E2F1 Induces Tumor Cell Survival Via Nuclear Factor-KB–Dependent Induction of EGR1 Transcription in Prostate Cancer Cells

Published OnlineFirst March 10, 2009; DOI: 10.1158/0008-5472.CAN-08-4113

Research Article

E2F1 Induces Tumor Cell Survival via Nuclear Factor-KB–Dependent Induction of EGR1 Transcription in Prostate Cancer Cells

Chaogu Zheng,1 Zijia Ren,1 Hetian Wang,1 Weici Zhang,1 Dhananjaya V. Kalvakolanu,2 Zhigang Tian,1 and Weihua Xiao1

1Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, Peoples Republic of China and 2Marlene and Stewart Greenebaum Cancer Center, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland

Abstract by E2F1 (14). However, tumors induced by the neu(ErbB-2) À/À Transcription factor E2F1 has been implicated in both oncogene in Gab2 background showed normal Akt activities, apoptosis-promoting and apoptosis-suppressing effects. How- suggesting a dispensable role of Gab2 in activating Akt in tumor ever, factors that mediate its antiapoptotic effects are still not (15). Thus, it is still not clear how E2F1 enhances PI3K/Akt identified. Using prostate tumor–derived cell lines, we showed signaling. here that E2F1 activated the expression of transcription factor Clinical studies showed that EGR1 expression level was EGR1 for promoting cell survival. E2F1 up-regulated the significantly increased in prostate cancers and directly correlated production of EGR1-induced growth factors, epidermal with Gleason score and tumor grade (16). This oncogenic role does growth factor, platelet-derived growth factor, and insulin-like not seem to extend to other tumor types, such as skin tumor, fibrosarcoma, and glioblastoma in which EGR1 acts as a tumor growth factor II, which in turn activated the phosphoinositide-3-kinase/Akt pathway to resist drug-induced apoptosis. suppressor gene through multiple mechanisms, including inducing Moreover, E2F1 directly induced the transcription of the Egr1 p53 and PTEN expression (17). Indeed, EGR1 deficiency results in a gene using the KB site located in its proximal promoter. significant delay in tumor initiation, mortality, and progression E2F1 physically interacted with the RelA subunit of nuclear from prostatic intraepithelial neoplasia to invasive carcinoma; factor-KB and modulated its transactivity to fully activate and inhibition of EGR1 expression reverses the transformation of EGR1 transcription. Together, these studies uncovered a novel prostate carcinoma both in vivo and in vitro (18, 19). Moreover, mechanism for E2F1-induced suppression of apoptosis in EGR1 physically interacts with androgen receptor and induces the prostate cancer. [Cancer Res 2009;69(6):2324–31] prostatic intraepithelial neoplasia carcinoma transition via transcriptionally activating growth and angiogenic factors such as platelet-derived growth factor (PDGF), insulin-like growth factor Introduction (IGF), epidermal growth factor (EGF), fibroblast growth factor, and Transcription factor E2F1 not only promotes cell proliferation vascular endothelial growth factor (20, 21), all of which contribute but also induces apoptosis in cancer. For example, both reduced to hormones refractory to prostate cancer (22, 23). Together, these proliferative and apoptotic rates were evidenced in SV40 T121– observations indicate a critical role for EGR1 in promoting prostate induced tumors in E2F1 knockout mice, suggesting a paradoxical cancer development. However, what causes EGR1 overexpression role for E2F1 in tumors (1). Therefore, it was considered as both and which factors control its expression in prostate cancer are still oncogene and tumor suppressor (2–4). The antiapoptotic function poorly understood. n of E2F1 was first observed to protect cells from UV-induced Here, we show that E2F1, by associating with nuclear factor- B n apoptosis in p53-deficient mice (5); however, the mechanism (NF- B), maximally induces the expression of EGR1. Elevated EGR1 governing such apoptosis-suppressing action of E2F1 is still then induces the production of a panel of growth factors, including unclear. Phosphoinositide-3-kinase (PI3K)/Akt signaling is a major at least EGF, PDGF, and IGF-II, etc. In turn, these growth factors survival pathway that can be activated by various stimuli such as activate PI3K/Akt and promote resistance to drug-induced growth factors (6–9) and is widely involved in the inhibition of apoptosis. Together, our study has identified a novel E2F1-induced apoptosis via regulating the activity of apoptotic factors such as cell survival pathway. BAD, caspase-9, and Mdm2 (10–12). It has been reported recently that E2F1 inhibited c-Myc–driven apoptosis via inducing a Materials and Methods PIK3CA/Akt/mTORcascade in human liver cancer (13), implicat- Reagent and antibodies. All reagents, chemicals, and affinity-purified ing a potential link between the oncogenic function of E2F1 and anti-rabbit IgG, anti-mouse IgG, and anti-goat IgG conjugated with the activation of Akt. Grb-associated binder 2 (Gab2), a receptor horseradish peroxidase or fluorescent tags were purchased from Sigma. n docking protein in the PI3K/Akt signaling pathway, is identified as The following specific antibodies were used in this study: anti–NF- B p65 n a target gene of E2F1, which is responsible for the activation of Akt and p50 (Upstate Biotech); anti-E2F1, EGR1, DP-1, I B, Akt, histone H1, and actin (Santa Cruz Biotech); and anti-BCL2, BCL-XL, phosphorylated Akt (p-Ser473), and phosphorylated InBa (p-Ser32/36; Cell Signaling). Short hairpin RNA expression, small interfering RNA duplex, and Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org). reporter gene constructs. The pU6+27, short hairpin RNA (shRNA) Requests for reprints: Weihua Xiao, Institute of Immunology, School of Life control, and shRNA p65 vectors were purchased from Panomics Sciences, University of Science and Technology of China, 443 Huangshan Road, Hefei Corporation. A 21-nucleotide sequence coding for amino acids 125 to 131 230027, P.R. China. Phone: 86-551-3600536; Fax: 86-551-3600535; E-mail: xiaow@ of human E2F1 and 413 to 419 of human EGR1 were selected as the targets ustc.edu.cn. I2009 American Association for Cancer Research. for RNAi according to the instructions of the manufacturer. In all cases, the doi:10.1158/0008-5472.CAN-08-4113 corresponding sequences were scrambled to generate a control vector. In

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E2F1 and Prostate Cancer initial studies, we used empty vector (pU6+27) and scrambled shRNA as Cell culture and transfection. DU145 and PC3, human prostate cancer controls. Double-stranded small interfering RNAs (siRNA) targeting the cell lines, were purchased from American Type Culture Collection and CDS: 891 to 909 of NF-nB (p65) and scrambled control was synthesized cultured at 37jC in DMEM supplemented with 10% fetal bovine serum and using the following sequences: siRNA-p65; forward, 5¶-GAU UGA GGA GAA 2 mmol/L of L-glutamine. LipofectAMINE 2000 (Invitrogen Corporation) ACG UAA AdTdT; reverse, 5¶-UUU ACG UUU CUC CUC AAU CdTdT. siRNA- was used for transfection. Stably transfected cell lines were obtained after scramble; forward, 5¶-AUG AAC GUG AAU UGC UCA AdTdT; reverse, being selectively screened by G418 (700 Ag/mL; Life Technologies) for 3 to 5¶-UUG AGC AAU UCA CGU UCA UdTdT. 4 weeks. To construct the EGR1 promoter-driven luciferase reporter, a 760-bp Immunoblot analyses. Whole cell extract were prepared by lysing cells DNA fragment from the promoter region of the EGR1 gene, including À70 in the NP40 lysis buffer containing 50 mmol/L of Tris-Cl (pH 6.8), to À846 nucleotides upstream of the start ATG, was obtained by PCRusing 150 mmol/L of NaCl, 1 mmol/L of EGTA, 1% NP40, and freshly added human genomic DNA as the template and primers EGR1-pf, 5¶-gaC TCG proteinase inhibitors including 1 mmol/L of phenylmethylsulfonyl fluoride, AGG CTC ACT GCT ATA CAG TGT C-3¶; and EGR1-pr, 5¶-cgA AGC TTTACA 1 Ag/mL of leupeptin (Sigma), 1 Ag/mL of pepstatin (Sigma), and 1 Ag/mL TGG CAT ATA TGG GAA GC-3¶, where the restriction enzyme sites for of aprotinin (Sigma). For preparing the nuclear extract, cytoplasmic cloning are indicated by italics. The PCRproducts were inserted into the membranes were disrupted in 0.5% NP40, 25 mmol/L of Hepes (pH 7.5), pGL3-basic vector (Promega) and designated as pGL3-Egr1-wt. The nB-like 5 mmol/L of KCl, 0.5 mmol/L of MgCl2, 1 mmol/L of DTT, and proteinase motif was mutated using site-directed mutagenesis correspondingly to two inhibitors; then nuclei were collected by centrifuging at 2,500 rpm for possibly overlapped NF-nB binding sites (indicated in Fig. 4A). All the 1 min and lysed in 25 mmol/L of Hepes (pH 7.5), 10% sucrose, 0.01% constructs were verified by sequencing. NP40, 350 mmol/L of NaCl, 1 mmol/L of DTT, and proteinase inhibitors. The pcDNA3 expression vectors with E2F1, E2F2, and E2F3 expressed were Lysates were subjected to immunoblot analysis. Horseradish peroxidase– provided by Dr. Mian Wu (University of Science & Technology of China, Hefei, conjugated secondary antibodies and enhanced chemiluminescence China) and described previously (24). EGR1 expression plasmids were detection (Pierce Chemical, Co.) was used to detect the specific immu- generous gifts from Dr. Jie Du (Baylor College of Medicine, Houston, TX). noreactive proteins.

Figure 1. E2F1 suppressed drug-induced apoptosis in prostate cancer cells and was correlated with EGR1 expression. A, expression levels of E2F1 and EGR1 in the parental PC3 and DU145 cells were assessed by RT-PCR and immunoblot assays, and the cellular sensitivity to 5-FU–induced apoptosis was determinedby Annexin V/PI staining after exposing the cells to 0.5 mg/mL of 5-FUfor the indicated times. 5-FU–induced inhibition of cell growth was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after exposing the cells with the indicated dosage of 5-FUfor 48 h. The same assays performed in A were applied on the DU145-derived cells (B) and PC3-derived cells (C). www.aacrjournals.org 2325 Cancer Res 2009; 69: (6). March 15, 2009

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Figure 2. Neither NF-nB nor BCL-2 expression were altered in E2F1 knockdown cells. The whole cell lysates (WCL), nuclear extracts (NE), and cytoplasmic extracts (CP) from cells were used for the immunoblot assays with the specific antibodies (A). Fluorescent immunostaining with either anti–NF-nB p65 or p50 followed Cy3-conjugated secondary antibody was performed on the indicated cells (B). Whole cell lysates from the cells treated with 0.5 mg/mL of 5-FUfor the indicated times were immunoblotted with the specific antibodies (C).

Reverse transcription-PCR. One microgram of isolated total RNA was DNA fragments were eluted and purified. DNA from these samples was converted to cDNA using Superscript III reverse transcriptase (Invitrogen). subjected to PCRanalyses with NF- nB-BS-pf, 5¶-GCG GCT AGA GCT CTA Gene-specific PCRwas conducted using the primer sets indicated in GGC-3¶;andNF-nB-BS-pr, 5¶-GCA GAA GCC CTA ATA TGG C-3¶; Supplementary Table S1. The PCRproducts were resolved on a 1.5% agarose glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-pf, 5¶-GTA TTC CCC gel and then visualized by ethidium bromide staining. CAG GTT TAC AT-3¶; and GAPDH-pr, 5¶-TTC TGT CTT CCA CTC ACT Immunoprecipitation. Cellular lysates were prepared using a radio- CCT-3¶. The PCRproducts were separated on 2% agarose gel and visualized immunoprecipitation assay buffer [50 mmol/L Tris-Cl (pH 7.4), 1% NP40, by ethidium bromide staining. The GAPDH promoter fragment acted as the 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L internal negative control for this assay. phenylmethylsulfonyl fluoride, 1 Ag/mL aprotinin, 1 Ag/mL leupeptin, Luciferase reporter assays. Briefly, an appropriate amount of the Egr1 A 1 g/mL pepstatin, 1 mmol/L Na3VO4, and 1 mmol/L NaF]. An equivalent promoter-luciferase reporters, together with Renilla luciferase plasmids, amount of total proteins were incubated with 2 Ag of specific antibody or which served as the internal control, were cotransfected into cells. Two days control IgG at 4jC overnight. The immunocomplexes were captured by later, cellular lysates were subjected to a dual-luciferase reporter assay incubation with protein A-agarose at 4jC for 2 h and the immunopreci- (Promega) according to the instructions of the manufacturer. The luciferase pitates were subjected to Western blotting with specific antibodies. activities for the promoter reporters were normalized to Renilla luciferase Annexin V staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte- activities. The data represented at least three independent experiments, and trazolium bromide assay. For apoptotic analysis, cell apoptosis detection in each experiment, tests were done in triplicate for all the samples. kit (BD Bioscience) was used. After treatment with 0.5 mg/mL of Immunofluorescence. Cells grown on coverslips were fixed with 5-fluorouracil (5-FU) for the indicated times, cells were washed with acetone/methanol (1:1) for 1 min After blocking with 5% normal goat 1Â binding buffer [10 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 5 mmol/L serum for 30 min at 37jC, they were incubated with anti-p65 or anti-p50 j KCl, 1 mmol/L MgCl2, and 1.8 mmol/L CaCl2], then incubated with FITC- overnight at 4 C. They were then incubated with Cy3-conjugated anti- conjugated Annexin V and 5 Ag/mL of propidium iodide (PI) in the dark rabbit IgG (Sigma) for 30 min at 37jC and photographs were captured with for 30 min, and analyzed with FACSCalibur (BD Bosciences) immediately. a confocal laser scanning microscope. Standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to measure the proliferation of 104 cells treated with drugs for 48 h in 96-well plates. These data represent at least three Results independent tests and each test with triplicate samples. E2F1 expression levels were correlated with resistance to Chromatin immunoprecipitation assay. Cells were seeded 24 h prior drug-induced apoptosis in prostate cancer cells. To examine to fixation with 1% formaldehyde at 37jC for 7 min. The cells were harvested in lysis buffer [50 mmol/L Tris-Cl (pH 8.1), 10 mmol/L EDTA, and the influence of E2F1 on prostate cancer cell survival in response to 1% SDS] and sonicated to shear the chromatin (f500 bp). The soluble chemotherapeutic drugs, two of the most commonly used fraction was collected by centrifugation and incubated with specific androgen-independent prostate cancer cell lines, DU145 and PC3, antibodies or control IgG at 4jC overnight. The immune complexes were were screened for the expression of E2F1 by reverse transcription- captured with protein A-agarose beads. After extensive washing, the bound PCR(RT-PCR)and immunoblot analyses (Fig. 1 A). Because our

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E2F1 and Prostate Cancer recent microarray analyses identified EGR1 as a potential target diminished 5-FU–induced apoptosis and promoted cell prolifera- of E2F1 (data not shown), we also monitored EGR1 expression in tion (Fig. 1C). Similar results were obtained with cisplatin the same samples. Both E2F1 and EGR1 were expressed at a treatment (data not shown). Cell cycle analyses showed a significantly higher level in DU145 than in PC3 cells. We next significant delay in G1-S transition upon the loss of E2F1, but not determined if these cells were differentially sensitive to the growth- EGR1, in DU145 cells (data not shown). In sum, E2F1 levels suppressive action of 5-FU, a commonly used cancer therapeutic. controlled the expression of EGR1 in prostate cancer cells and Consistent with the higher levels of E2F1 and EGR1 expression, determined their sensitivity to drug-induced apoptosis. DU145 cells were more resistant to 5-FU–induced apoptosis than Neither NF-KB nor BCL-2 expression were altered in E2F1 PC3 cells (Fig. 1A). Similar results were obtained when cells were knockdown cells. Previous studies have shown that E2F1 could treated with another DNA-damaging drug, cisplatin (data not inhibit tumor necrosis factor-a–induced NF-nB activation by shown). Next, we investigated whether the insensitivity to drug- stabilizing its inhibitor InBa in endothelial cells (25). NF-nB has induced apoptosis was due to E2F1. Plasmids expressing shRNA been implicated in inhibiting apoptosis through a transcriptional specifically against E2F1 and wild-type E2F1 protein, respectively, induction of multiple antiapoptotic factors, including BCL-2 family were used for establishing the stably transfected cell lines proteins (26). Moreover, BCL-2 gene expression was also found to (knockdown in DU145 and overexpression in PC3, respectively) be up-regulated by E2F1 in a large-scale microarray analysis (27). that were validated by RT-PCR and immunoblot (Fig. 1B and C). Therefore, we tested whether changes in the activation of NF-nB Consistent with the data shown in Fig. 1A, knockdown of E2F1 pathways or expression levels of BCL-2 family protein could resulted in the down-regulation of EGR1, whereas overexpression account for these antiapoptotic actions. Whole cell lysates, nuclear of E2F1 resulted in elevated EGR1. The apoptosis and proliferation extracts, and cytoplasmic extracts from DU145 expressing a assays showed that inhibition of E2F1 expression in DU145 scrambled or E2F1-specific shRNA were subjected to immunoblot increased sensitivity to 5-FU–induced apoptosis and reduced analyses with specific antibodies (Fig. 2A). Although E2F1 was proliferation compared with the scrambled control (Fig. 1B). knocked down efficiently, NF-nB pathway proteins, as well as DP1, Conversely, elevation of endogenous E2F1 in PC3 cells significantly the dimerization partner of E2F1, was unaffected (Fig. 2A). These

Figure 3. EGR1 mediated E2F1-induced suppression of apoptosis through up-regulating growth factor production and Akt activity. Cells were treated with 0.5 mg/mL of 5-FUfor the indicated times before examining ( A) the expression of EGR1 and E2F1 protein levels by immunoblot or apoptotic assays by Annexin V/PI staining. B, DU145/Scramble and DU145/EGR1-RNAi were tested for EGR1 expression by immunoblot assay and then treated with 0.5 mg/mL of 5-FU for the indicated times before apoptotic analysis. C, the transcript levels of EGF, IGF-II, and PDGF-a in the indicated cells treated or not treated with 0.5 mg/mL of 5-FUfor 12 h were determined by RT-PCR. D, Akt activation was examined by immunoblot assays with anti–phosphorylated Akt following 0.5 mg/mL of 5-FUtreatment for 12 h. www.aacrjournals.org 2327 Cancer Res 2009; 69: (6). March 15, 2009

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Figure 4. E2F1 and NF-nB cooperatively regulated Egr1 transcription through a nB motif within the Egr1 promoter. A, human Egr1 promoter luciferase reporters used in this study. The mutated nucleotides corresponding to nB-Mut1 and nB-Mut2 are indicated in italics. B, these reporters were used for luciferase assays in PC3-derived cells. C, equal amounts of the expression plasmids for E2F1, E2F2, E2F3, or empty vector pcDNA3.1 were cotransfected with the pGL3-Egr1-wt reporter into DU145, and luciferase activities were determined 36 h later. D, NF-nB p65 knockdown was confirmed in DU145 stably transfected with a plasmid expressing either a scrambled shRNA control or a NF-nB p65-shRNA by immunoblot and RT-PCR. Promoter luciferase reporter assays were then performed as described in B. biochemical studies were then confirmed by an immunofluorescent when compared with E2F1 knockdown cells (compare Fig. 3B staining of NF-nB p65 and p50 and the steady state nuclear with Fig. 1B). Together, these data indicated that EGR1 is a critical translocation of NF-nB was found to be unaffected by the downstream effector of E2F1-induced suppression of apoptosis. knockdown of E2F1 (Fig. 2B). Together, these data indicated that EGR1 regulates the expression of a number of growth factors E2F1 did not influence the constitutive activation of NF-nB in these including EGF, PDGF-a, and IGF-II. Therefore, we tested whether cells. Additionally, 5-FU treatment did not cause any discernible the expression of these growth factors was affected by the changes on the expression of BCL-2 and BCL-XL (Fig. 2C), depletion of E2F1 or EGR1 and 5-FU induction (Fig. 3C). These regardless of the E2F1 status in these cells. results showed that regardless of 5-FU treatment, the expression of Suppression of drug-induced apoptosis by E2F1 is mediated all these growth factors was significantly inhibited upon the by EGR1. We next investigated the mechanism that mediates the knockdown of EGR1 and E2F1. Consistent with the loss of growth antiapoptotic function of E2F1. EGR1 has been shown to promote factor expression, depletion of E2F1 or EGR1 significantly inhibited prostate cancer progression by inducing the expression of growth both constitutive and 5-FU–induced activation of Akt, whereas factors such as transforming growth factor-h, EGF, IGF, and PDGF total Akt proteins remained unchanged (Fig. 3D). These observa- (17, 20, 21), which are critical for tumor development, growth, and tions suggest that the loss of E2F1 and EGR1 significantly impaired survival through activating the PI3K/Akt pathway (6, 8, 9). Because the production of growth factors and the activation of Akt in both we observed a close correlation between E2F1 and EGR1 levels and 5-FU–induced and basal levels in prostate cancer cells. that 5-FU induced stronger apoptosis in the presence of E2F1- E2F1 regulated EGR1 expression through a KB element. shRNA (Fig. 1), we next tested if restoring EGR1 levels could Next, we examined whether E2F1 directly regulated Egr1 tran- diminish the apoptotic response in DU145 cells expressing E2F1- scription. Surprisingly, no E2F1 binding sites were found in Egr1 shRNA (Fig. 3A). Using this strategy, we could avoid the additional promoters using computational analysis and prediction. However, regulatory pathways induced by E2F1. The expression of EGR1 previous studies have implicated that EGR1 can be transcription- in cells was confirmed by immunoblot analysis (Fig. 3A). More ally regulated by NF-nB (28) through the proximal nB-like importantly, reintroducing EGR1 in cells lacking E2F1 abrogated regulatory motif located between À195 and À212 upstream of enhanced drug-induced apoptosis (Fig. 3A). Furthermore, knock- the start codon of the human Egr1 gene (Fig. 4A). A careful analysis down of EGR1 in DU145 cells using a specific shRNA-expressing of this region identified two potential overlapped nB-like sites, vector led to an enhancement of drug-induced apoptosis compared Egr1-nB-1 and Egr1-nB-2, located between positions À212 to À203 with the control cells (Fig. 3B), showing the survival-promoting role (5¶-GGGCGCCTGG-3¶) and À204 to À195 (5¶-GGGATGCGGG-3¶), of EGR1. Notably, a greater sensitivity and a time delay in response respectively (Fig. 4A). Additionally, E2F1 has been shown to to drug-induced apoptosis were observed in EGR1 knockdown cells interact with NF-nB p65 and promote its transcriptional activity on

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E2F1 and Prostate Cancer a subset of NF-nB–driven genes (29). Thus, it is possible that E2F1 fected with wild-type Egr1 promoter luciferase reporter into DU145 may regulate EGR1 expression by interacting with NF-nB and using cells (Fig. 4C) and luciferase activity was monitored. These the identified nB sites. experiments showed that the Egr1 promoter responded only to To elucidate such a hypothesis, we mutated nB-1 and nB-2 sites E2F1, but not to E2F2 and E2F3a, which indicates a specific effect (Fig. 4A) and tested the effect of E2F1. Luciferase reporters driven of E2F1. either by the wild-type or mutated Egr1 promoters were trans- Having asserted the importance of the nB-1 site for Egr1 gene fected into PC3 cells expressing E2F1 or a control vector and their expression, we next determined if loss of NF-nB affected the expressions were monitored (Fig. 4B). Mutation of the nB-1 site, expression of endogenous EGR1. Therefore, we knocked down the but not the nB-2 site, significantly reduced the basal expression of expression of NF-nB p65 subunit using vector-based RNAi in the reporter in PC3 cells. In the presence of E2F1 overexpression, DU145 cells. Loss of expression of p65 was confirmed by Western wild-type and nB-Mut2, but not nB-Mut1, expressed a higher level blot and RT-PCR analyses (Fig. 4D). Knockdown of p65 resulted in a of luciferase compared with those cells transfected with empty dramatically decreased expression of endogenous EGR1, even vector (Fig. 4B). These data show that E2F1-stimulated expression though these cells expressed comparable levels of E2F1, indicating of EGR1 requires the nB-1 site. a requirement for NF-nB in regulating Egr1 expression. To examine We then examined if other members of the E2F family also if the nB-1 site within the Egr1 promoter was required for E2F1- induce the EGR1 promoter. Based on the structural and functional and NF-nB–dependent expression of luciferase, promoter-reporters considerations, E2F superfamily members can be divided into two shown in Fig. 3A were transfected into DU145 cells stably subclasses: E2F1, E2F2, and E2F3a as transcription activators, and expressing scrambled or E2F1-specific or p65-specific shRNA E2F3b, E2F4, E2F5, E2F6, E2F7, and E2F8 as negative regulators (Fig. 4D). Although the wild-type and nB-2 mutant were expressed of gene expression (30). Therefore, we chose the transcriptional normally, they failed to express in cells expressing either E2F1- activators, E2F2 and E2F3a, along with E2F1, to test their specific or p65-specific shRNAs. The nB-1 mutant was not induced capabilities in stimulating Egr1 promoter-driven luciferase expres- in any of these cells. Thus, the nB-1 site, E2F1 and NF-nB p65 are sion. Expression vectors for E2F1, E2F2, or E2F3a were cotrans- required for inducing transcription from the Egr1 promoter.

Figure 5. NF-nB was required for the recruitment of E2F1 to Egr1 promoter. A, ChIP assays were performed with cell lysates from the indicated cells and anti–NF-nB p65, anti-p50, and anti-E2F1 antibodies. A pair of primers flanking the nB element within the Egr1 promoter was used in PCR. B, synthetic scrambled or NF-nB p65-specific siRNA duplex was transfected into DU145/Scramble and DU145/E2F1-RNAi cells. Extracts from these cells were used for immunoblot assays to test the expression of E2F1 and NF-nB p65 and ChIP assays (C), as performed in A. D, formation of the E2F1/NF-nB p65 complex was examined by coimmunoprecipitation using whole cell lysates from DU145 cells. Lysates that were not immunoprecipitated were used as the input to ensure equal loading and normal IgG was used as immunoprecipitation controls in A, B, and C. www.aacrjournals.org 2329 Cancer Res 2009; 69: (6). March 15, 2009

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Recruitment of E2F1 was dependent on the binding of NF- E2F1 promoted the development of skin carcinoma and protected KB p65 to Egr1 promoter. Because E2F1 and NF-nB p65 and the cells from UV-induced apoptosis (5, 31). It was also observed that nB-1 site were required for regulating EGR1 expression, we further E2F1 counteracted c-Myc–driven apoptosis and enhanced hepato- investigated whether these transcription factors bound to the cellular carcinoma development (13). However, the underlying endogenous EGR1 promoter. A chromatin immunoprecipitation mechanisms behind this apoptosis-suppressing action of E2F1 have (ChIP) assay was used for this purpose. A pair of primers flanking not been identified. Here, we have uncovered an E2F1-induced the Egr1-nB-1 site was used for detecting the presence of Egr1 survival pathway which suppressed chemotherapeutic drug– promoter in the immunoprecipitation products of DU145 cells induced apoptosis in prostate carcinomas. In this novel pathway, expressing E2F1-specific or scrambled shRNAs and PC3 cells E2F1 directly induces Egr1 gene transcription and then elevated overexpressing E2F1 or not. As shown in Fig. 5A, significantly EGR1 protein up-regulates the production of downstream growth reduced recruitment of E2F1 to Egr1 promoter was observed when factors which subsequently activate the PI3K/Akt pathway. E2F1 expression was lowered in DU145 cells, whereas considerably Therefore, we found that oncogenic E2F1 promotes tumor cell increased E2F1 was recruited when E2F1 was overexpressed in PC3 survival and attenuates apoptosis in human prostate cancer. cells. Under these conditions, recruitment of NF-nB p65 and p50 Recently, a widespread role of E2F1 in transcriptional regulation subunits was unaffected regardless of the E2F1 levels. The control has been indicated by an unbiased location analysis of E2F1 ChIP reaction using normal IgG did not produce any detectable binding sites in the human genome (32). It is surprising that only a bands suggesting the specificities of the ChIP assays. Therefore, small proportion (<15%) of E2F1-regulated gene promoters possess both E2F1 and NF-nB bind specifically to the nB site within the the canonical E2F-binding sites. Perplexingly, E2F1 is recruited to a Egr1 promoter. large fraction (f25%) of human gene promoters via a mechanism Because p65 bound to Egr1 promoter independently of E2F1, distinct from the recognition of consensus DNA-binding sites. Such yet E2F1 was required for EGR1 expression, we next determined if effects are likely to be mediated by the interactions of E2F1 with E2F1 binding to the EGR1 promoter was dependent on p65. other transcription factors, such as NF-nB. This hypothesis, DU145 cells expressing the scrambled or E2F1-specific shRNA although originally based on computational analyses, was verified were transfected with scrambled or specific siRNA capable of in recent studies. Lim and colleagues described that NF-nB (p50/ targeting p65. The p65-specific siRNA, but not the scrambled RelA) rapidly recruited E2F1 upon lipopolysaccharide stimulation, siRNA, suppressed the expression of p65 in both cell lines. The cell which was critical for full transcriptional activation of some line expressing E2F1-shRNA and transfected with p65-siRNA lost NF-nB–driven genes such as interleukin 1h, interleukin 23A, CCL-3, both E2F1 and p65 proteins (Fig. 5B). A parallel set of cells treated and tumor necrosis factor-a (29). Furthermore, NF-nB has been similarly with siRNAs were used for ChIP assays. Although a found to be involved in the regulation of more than 200 genes in normal recruitment of E2F1 and p65 occurred in DU145 cells, response to diverse stimuli, and could interact with more than a significant loss of E2F1 recruitment to the Egr1 promoter was 150 cellular proteins in various cells (33, 34). However, given a observed in cells transfected with p65-specific siRNA, indicating certain condition, NF-nB only specifically regulates a subset of the dependence of E2F1 binding on the availability of p65 protein. genes, implicating that NF-nB–mediated transcriptional regulation As expected, E2F1 was not strongly recruited to the promoter has promoter-specificity and cell-specificity that may be restrained when cells expressed E2F1-specific shRNA (Fig. 5C). Thus, the or facilitated by other cofactors such as E2F1 (35–38). binding of E2F1 to the nB site of Egr1 promoter requires NF-nB In this study, an example that supports the above hypothesis p65. was presented in which E2F1 regulated Egr1 transcription by In the next experiment, we investigated whether the endogenous facilitating the transactivity of NF-nB and by using the nB site. We NF-nB p65 and E2F1 proteins actually formed a complex in DU145 showed that recruitment of E2F1 to the Egr1 promoter via the cells by coimmunoprecipitation. Lysates were first immunopreci- inverted mechanism proposed by Bieda and colleagues (32) was pitated with p65-specific or a control antibody. The immunopre- dependent on the binding of the NF-nB to the nB site. However, cipitation products were subjected to immunoblot analysis with recruitment of NF-nB alone was insufficient to fully activate Egr1 E2F1-specific antibodies. Indeed, the p65-specific, but not the transcription. Only when both NF-nB and E2F1 were concurrently control IgG, coimmunoprecipitated E2F1. The input samples from available could the induction of Egr1 expression occur. Thus, each reaction had equal amounts of E2F1 (Fig. 5D). Together, these through modulating the transcriptional activity of NF-nB, E2F1 experiments suggested the formation of a transcriptional complex may participate in far more cellular functions beyond controlling consisting of E2F1/p65, which associated with the nB-1 site of the cell cycle transition and inducing apoptosis. In another view, our Egr1 promoter. report also pointed out the requirement for E2F1 as a coactivator for full activation of some NF-nB–driven genes, which explained how NF-nB differentially regulated target genes in response to Discussion various stimuli. E2F1 is known to exert diverse effects on cell growth and Contrary to our study, Hoffman’s lab recently reported that EGR1 apoptosis depending on the cell context. In normal cells, transient abrogated the E2F1-induced block in terminal myeloid differenti- activation of E2F1 promotes cell growth by driving cell cycle ation for suppressing leukemia (39). This tissue-specific oncogenic transition under the control of pRb, whereas its ectopic expression effect of EGR1 could be partially explained by frequent loss of causes E2F1-dependent apoptosis. In cancer cells, loss or its downstream genes, p53 and PTEN (17), and natural inhibitor, inactivation of pRb, the regulatory switch, leads to an uncontrolled NAB2 (40), in prostate cancer and its capability to induce the activation of E2F1. Owing to the dual role of promoting both expression of tumor-promoting growth factors. Indeed, E2F1, proliferation and apoptosis, the function of E2F1 in oncogenesis NF-nB, and EGR1 have been found to be overexpressed in prostate remained controversial and highly dependent on certain regulators cancer concurrently and play critical roles in proliferation, survival, À À such as p53 and c-Myc. In a p53 / background, overexpression of and metastasis (18, 19, 21, 41–43). NF-nB is constitutively expressed

Cancer Res 2009; 69: (6). March 15, 2009 2330 www.aacrjournals.org

E2F1 and Prostate Cancer and activated in prostate cancers, which parallels with tumor Disclosure of Potential Conflicts of Interest development and progression (44, 45), whereas the expression No potential conflicts of interest were disclosed. levels of E2F1 and EGR1 are gradually increased as the cancer progresses, particularly during conversion into advanced and Acknowledgments metastatic stage (16, 41, 43). Furthermore, growth factors including Received 10/24/2008; revised 12/11/2008; accepted 1/8/2009; published OnlineFirst EGF, PDGF, and IGF have also been found to closely correlate with 3/10/09. Gleason scores in clinical prostate cancers (46). Together, these Grant support: National Natural Science Foundation of China (30570704, 30721002, and 30528020), National Basic Research Program of China (973 Program; data implicated that a possible cross-talk among these oncogenic 2007CB914503), Ministry of Science and Technology of China (KSCX1-YW-R-58), and factors may contribute to prostate cancer progression. Our findings China Ministry of Education (20060358019). Funding to pay the Open Access publication charges for this article was provided by grant 2007CB914503 from the provide the first molecular evidence for how these interactions National Basic Research Program of China (W. Xiao), and by U.S. National Cancer result in the loss of control over cell growth and the development Institute grants CA78282 and CA105005 (D.V. Kalvakolanu). The costs of publication of this article were defrayed in part by the payment of page of drug resistance, which is commonly seen in most advanced charges. This article must therefore be hereby marked advertisement in accordance prostate cancers. with 18 U.S.C. Section 1734 solely to indicate this fact.

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www.aacrjournals.org 2331 Cancer Res 2009; 69: (6). March 15, 2009

E2F1 Induces Tumor Cell Survival via Nuclear Factor-κB− Dependent Induction of EGR1 Transcription in Prostate Cancer Cells

Chaogu Zheng, Zijia Ren, Hetian Wang, et al.

Cancer Res 2009;69:2324-2331. Published OnlineFirst March 10, 2009.

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