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

Transcription factor E2F1 not only promotes cell proliferation but also induces apoptosis in cancer. For example, both reduced proliferative and apoptotic rates were evidenced in SV40 T121–induced tumors in E2F1 knockout mice, suggesting a paradoxical role for E2F1 in tumors (1). Therefore, it was considered as both oncogene and tumor suppressor (2–4). The antiapoptotic function of E2F1 was first observed to protect cells from UV-induced apoptosis in p53-deficient mice (5); however, the mechanism governing such apoptosis-suppressing action of E2F1 is still unclear. Phosphoinositide-3-kinase (PI3K)/Akt signaling is a major survival pathway that can be activated by various stimuli such as growth factors (6–9) and is widely involved in the inhibition of apoptosis via regulating the activity of apoptotic factors such as BAD, caspase-9, and Mdm2 (10–12). It has been reported recently that E2F1 inhibited c-Myc–driven apoptosis via inducing a PIK3CA/Akt/mTOR cascade in human liver cancer (13), implicating a potential link between the oncogenic function of E2F1 and the activation of Akt. Grb-associated binder 2 (Gab2), a receptor docking protein in the PI3K/Akt signaling pathway, is identified as a target gene of E2F1, which is responsible for the activation of Akt by E2F1 (14). However, tumors induced by the neu(ErbB-2) oncogene in Gab2^−/− background showed normal Akt activities, suggesting a dispensable role of Gab2 in activating Akt in tumor (15). Thus, it is still not clear how E2F1 enhances PI3K/Akt signaling.

Clinical studies showed that EGR1 expression level was significantly increased in prostate cancers and directly correlated with Gleason score and tumor grade (16). This oncogenic role does not seem to extend to other tumor types, such as skin tumor, fibrosarcoma, and glioblastoma in which EGR1 acts as a tumor suppressor gene through multiple mechanisms, including inducing p53 and PTEN expression (17). Indeed, EGR1 deficiency results in a significant delay in tumor initiation, mortality, and progression from prostatic intraepithelial neoplasia to invasive carcinoma; and inhibition of EGR1 expression reverses the transformation of prostate carcinoma both in vivo and in vitro (18, 19). Moreover, EGR1 physically interacts with androgen receptor and induces the prostatic intraepithelial neoplasia carcinoma transition via transcriptionally activating growth and angiogenic factors such as platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), fibroblast growth factor, and vascular endothelial growth factor (20, 21), all of which contribute to hormones refractory to prostate cancer (22, 23). Together, these observations indicate a critical role for EGR1 in promoting prostate cancer development. However, what causes EGR1 overexpression and which factors control its expression in prostate cancer are still poorly understood.

Here, we show that E2F1, by associating with nuclear factor-κB (NF-κB), maximally induces the expression of EGR1. Elevated EGR1 then induces the production of a panel of growth factors, including at least EGF, PDGF, and IGF-II, etc. In turn, these growth factors activate PI3K/Akt and promote resistance to drug-induced apoptosis. Together, our study has identified a novel E2F1-induced cell survival pathway.

Reagent and antibodies. All reagents, chemicals, and affinity-purified anti-rabbit IgG, anti-mouse IgG, and anti-goat IgG conjugated with horseradish peroxidase or fluorescent tags were purchased from Sigma. The following specific antibodies were used in this study: anti–NF-κB p65 and p50 (Upstate Biotech); anti-E2F1, EGR1, DP-1, IκB, Akt, histone H1, and actin (Santa Cruz Biotech); and anti-BCL2, BCL-X_l, phosphorylated Akt (p-Ser⁴⁷³), and phosphorylated IκBα (p-Ser^32/36; Cell Signaling).

Short hairpin RNA expression, small interfering RNA duplex, and reporter gene constructs. The pU6+27, short hairpin RNA (shRNA) control, and shRNA p65 vectors were purchased from Panomics Corporation. A 21-nucleotide sequence coding for amino acids 125 to 131 of human E2F1 and 413 to 419 of human EGR1 were selected as the targets for RNAi according to the instructions of the manufacturer. In all cases, the corresponding sequences were scrambled to generate a control vector. In initial studies, we used empty vector (pU6+27) and scrambled shRNA as controls. Double-stranded small interfering RNAs (siRNA) targeting the CDS: 891 to 909 of NF-κB (p65) and scrambled control was synthesized using the following sequences: siRNA-p65; forward, 5′-GAU UGA GGA GAA ACG UAA AdTdT; reverse, 5′-UUU ACG UUU CUC CUC AAU CdTdT. siRNA-scramble; forward, 5′-AUG AAC GUG AAU UGC UCA AdTdT; reverse, 5′-UUG AGC AAU UCA CGU UCA UdTdT.

To construct the EGR1 promoter-driven luciferase reporter, a 760-bp DNA fragment from the promoter region of the EGR1 gene, including −70 to −846 nucleotides upstream of the start ATG, was obtained by PCR using human genomic DNA as the template and primers EGR1-pf, 5′-gaC TCG AGG CTC ACT GCT ATA CAG TGT C-3′; and EGR1-pr, 5′-cgA AGC TTT ACA TGG CAT ATA TGG GAA GC-3′, where the restriction enzyme sites for cloning are indicated by italics. The PCR products were inserted into the pGL3-basic vector (Promega) and designated as pGL3-Egr1-wt. The κB-like motif was mutated using site-directed mutagenesis correspondingly to two possibly overlapped NF-κB binding sites (indicated in Fig. 4A). All the constructs were verified by sequencing.

The pcDNA3 expression vectors with E2F1, E2F2, and E2F3 expressed were provided by Dr. Mian Wu (University of Science & Technology of China, Hefei, China) and described previously (24). EGR1 expression plasmids were generous gifts from Dr. Jie Du (Baylor College of Medicine, Houston, TX).

Cell culture and transfection. DU145 and PC3, human prostate cancer cell lines, were purchased from American Type Culture Collection and cultured at 37°C in DMEM supplemented with 10% fetal bovine serum and 2 mmol/L of l-glutamine. LipofectAMINE 2000 (Invitrogen Corporation) was used for transfection. Stably transfected cell lines were obtained after being selectively screened by G418 (700 μg/mL; Life Technologies) for 3 to 4 weeks.

Immunoblot analyses. Whole cell extract were prepared by lysing cells in the NP40 lysis buffer containing 50 mmol/L of Tris-Cl (pH 6.8), 150 mmol/L of NaCl, 1 mmol/L of EGTA, 1% NP40, and freshly added proteinase inhibitors including 1 mmol/L of phenylmethylsulfonyl fluoride, 1 μg/mL of leupeptin (Sigma), 1 μg/mL of pepstatin (Sigma), and 1 μg/mL of aprotinin (Sigma). For preparing the nuclear extract, cytoplasmic membranes were disrupted in 0.5% NP40, 25 mmol/L of Hepes (pH 7.5), 5 mmol/L of KCl, 0.5 mmol/L of MgCl₂, 1 mmol/L of DTT, and proteinase inhibitors; then nuclei were collected by centrifuging at 2,500 rpm for 1 min and lysed in 25 mmol/L of Hepes (pH 7.5), 10% sucrose, 0.01% NP40, 350 mmol/L of NaCl, 1 mmol/L of DTT, and proteinase inhibitors. Lysates were subjected to immunoblot analysis. Horseradish peroxidase–conjugated secondary antibodies and enhanced chemiluminescence detection (Pierce Chemical, Co.) was used to detect the specific immunoreactive proteins.

Reverse transcription-PCR. One microgram of isolated total RNA was converted to cDNA using Superscript III reverse transcriptase (Invitrogen). Gene-specific PCR was conducted using the primer sets indicated in Supplementary Table S1. The PCR products were resolved on a 1.5% agarose gel and then visualized by ethidium bromide staining.

Immunoprecipitation. Cellular lysates were prepared using a radioimmunoprecipitation assay buffer [50 mmol/L Tris-Cl (pH 7.4), 1% NP40, 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 1 mmol/L Na₃VO₄, and 1 mmol/L NaF]. An equivalent amount of total proteins were incubated with 2 μg of specific antibody or control IgG at 4°C overnight. The immunocomplexes were captured by incubation with protein A-agarose at 4°C for 2 h and the immunoprecipitates were subjected to Western blotting with specific antibodies.

Annexin V staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. For apoptotic analysis, cell apoptosis detection kit (BD Bioscience) was used. After treatment with 0.5 mg/mL of 5-fluorouracil (5-FU) for the indicated times, cells were washed with 1× binding buffer [10 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl₂, and 1.8 mmol/L CaCl₂], then incubated with FITC-conjugated Annexin V and 5 μg/mL of propidium iodide (PI) in the dark for 30 min, and analyzed with FACSCalibur (BD Bosciences) immediately. 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 independent tests and each test with triplicate samples.

Chromatin immunoprecipitation assay. Cells were seeded 24 h prior to fixation with 1% formaldehyde at 37°C for 7 min. The cells were harvested in lysis buffer [50 mmol/L Tris-Cl (pH 8.1), 10 mmol/L EDTA, and 1% SDS] and sonicated to shear the chromatin (∼500 bp). The soluble fraction was collected by centrifugation and incubated with specific antibodies or control IgG at 4°C overnight. The immune complexes were captured with protein A-agarose beads. After extensive washing, the bound DNA fragments were eluted and purified. DNA from these samples was subjected to PCR analyses with NF-κB-BS-pf, 5′-GCG GCT AGA GCT CTA GGC-3′; and NF-κB-BS-pr, 5′-GCA GAA GCC CTA ATA TGG C-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-pf, 5′-GTA TTC CCC CAG GTT TAC AT-3′; and GAPDH-pr, 5′-TTC TGT CTT CCA CTC ACT CCT-3′. The PCR products were separated on 2% agarose gel and visualized by ethidium bromide staining. The GAPDH promoter fragment acted as the internal negative control for this assay.

Luciferase reporter assays. Briefly, an appropriate amount of the Egr1 promoter-luciferase reporters, together with Renilla luciferase plasmids, which served as the internal control, were cotransfected into cells. Two days later, cellular lysates were subjected to a dual-luciferase reporter assay (Promega) according to the instructions of the manufacturer. The luciferase activities for the promoter reporters were normalized to Renilla luciferase activities. The data represented at least three independent experiments, and in each experiment, tests were done in triplicate for all the samples.

Immunofluorescence. Cells grown on coverslips were fixed with acetone/methanol (1:1) for 1 min After blocking with 5% normal goat serum for 30 min at 37°C, they were incubated with anti-p65 or anti-p50 overnight at 4°C. They were then incubated with Cy3-conjugated anti-rabbit IgG (Sigma) for 30 min at 37°C and photographs were captured with a confocal laser scanning microscope.

E2F1 expression levels were correlated with resistance to drug-induced apoptosis in prostate cancer cells. To examine the influence of E2F1 on prostate cancer cell survival in response to chemotherapeutic drugs, two of the most commonly used androgen-independent prostate cancer cell lines, DU145 and PC3, were screened for the expression of E2F1 by reverse transcription-PCR (RT-PCR) and immunoblot analyses (Fig. 1A). Because our recent microarray analyses identified EGR1 as a potential target of E2F1 (data not shown), we also monitored EGR1 expression in the same samples. Both E2F1 and EGR1 were expressed at a significantly higher level in DU145 than in PC3 cells. We next determined if these cells were differentially sensitive to the growth-suppressive action of 5-FU, a commonly used cancer therapeutic. Consistent with the higher levels of E2F1 and EGR1 expression, DU145 cells were more resistant to 5-FU–induced apoptosis than PC3 cells (Fig. 1A). Similar results were obtained when cells were treated with another DNA-damaging drug, cisplatin (data not shown). Next, we investigated whether the insensitivity to drug-induced apoptosis was due to E2F1. Plasmids expressing shRNA specifically against E2F1 and wild-type E2F1 protein, respectively, were used for establishing the stably transfected cell lines (knockdown in DU145 and overexpression in PC3, respectively) that were validated by RT-PCR and immunoblot (Fig. 1B and C). Consistent with the data shown in Fig. 1A, knockdown of E2F1 resulted in the down-regulation of EGR1, whereas overexpression of E2F1 resulted in elevated EGR1. The apoptosis and proliferation assays showed that inhibition of E2F1 expression in DU145 increased sensitivity to 5-FU–induced apoptosis and reduced proliferation compared with the scrambled control (Fig. 1B). Conversely, elevation of endogenous E2F1 in PC3 cells significantly diminished 5-FU–induced apoptosis and promoted cell proliferation (Fig. 1C). Similar results were obtained with cisplatin treatment (data not shown). Cell cycle analyses showed a significant delay in G₁-S transition upon the loss of E2F1, but not EGR1, in DU145 cells (data not shown). In sum, E2F1 levels controlled the expression of EGR1 in prostate cancer cells and determined their sensitivity to drug-induced apoptosis.

Neither NF-κB nor BCL-2 expression were altered in E2F1 knockdown cells. Previous studies have shown that E2F1 could inhibit tumor necrosis factor-α–induced NF-κB activation by stabilizing its inhibitor IκBα in endothelial cells (25). NF-κB has been implicated in inhibiting apoptosis through a transcriptional induction of multiple antiapoptotic factors, including BCL-2 family proteins (26). Moreover, BCL-2 gene expression was also found to be up-regulated by E2F1 in a large-scale microarray analysis (27). Therefore, we tested whether changes in the activation of NF-κB pathways or expression levels of BCL-2 family protein could account for these antiapoptotic actions. Whole cell lysates, nuclear extracts, and cytoplasmic extracts from DU145 expressing a scrambled or E2F1-specific shRNA were subjected to immunoblot analyses with specific antibodies (Fig. 2A). Although E2F1 was knocked down efficiently, NF-κB pathway proteins, as well as DP1, the dimerization partner of E2F1, was unaffected (Fig. 2A). These biochemical studies were then confirmed by an immunofluorescent staining of NF-κB p65 and p50 and the steady state nuclear translocation of NF-κB was found to be unaffected by the knockdown of E2F1 (Fig. 2B). Together, these data indicated that E2F1 did not influence the constitutive activation of NF-κB in these cells. Additionally, 5-FU treatment did not cause any discernible changes on the expression of BCL-2 and BCL-X_l (Fig. 2C), regardless of the E2F1 status in these cells.

Suppression of drug-induced apoptosis by E2F1 is mediated by EGR1. We next investigated the mechanism that mediates the antiapoptotic function of E2F1. EGR1 has been shown to promote prostate cancer progression by inducing the expression of growth factors such as transforming growth factor-β, EGF, IGF, and PDGF (17, 20, 21), which are critical for tumor development, growth, and survival through activating the PI3K/Akt pathway (6, 8, 9). Because we observed a close correlation between E2F1 and EGR1 levels and that 5-FU induced stronger apoptosis in the presence of E2F1-shRNA (Fig. 1), we next tested if restoring EGR1 levels could diminish the apoptotic response in DU145 cells expressing E2F1-shRNA (Fig. 3A). Using this strategy, we could avoid the additional regulatory pathways induced by E2F1. The expression of EGR1 in cells was confirmed by immunoblot analysis (Fig. 3A). More importantly, reintroducing EGR1 in cells lacking E2F1 abrogated enhanced drug-induced apoptosis (Fig. 3A). Furthermore, knockdown of EGR1 in DU145 cells using a specific shRNA-expressing vector led to an enhancement of drug-induced apoptosis compared with the control cells (Fig. 3B), showing the survival-promoting role of EGR1. Notably, a greater sensitivity and a time delay in response to drug-induced apoptosis were observed in EGR1 knockdown cells when compared with E2F1 knockdown cells (compare Fig. 3B with Fig. 1B). Together, these data indicated that EGR1 is a critical downstream effector of E2F1-induced suppression of apoptosis.

EGR1 regulates the expression of a number of growth factors including EGF, PDGF-α, and IGF-II. Therefore, we tested whether the expression of these growth factors was affected by the depletion of E2F1 or EGR1 and 5-FU induction (Fig. 3C). These results showed that regardless of 5-FU treatment, the expression of all these growth factors was significantly inhibited upon the knockdown of EGR1 and E2F1. Consistent with the loss of growth factor expression, depletion of E2F1 or EGR1 significantly inhibited both constitutive and 5-FU–induced activation of Akt, whereas total Akt proteins remained unchanged (Fig. 3D). These observations suggest that the loss of E2F1 and EGR1 significantly impaired the production of growth factors and the activation of Akt in both 5-FU–induced and basal levels in prostate cancer cells.

E2F1 regulated EGR1 expression through a κB element. Next, we examined whether E2F1 directly regulated Egr1 transcription. Surprisingly, no E2F1 binding sites were found in Egr1 promoters using computational analysis and prediction. However, previous studies have implicated that EGR1 can be transcriptionally regulated by NF-κB (28) through the proximal κB-like regulatory motif located between −195 and −212 upstream of the start codon of the human Egr1 gene (Fig. 4A). A careful analysis of this region identified two potential overlapped κB-like sites, Egr1-κB-1 and Egr1-κB-2, located between positions −212 to −203 (5′-GGGCGCCTGG-3′) and −204 to −195 (5′-GGGATGCGGG-3′), respectively (Fig. 4A). Additionally, E2F1 has been shown to interact with NF-κB p65 and promote its transcriptional activity on a subset of NF-κB–driven genes (29). Thus, it is possible that E2F1 may regulate EGR1 expression by interacting with NF-κB and using the identified κB sites.

To elucidate such a hypothesis, we mutated κB-1 and κB-2 sites (Fig. 4A) and tested the effect of E2F1. Luciferase reporters driven either by the wild-type or mutated Egr1 promoters were transfected into PC3 cells expressing E2F1 or a control vector and their expressions were monitored (Fig. 4B). Mutation of the κB-1 site, but not the κB-2 site, significantly reduced the basal expression of the reporter in PC3 cells. In the presence of E2F1 overexpression, wild-type and κB-Mut2, but not κB-Mut1, expressed a higher level of luciferase compared with those cells transfected with empty vector (Fig. 4B). These data show that E2F1-stimulated expression of EGR1 requires the κB-1 site.

We then examined if other members of the E2F family also induce the EGR1 promoter. Based on the structural and functional considerations, E2F superfamily members can be divided into two subclasses: E2F1, E2F2, and E2F3a as transcription activators, and E2F3b, E2F4, E2F5, E2F6, E2F7, and E2F8 as negative regulators of gene expression (30). Therefore, we chose the transcriptional activators, E2F2 and E2F3a, along with E2F1, to test their capabilities in stimulating Egr1 promoter-driven luciferase expression. Expression vectors for E2F1, E2F2, or E2F3a were cotransfected with wild-type Egr1 promoter luciferase reporter into DU145 cells (Fig. 4C) and luciferase activity was monitored. These experiments showed that the Egr1 promoter responded only to E2F1, but not to E2F2 and E2F3a, which indicates a specific effect of E2F1.

Having asserted the importance of the κB-1 site for Egr1 gene expression, we next determined if loss of NF-κB affected the expression of endogenous EGR1. Therefore, we knocked down the expression of NF-κB p65 subunit using vector-based RNAi in DU145 cells. Loss of expression of p65 was confirmed by Western blot and RT-PCR analyses (Fig. 4D). Knockdown of p65 resulted in a dramatically decreased expression of endogenous EGR1, even though these cells expressed comparable levels of E2F1, indicating a requirement for NF-κB in regulating Egr1 expression. To examine if the κB-1 site within the Egr1 promoter was required for E2F1- and NF-κB–dependent expression of luciferase, promoter-reporters shown in Fig. 3A were transfected into DU145 cells stably expressing scrambled or E2F1-specific or p65-specific shRNA (Fig. 4D). Although the wild-type and κB-2 mutant were expressed normally, they failed to express in cells expressing either E2F1-specific or p65-specific shRNAs. The κB-1 mutant was not induced in any of these cells. Thus, the κB-1 site, E2F1 and NF-κB p65 are required for inducing transcription from the Egr1 promoter.

Recruitment of E2F1 was dependent on the binding of NF-κB p65 to Egr1 promoter. Because E2F1 and NF-κB p65 and the κB-1 site were required for regulating EGR1 expression, we further investigated whether these transcription factors bound to the endogenous EGR1 promoter. A chromatin immunoprecipitation (ChIP) assay was used for this purpose. A pair of primers flanking the Egr1-κB-1 site was used for detecting the presence of Egr1 promoter in the immunoprecipitation products of DU145 cells expressing E2F1-specific or scrambled shRNAs and PC3 cells overexpressing E2F1 or not. As shown in Fig. 5A, significantly reduced recruitment of E2F1 to Egr1 promoter was observed when E2F1 expression was lowered in DU145 cells, whereas considerably increased E2F1 was recruited when E2F1 was overexpressed in PC3 cells. Under these conditions, recruitment of NF-κB p65 and p50 subunits was unaffected regardless of the E2F1 levels. The control ChIP reaction using normal IgG did not produce any detectable bands suggesting the specificities of the ChIP assays. Therefore, both E2F1 and NF-κB bind specifically to the κB site within the Egr1 promoter.

Because p65 bound to Egr1 promoter independently of E2F1, yet E2F1 was required for EGR1 expression, we next determined if E2F1 binding to the EGR1 promoter was dependent on p65. DU145 cells expressing the scrambled or E2F1-specific shRNA were transfected with scrambled or specific siRNA capable of targeting p65. The p65-specific siRNA, but not the scrambled siRNA, suppressed the expression of p65 in both cell lines. The cell line expressing E2F1-shRNA and transfected with p65-siRNA lost both E2F1 and p65 proteins (Fig. 5B). A parallel set of cells treated similarly with siRNAs were used for ChIP assays. Although a normal recruitment of E2F1 and p65 occurred in DU145 cells, a significant loss of E2F1 recruitment to the Egr1 promoter was observed in cells transfected with p65-specific siRNA, indicating the dependence of E2F1 binding on the availability of p65 protein. As expected, E2F1 was not strongly recruited to the promoter when cells expressed E2F1-specific shRNA (Fig. 5C). Thus, the binding of E2F1 to the κB site of Egr1 promoter requires NF-κB p65.

In the next experiment, we investigated whether the endogenous NF-κB p65 and E2F1 proteins actually formed a complex in DU145 cells by coimmunoprecipitation. Lysates were first immunoprecipitated with p65-specific or a control antibody. The immunoprecipitation products were subjected to immunoblot analysis with E2F1-specific antibodies. Indeed, the p65-specific, but not the control IgG, coimmunoprecipitated E2F1. The input samples from each reaction had equal amounts of E2F1 (Fig. 5D). Together, these experiments suggested the formation of a transcriptional complex consisting of E2F1/p65, which associated with the κB-1 site of the Egr1 promoter.

E2F1 is known to exert diverse effects on cell growth and apoptosis depending on the cell context. In normal cells, transient activation of E2F1 promotes cell growth by driving cell cycle transition under the control of pRb, whereas its ectopic expression causes E2F1-dependent apoptosis. In cancer cells, loss or inactivation of pRb, the regulatory switch, leads to an uncontrolled activation of E2F1. Owing to the dual role of promoting both proliferation and apoptosis, the function of E2F1 in oncogenesis remained controversial and highly dependent on certain regulators such as p53 and c-Myc. In a p53^−/− background, overexpression of E2F1 promoted the development of skin carcinoma and protected cells from UV-induced apoptosis (5, 31). It was also observed that E2F1 counteracted c-Myc–driven apoptosis and enhanced hepatocellular carcinoma development (13). However, the underlying mechanisms behind this apoptosis-suppressing action of E2F1 have not been identified. Here, we have uncovered an E2F1-induced survival pathway which suppressed chemotherapeutic drug–induced apoptosis in prostate carcinomas. In this novel pathway, E2F1 directly induces Egr1 gene transcription and then elevated EGR1 protein up-regulates the production of downstream growth factors which subsequently activate the PI3K/Akt pathway. Therefore, we found that oncogenic E2F1 promotes tumor cell survival and attenuates apoptosis in human prostate cancer.

Recently, a widespread role of E2F1 in transcriptional regulation has been indicated by an unbiased location analysis of E2F1 binding sites in the human genome (32). It is surprising that only a small proportion (<15%) of E2F1-regulated gene promoters possess the canonical E2F-binding sites. Perplexingly, E2F1 is recruited to a large fraction (∼25%) of human gene promoters via a mechanism distinct from the recognition of consensus DNA-binding sites. Such effects are likely to be mediated by the interactions of E2F1 with other transcription factors, such as NF-κB. This hypothesis, although originally based on computational analyses, was verified in recent studies. Lim and colleagues described that NF-κB (p50/RelA) rapidly recruited E2F1 upon lipopolysaccharide stimulation, which was critical for full transcriptional activation of some NF-κB–driven genes such as interleukin 1β, interleukin 23A, CCL-3, and tumor necrosis factor-α (29). Furthermore, NF-κB has been found to be involved in the regulation of more than 200 genes in response to diverse stimuli, and could interact with more than 150 cellular proteins in various cells (33, 34). However, given a certain condition, NF-κB only specifically regulates a subset of genes, implicating that NF-κB–mediated transcriptional regulation has promoter-specificity and cell-specificity that may be restrained or facilitated by other cofactors such as E2F1 (35–38).

In this study, an example that supports the above hypothesis was presented in which E2F1 regulated Egr1 transcription by facilitating the transactivity of NF-κB and by using the κB site. We showed that recruitment of E2F1 to the Egr1 promoter via the inverted mechanism proposed by Bieda and colleagues (32) was dependent on the binding of the NF-κB to the κB site. However, recruitment of NF-κB alone was insufficient to fully activate Egr1 transcription. Only when both NF-κB and E2F1 were concurrently available could the induction of Egr1 expression occur. Thus, through modulating the transcriptional activity of NF-κB, E2F1 may participate in far more cellular functions beyond controlling cell cycle transition and inducing apoptosis. In another view, our report also pointed out the requirement for E2F1 as a coactivator for full activation of some NF-κB–driven genes, which explained how NF-κB differentially regulated target genes in response to various stimuli.

Contrary to our study, Hoffman's lab recently reported that EGR1 abrogated the E2F1-induced block in terminal myeloid differentiation for suppressing leukemia (39). This tissue-specific oncogenic effect of EGR1 could be partially explained by frequent loss of its downstream genes, p53 and PTEN (17), and natural inhibitor, NAB2 (40), in prostate cancer and its capability to induce the expression of tumor-promoting growth factors. Indeed, E2F1, NF-κB, and EGR1 have been found to be overexpressed in prostate cancer concurrently and play critical roles in proliferation, survival, and metastasis (18, 19, 21, 41–43). NF-κB is constitutively expressed and activated in prostate cancers, which parallels with tumor development and progression (44, 45), whereas the expression levels of E2F1 and EGR1 are gradually increased as the cancer progresses, particularly during conversion into advanced and metastatic stage (16, 41, 43). Furthermore, growth factors including EGF, PDGF, and IGF have also been found to closely correlate with Gleason scores in clinical prostate cancers (46). Together, these data implicated that a possible cross-talk among these oncogenic factors may contribute to prostate cancer progression. Our findings provide the first molecular evidence for how these interactions result in the loss of control over cell growth and the development of drug resistance, which is commonly seen in most advanced prostate cancers.

No potential conflicts of interest were disclosed.

Grant support: National Natural Science Foundation of China (30570704, 30721002, and 30528020), National Basic Research Program of China (973 Program; 2007CB914503), Ministry of Science and Technology of China (KSCX1-YW-R-58), and China Ministry of Education (20060358019). Funding to pay the Open Access publication charges for this article was provided by grant 2007CB914503 from the National Basic Research Program of China (W. Xiao), and by U.S. National Cancer Institute grants CA78282 and CA105005 (D.V. Kalvakolanu).

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.