibitingdrugsTo prove that E2F3a upregulation is mediated via EGFR

activation, we evaluated whether direct inhibition of EGFRby means of cetuximab, erlotinib, or lapatinib is able to



reverse this effect. In HOC-7 and A2780, all three EGFRinhibitors completely abolished the EGF-induced increasein E2F3a mRNA (Fig. 3A and B). However, in HTB-77, 30 μg/mLcetuximab did not abrogate the increase in E2F3a mRNA. Ce-tuximab abolished E2F3a upregulation in HTB-77 when theEGF dose was lowered to 10 ng/mL (Fig. 3C). Again, E2F3btranscript levels were not influenced by EGFR inhibitors,and the EGF-induced downregulation of E2F3b observed inHOC-7 was also not counteracted by EGFR inhibition. Aboutcell growth, erlotinib and lapatinib were able to neutralizethe proliferative effect of EGF in all cell lines, whereasEGF-induced cell proliferation remained unaffected in thepresence of 30 μg/mL cetuximab in HTB-77 (Fig. 3D).

siRNA-mediated knockdown of E2F3a abolishesEGF-induced proliferationHOC-7 cells were transiently transfected with siRNAs

targeting E2F3a. Transient siRNA-based downregulation ofE2F3a yielded a 70% knockdown of E2F3a protein in HOC-7(Fig. 4A). As expected, knockdown of E2F3a was found to

Figure 2. A, growth induction of ovarian cancer cell lines and the mesothelioma cell line CRL-5820 under EGF. Percentage of growth enhancement isshown normalized to untreated controls (set as 1.0). Asterisks indicate statistical significance. B, E2F3a and E2F3b mRNA in SKOV-6 cells undertime-dependent EGF exposure. C, EGF exposure of healthy OSE cells yielded an upregulation of proliferation-promoting E2F1 and E2F2 but not ofE2F3 isoforms. *, P < 0.05. D, EGF-mediated induction of E2F1 and E2F2 in healthy HPMC. E, EGF treatment of CRL-5820 did not alter E2F isoformtranscript levels.

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inhibit the basal proliferation of HOC-7 cells. Moreover, thisproliferation arrest could not be overcome by treatment with100 ng/mL EGF (Fig. 4B). Furthermore, E2F1 or E2F2 mRNAlevels remained unaffected by EGF under E2F3a depletion,revealing that blockage of the EGF-E2F3a pathway cannotbe bypassed by other E2F cell cycle promoters. This temptedus to speculate that E2F3a is crucially involved in EGF-induced proliferation in ovarian cancer cells.

Intratumoral E2F3a mRNA expression correlates withactivated EGFR statusIn light of our in vitro data pointing to a pivotal role of the

EGFR-mediated induction of E2F3a expression, we investigat-ed whether primary ovarian cancer samples (n = 32) exhibitinghigh E2F3a expression levels would also show an activatedEGFR status. Clinicopathologic parameters of patients are giv-en in Table 1. Indeed, E2F3a mRNA levels correlated strongly



with the immunohistologic score of phospho-EGFR (r =0.5053; P = 0.001) in the tumor specimens (Fig. 4C).

Signaling pathway governing the EGFR-E2F3a axisTo elucidate the putative pathway downstream from the

EGFR-regulating E2F3a transcription, we first inhibitedknown pathways of the EGF signaling network (JAK/STAT1/3,protein kinase C, and MAPK kinase). Only inhibition of theJAK/STAT1/3 pathway using pyridone-6 or AG490 promptedsignificant changes in the expression of E2F3a transcripts interms of E2F3a upregulation, thus pointing to a selectiveinvolvement of downstream effectors of the JAK/STAT1/3pathway in EGF-mediated E2F3a control (Fig. 4D). To delimitthe number of candidate molecules participating in EGFR-JAK/STAT1/3-E2F3a regulation, we compared the first 2,000bp of the 5′-untranslated region (UTR) of the E2F1, E2F2,and E2F3 genes with regard to potential transcription factor

Figure 3. Inhibition of EGFR by means of cetuximab, erlotinib, or lapatinib reverses EGF-induced E2F3a upregulation. E2F3a and E2F3b mRNA levelsafter EGFR inhibition in HOC-7 (A) and A2780 (B). C, 30 μg/mL cetuximab did not abrogate E2F3a enhancement in HTB-77 at an EGF concentration of100 ng/mL. Small box, when EGF concentration was lowered to 10 ng/mL, E2F3a expression was abolished with cetuximab. */†, P < 0.05; **/††, P < 0.01.D, ovarian cancer cells grown under EGF or in medium containing EGF and EGF inhibitors. The percentage of growth induction or inhibition was estimatedby comparison with untreated cells grown over the same time period (set at 0). The arrow indicates the resistance of HTB-77 to 30 μg/mL cetuximab.

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binding sites (TFB). At an 85% profile score threshold, 36 dif-ferent putative TFBs were identified within the investigated5′-UTRs (Supplementary Table S3). Overall homogeneitywas 92% and the 5′-UTR of E2F3a differed only in the occur-rence of two additional TFBs, namely, a vitamin D3 receptor(VDR) recognition site (−1055) and an IRF-2 site (−791) locat-ed within an IRF-1 binding site (Supplementary Fig. S2). Incontrast, IRF-1 sites were detectable within the 5′-UTRs ofall three E2F transcription factors investigated. Because theIRFs are known downstream effectors of the JAK/STAT1/3pathway, we assumed the IRF-1/IRF-2 interplay to be involved



in the regulation of the EGF-E2F3a axis. In fact, EGF treat-ment resulted in an opposite effect on IRF-1 and IRF-2 expres-sion during the first 60 minutes of incubation such that IRF-1was downregulated and IRF-2 was upregulated (Fig. 5A and B),thereby causing a significant shift in the IRF-1/IRF-2 ratio infavor of IRF-2. Considering the protein level, the EGF-induced50-kDa IRF-2 protein did not change, whereas the 43-kDa IRF-1 protein band showed a significant decrease as early as after20 minutes of exposure (Fig. 5C and D). In these cell lines, re-producible transient increases in IRF-1 mRNA and proteinwere revealed at 60 minutes of EGF treatment. Similar but less

Figure 4. Effects of siRNA-based downregulation of E2F3a on EGF-induced proliferation in HOC-7. A, silencing efficiency of siRNA oligonucleotidestargeting E2F3a in HOC-7. Shown is a representative blot of three independent experiments. B, growth of siRNA-transfected HOC-7 cells oruntransfected controls cultured under EGF-free conditions or in medium containing EGF over various time periods. Points, mean of three independentexperiments; bars, SD. Knockdown of E2F3a (•) resulted in an inhibition of HOC-7 cell growth. This effect was not overcome by EGF exposure (○).C, immunohistochemical detection of activated EGFR in 32 ovarian cancer primary tumors. Representative examples of anti–phospho-EGFR stainingin ovarian cancer specimens with different status of activated EGFR are shown. Correlation between histologic score [percentage × intensity of staining(0–3)] of activated EGFR and E2F3a mRNA levels yielded a high correlation between activated EGFR and E2F3a mRNA levels (P < 0.001).Clinicopathologic parameters of the collective (Table 1). D, E2F3a transcript levels in ovarian cancer cell lines after selective inhibition of EGFR pathways.*, P < 0.05; **, P < 0.01.

Cancer Research






prominent results on IRF-1 and IRF-2 mRNA and protein wereobtained in EGF-treated A2780 cells.To corroborate our hypothesis that IRF-1 mediates a nega-

tive control on E2F3a expression, we assessed E2F3a transcriptlevels in an IRF-1–enriched intracellular context. IFNγ(10 ng/mL), which was shown to induce fast upregulation ofIRF-1 (e.g., up to 54-fold in HTB-77) accompanied bymoderate upregulation of IRF-2 (e.g., up to 4-fold in HTB-77),yielded significant time-dependent downregulation of E2F3a(Fig. 6A) but not of E2F3b (Supplementary Fig. S3A). Theseeffects were paralleled by a significant inhibition of cell growth(Fig. 6B). Moreover, 10 ng/mL IFNγ was able to neutralize theEGF-induced E2F3a increase in the three cell lines investigated(Fig. 6C), but no effect of EGF/IFNγ coincubation was ob-served for E2F3b expression (Supplementary Fig. S3B).

siRNA-based knockdown of IRF-1 and IRF-2 influencesE2F3a expressionMotivated by these results, we assumed that EGF-induced

shifting of the IRF-1/IRF-2 ratio toward IRF-2 is pivotal forE2F3a induction. Therefore, HOC-7 cells were subjected to



siRNA-based knockdown of IRF-1 and IRF-2, either separate-ly or both together. Silencing efficiency of IRF-1 and IRF-2siRNAs was 0.7 and 0.9, respectively (Fig. 6C). In untreatedcells, IRF-1 knockdown yielded a minor upregulation of theE2F3a protein, whereas IRF-2 knockdown caused a signifi-cant decrease in E2F3a. EGF treatment of IRF-1 knockdowncells resulted in an overwhelming increase in E2F3a protein,whereas EGF treatment of IRF-2 knockdown and concomi-tant IRF-1 + IRF-2 knockdown cells abolished the E2F3adecrease observed in untreated cells (Fig. 6D). E2F3b expres-sion remained unchanged after IRF-1 or IRF-2 knockdown.

Discussion

Activation of the proto-oncogene encoding the EGFR is animportant step in tumorigenesis and an essential drivingforce toward aggressive growth behavior of tumor cells(19). Because EGF and other growth factors are known toaffect key transition points in the cell cycle (5, 6), we inves-tigated whether the proliferation-promoting members of theE2F family involved in regulation of G0-G1 transition (20) arepotential effectors in EGF-mediated cell proliferation.We showed for the first time that in ovarian cancer cells

EGF or TGF-α treatment selectively induces E2F3a ex-pression (Fig. 1A–C; Supplementary Fig. S1A and B) withoutaffecting especially E2F1 or E2F2 expression or any of theother E2F family members. Our observation that EGF treat-ment induced not only E2F3a mRNA but also E2F3a proteinlevels (Fig. 1D) points to a relevant functional role of E2F3ainduction in ovarian cancer cells. This selective effecton E2F3a was completely abolished by EGFR blockade(Fig. 3A–C), substantiating that E2F3a induction is mediatedby EGFR signaling. However, SKOV-6, although EGFR over-expressing, was the only ovarian cancer cell line investigatedin which E2F3a mRNA was not inducible by EGF or TGF-α.Furthermore, these cells were found to exhibit a very lowconstitutive expression of both E2F3 isoforms in comparisonwith the other cell lines (Supplementary Fig. S3C). The linkbetween EGFR and E2F3a was further corroborated by theobservation that in primary ovarian cancer specimens a highcorrelation between activated EGFR status and E2F3a mRNAlevels was revealed (Fig. 4C).As EGF or TGF-α treatment did not affect E2F3a ex-

pression in normal OSE and HPMC, which both are embry-ologically and histomorphologically related (Fig. 2C and D;refs. 21, 22), we postulate that the EGFR-E2F3a molecularpathway is an unphysiologic axis, which may not be exclusive(see SKOV-6) but seems to be very common in ovariancancer cells. In contrast, in healthy OSE and HPMC,EGF-induced proliferation was found to be driven via up-regulation of E2F1 and E2F2 (Fig. 2C). This shift in EGF-induced cell cycle promotion from E2F1 and E2F2 towardE2F3a during malignant transformation is in line with datafrom Chen and Wells (23), who found that E2F3a exhibits thehighest oncogenic potential in fibroblasts compared withthe weak oncogenicity of the other E2F family members. Thepivotal role of E2F3a in cell cycle control of malignant cells is

Table 1. Clinicopathologic parameters of ovar-ian cancer patients (n = 32)

n (%)

International Federation of Gynecologists and Obstetri-cians stage

I
7 (21.9) II 2 (6.2) III 15 (46.9) IV 8 (25.0)
Histologic subtype
Serous 16 (50.0) Mucinous 6 (18.8) Endometrioid 8 (25.0) Undifferentiated 2 (6.2)
Grading*
1 3 (9.7) 2 13 (41.9) 3 15 (48.4)
EGFR score†

Negative to ≤10
17 (53.1) 11–100 10 (31.3) ≥101 5 (15.6)
E2F3a expression
Negative 0 (0.0) ≤25th percentile (1.07) 8 (25.0) ≤50th percentile (2.62) 7 (21.9) ≤75th percentile (4.72) 9 (28.1) ≥75th percentile 8 (25.0)
*Grading in one patient was not available due to incom-plete follow-up.†EGFR score was defined as the product of percentage ofstained cells and the staining intensity.

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further underlined by the fact that E2F3a knockdown yieldeda significant inhibition of cancer cell proliferation, which wasnot overcome by EGF treatment and even more could notbe bypassed by upregulation of the other proliferation-promoting E2F transcription factors (i.e., E2F1 or E2F2).



This leads us to postulate the existence of an integral EGF-E2F3a “fast track” in ovarian cancer cells. This hypothesis isfurther corroborated by two additional observations. (a) Inthe five EGF-responsive cell lines investigated, E2F3a induc-ibility mirrored the extent of EGF-triggered proliferative

Figure 5. EGF-induced shift in IRF-1 and IRF-2 expression. A and B, IRF-1 and IRF-2 mRNA levels in HOC-7 and HTB-77 cells. *, P < 0.05;**, P < 0.01. Changes in IRF-1 and IRF-2 mRNA levels occurred fast, yielding a shift in IRF-1/IRF-2 ratio toward IRF-2 within the first 60 min of EGFtreatment. C and D, EGF-induced changes in IRF-1 and IRF-2 protein in HOC-7 and HTB-77. The arrows indicate the 43-kDa IRF-1 and the 50-kDaIRF-2 band, respectively. Semiquantitative evaluation of changes in IRF-1 and IRF-2 protein in the Western blots is indicated as gray bars inthe right panels of C and D.

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stimulation. (b) EGF treatment of ovarian cancer SKOV-6and malignant mesothelioma CRL-5820 cells did not causean increase in E2F3a expression, and in fact, proliferationof these cells also remained unaffected on EGF or TGF-αstimulation. In these EGF-refractory cell lines, the EGFR-E2F3a axis seems to be defective either at the level of theEGFR itself or at its downstream signaling. Whereas in ma-lignant mesothelioma the potential role of the E2F family inEGFR-driven cell cycle promotion remains to be furtherestablished, our data on ovarian cancer cells neverthelessargue in favor of a substantial involvement of E2F3a inEGF-mediated mitogenic signaling. As the increase inE2F3a following EGF treatment was transient and ofshort duration at both the mRNA and the protein level andmeasurable EGF-induced cell growth was seen only after



E2F3a expression had returned to baseline, it is temptingto speculate that EGF-mediated E2F3a induction is aninitial but crucial trigger in the G1-S–phase transition.Interestingly, the EGF-driven mitogenic signaling in ovariancancer cells does not seem to be mediated by downregulationof the growth-inhibiting E2F3b isoform or other cell cycle–inhibiting E2F family members. Only in HOC-7 was a significantdecrease in E2F3b achieved with EGF treatment (Fig. 1A), which,however, was not abrogated by EGFR (Fig. 3A). Thus, we areunable to explain this unspecific but reproducible effect onE2F3b observed in that particular cell line.Analyses of the 5′-UTRs of all proliferation-promoting E2F

family members revealed a high homogeneity of the TFB profiles(Supplementary Table S3). Thus, it seems that regulation of theirexpression involves similar transducers. c-myc was shown to be

Figure 6. Effect of 10 ng/mL IFNγ on E2F3a and EGF-induced E2F3a expression. A, HOC-7, HTB-77, and A2780 were incubated with 10 ng/mL IFNγover various time periods, and quantitative expression of E2F3a mRNA was determined with RT-PCR. *, P < 0.05. B, IFNγ-mediated growth inhibitionof ovarian cancer cell lines. C, coincubation of ovarian cancer cells with EGF and IFNγ. *, P < 0.05. E2F3a expression in HOC-7 after siRNA-basedknockdown of IRF-1 and IRF-2 or simultaneous downregulation of both IRFs. D, silencing efficiency of siRNA oligonucleotides targeting IRF-1 andIRF-2 expression in HOC-7. Arrows indicate 43-kDa IRF-1 and 50-kDa IRF-2 immunoreactivity. Semiquantitative evaluation of protein levels is indicatedas gray bars. E, changes in E2F3a protein levels in untreated HOC-7 (control) and HOC-7 knockdowns cultured under EGF-free conditions (untreated) or inmedium containing EGF for 12 h.

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Reimer et al.

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substantially involved in activation of E2F1, E2F2, and E2F3a(13, 24, 25), and c-myc recognition sites are present in the 5′-UTRsof all E2F family members investigated. Although c-myc is a well-known downstream effector of the EGFR pathway and was cru-cially linked to carcinogenesis (26), the selective induction ofE2F3a by EGF clearly argues against a direct involvement ofc-myc in the EGFR-E2F3a molecular pathway. However, the5′-UTR of the E2F3 gene contains two recognition sites that areabsent in all other investigated E2F family members (i.e., that forIRF-2 and VDR). The observation that of the various EGFR down-stream pathways only inhibition of the JAK/STAT1/3 pathwayrevealed changes in E2F3a expression in terms of an E2F3aupregulation (Fig. 4D), together with the knowledge that JAK/STAT1/3 participates at least in the regulation of IRF-1 expres-sion (27), gave rise to the hypothesis that IRF-1 could act as arepressor and IRF-2 as an activator of E2F3a expression. On theother hand, VDR is not a known component of the JAK/STAT1/3pathway, and furthermore, treatment of ovarian cancer cell lineswith 1α, 25-dihydroxyvitamin D3, the most active metabolite ofvitamin D3, did not affect E2F3a mRNA levels.6

The effect of a balanced ratio between the two mutuallyantagonistic transcription factors IRF-1 and IRF-2 was shownearlier (28–30). Whereas overexpression of IRF-2 inducedoncogenic transformation of NIH3T3 cells, stable coexpres-sion of IRF-1 caused these cells to revert to a nontransformedphenotype (28). Moreover, unstimulated ovarian cancer cellsshowed an IRF-1/IRF-2 ratio in favor of IRF-1 (31), and EGFtreatment yielded rapid transient upregulation of IRF-2mRNAaccompanied by a long-lasting IRF-1 decrease (Fig. 5A and B).This IRF-1 decline was confirmed at the protein level, whereasIRF-2 protein remained at elevated levels during the wholeincubation period (Fig. 5C and D). These differences betweenEGF-induced transcript and protein changes in IRF-2 can beexplained by the longer protein stability of IRF-2 (half-life, 8 h)compared with IRF-1 (half-life, 30 min; refs. 28, 32).The crucial involvement of IRF-1 and IRF-2 in EGFR-

directed regulation of E2F3a is further corroborated bysiRNA knockdown (Fig. 6C and D). Knockdown of IRF-1 re-vealed an increase in E2F3a expression, which was impres-sively enhanced by EGF. On the other hand, knockdown ofIRF-2 or both IRFs concomitantly yielded significant down-regulation of E2F3a protein. The observation that EGF treat-ment restored E2F3a levels to baseline in these cells wouldpoint to an induction of residual IRF-2 activity caused byEGF exposure. Taken together, our findings indicate that

6 Unpublished observation.



IRF-2 is the principal inductor of E2F3a, whereas IRF-1 exhi-bits inhibitory properties on its expression, and that conse-quently the observed EGF-induced shift in the IRF-1/IRF-2ratio toward IRF-2 is responsible for E2F3a upregulation. Incontrast, the IFNγ-mediated growth inhibition observed inall the ovarian cancer cell lines was associated with a ratioshift toward IRF-1, paralleled by downregulation of E2F3a.Thus, our investigations delineate both antagonistic inter-

playing factors, IRF-1 and IRF-2, as a crucial point of inter-section linking two central signaling pathways (i.e., theproliferation-inducing EGF and the growth-inhibiting IFNγpathway) in malignant cells. This was underscored by theneutralizing effects of IFNγ treatment on EGF-inducedE2F3a upregulation and growth stimulation. All these find-ings would implicate that in vivo the herein described EGF-E2F3a axis might be significantly influenced by immunologicphenomena originating from the tumor microenvironment.In conclusion, we here describe for the first time a new

integral signaling of the EGFR-driven cell response, which,through its key player E2F3a, is essential in triggering prolif-eration in ovarian cancer cells. Furthermore, the mutuallyantagonistic factors IRF-1 and IRF-2 are found to be cruciallyinvolved in this molecular pathway downstream of the EGFR.However, we cannot completely exclude the possibility thatadditional intracellular constituents also participate ingoverning this axis. Nonetheless, the in vivo relevance ofthe described EGF-E2F3a model was evidenced by its highlysignificant clinical effect in a set of 32 ovarian cancer pa-tients. Modulation of this axis could pose an appealing newtherapeutic approach for ovarian cancer, one that deservesfurther consideration beyond the classic EGFR targeting.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank M. Fleischer, J. Rössler, A. Wiedemair, and M. Champson fortechnical assistance.

Grant Support

“Verein für Krebsforschung in der Frauenheilkunde,” Department of Obstet-rics and Gynecology, Innsbruck Medical University.

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

Received 09/24/2009; revised 02/05/2010; accepted 03/17/2010; publishedOnlineFirst 05/11/2010.

References
1. Lafky JM, Wilken JA, Baron AT, Maihle NJ. Clinical implications of
the ErbB/epidermal growth factor (EGF) receptor family and itsligands in ovarian cancer. Biochim Biophys Acta 2008;1785:232–65.

2. Stadlmann S, Gueth U, Reiser U, et al. Epithelial growth factor recep-tor status in primary and recurrent ovarian cancer. Mod Pathol 2006;19:607–10.

3. Psyrri A, Kassar M, Yu Z, et al. Effect of epidermal growth factorreceptor expression level on survival in patients with epithelialovarian cancer. Clin Cancer Res 2005;11:8637–43.

4. Meert AP, Martin B, Delmotte P, et al. The role of EGFR expressionon patient survival in lung cancer: a systematic review with meta-analysis. Eur Respir J 2002;20:975–81.

5. Lavoie JN, Rivard N, L'Allemain G, Pouysségur J. A temporal

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and biochemical link between growth factor-activated MAP kinases,cyclin D1 induction and cell cycle entry. Prog Cell Cycle Res 1996;2:49–58.

6. Chen YY, Rabinovitch PS. Altered cell cycle responses to insulin-likegrowth factor I, but not platelet-derived growth factor and epidermalgrowth factor, in senescing human fibroblasts. J Cell Physiol 1990;144:18–25.

7. Dimova DK, Dyson NJ. The E2F transcriptional network: oldacquaintances with new faces. Oncogene 2005;24:2810–26.

8. Stevens C, La Thangue N. E2F and cell cycle control: a double-edged sword. Arch Biochem Biophys 2003;412:157–69.

9. Black AR, Azizkhan-Clifford J. Regulation of E2F: a family of transcrip-tion factors involved in proliferation control. Gene 1999;237:281–302.

10. Takahashi Y, Rayman JB, Dynlacht BD. Analysis of promoterbinding by the E2F and pRB families in vivo: distinct E2F proteinsmediate activation and repression. Genes Dev 2000;14:804–16.

11. Attwooll C, Lazzerini Denchi E, Helin K. The E2F family: specificfunctions and overlapping interests. EMBO J 2004;23:4709–16.

12. Lazzerini Denchi E, Helin K. E2F1 is crucial for E2F-dependentapoptosis. EMBO Rep 2005;6:661–8.

13. Adams MR, Sears R, Nuckolls F, Leone G, Nevins JR. Complextranscriptional regulatory mechanisms control expression of theE2F3 locus. Mol Cell Biol 2000;20:3633–9.

14. He Y, Armanious MK, Thomas MJ, Cress WD. Identification ofE2F3B, an alternative form of E2F-3 lacking a conserved N-terminalregion. Oncogene 2000;19:3422–33.

15. Reimer D, Sadr S, Wiedemair A, et al. Heterogeneous cross-talkof E2F family members is crucially involved in growth modulatoryeffects of interferon-γ and EGF. Cancer Biol Ther 2006;5:771–6.

16. Auersperg N, Siemens CH, Myrdal SE. Human ovarian surfaceepithelium in primary culture. In Vitro 1984;20:743–55.

17. Reimer D, Sadr S, Wiedemair A, et al. Clinical relevance of E2Ffamily members in ovarian cancer—an evaluation in a trainingset of 77 patients. Clin Cancer Res 2007;13:144–51.

18. Loeffler-Ragg J, Skvortsov S, Sarg B, et al. Gefitinib-responsiveEGFR-positive colorectal cancers have different proteome profilesfrom non-responsive cell lines. Eur J Cancer 2005;41:2338–46.

19. Huang SM, Harari PM. Epidermal growth factor receptor inhibitionin cancer therapy: biology, rationale and preliminary clinical results.Invest New Drugs 1999;17:259–69.

20. DeGregori J. The genetics of the E2F family of transcription factors:shared functions and unique roles. Biochim Biophys Acta 2002;1602:131–50.



21. Blaustein A, Lee H. Surface cells of the ovary and pelvic peritoneum:a histochemical and ultrastructure comparison. Gynecol Oncol 1979;8:34–43.

22. Nicosia SV, Johnson JH. Surface morphology of ovarian meso-thelium (surface epithelium) and of other pelvic and extrapelvicmesothelial sites in the rabbit. Int J Gynecol Pathol 1984;3:249–60.

23. Chen C, Wells AD. Comparative analysis of E2F family memberoncogenic activity. PLoS One 2007;2:e912.

24. Leone G, DeGregori J, Sears R, Jakoi L, Nevins JR. Myc and Rascollaborate in inducing accumulation of active cyclin E/Cdk2 andE2F. Nature 1997;387:422–6.

25. Sears R, Ohtani K, Nevins JR. Identification of positively andnegatively acting elements regulating expression of the E2F2gene in response to cell growth signals. Mol Cell Biol 1997;17:5227–35.

26. Abdollahi A, Gruver BN, Patriotis C, Hamilton TC. Identification ofepidermal growth factor-responsive genes in normal rat ovariansurface epithelial cells. Biochem Biophys Res Commun 2003;307:188–97.

27. Andersen P, Pedersen MW, Woetmann A, et al. EGFR inducesexpression of IRF-1 via STAT1 and STAT3 activation leading togrowth arrest of human cancer cells. Int J Cancer 2008;122:342–9.

28. Harada H, Kitagawa M, Tanaka N, et al. Anti-oncogenic and onco-genic potentials of interferon regulatory factors-1 and -2. Science1993;259:971–4.

29. Yoshino A, Katayama Y, Watanabe T, et al. Therapeutic implica-tions of interferon regulatory factor (IRF)-1 and IRF-2 in diffuselyinfiltrating astrocytomas (DIA): response to interferon (IFN)-β inglioblastoma cells and prognostic value for DIA. J Neurooncol2005;74:249–60.

30. Taniguchi T. Transcription factors IRF-1 and IRF-2: linking theimmune responses and tumor suppression. J Cell Physiol 1997;173:128–30.

31. Zeimet AG, Reimer D, Wolf D, et al. Intratumoral interferon regulatoryfactor (IRF)-1 but not IRF-2 is of relevance in predicting patientoutcome in ovarian cancer. Int J Cancer 2009;124:2353–60.

32. Fujita T, Reis LF, Watanabe N, Kimura Y, Taniguchi T, Vilcek J.Induction of the transcription factor IRF-1 and interferon-β mRNAsby cytokines and activators of second-messenger pathways. ProcNatl Acad Sci U S A 1989;86:9936–40.

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Regulation of transcription factor E2F3a and its clinical relevance in ovarian cancer

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