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Elevated E2F1 Inhibits Transcription of the Androgen Receptor in Metastatic Hormone-Resistant Prostate Cancer

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Elevated E2F1 Inhibits Transcription of the Androgen Receptor in Metastatic Hormone-Resistant Prostate Cancer Research Article Elevated E2F1 Inhibits Transcription of the Androgen Receptor in Metastatic Hormone-Resistant Prostate Cancer Joanne N. Davis,1 Kirk J. Wojno,1 Stephanie Daignault,1 Matthias D. Hofer,2,3 Rainer Kuefer,3 Mark A. Rubin,3,4 and Mark L. Day1 1Department of Urology, University of Michigan, Ann Arbor, Michigan; 2Department of Urology, University of Ulm, Ulm, Germany; 3Department of Pathology, Brigham and Women’s Hospital; and 4Harvard University, School of Medicine, Boston, Massachusetts Abstract disease recurs in an estimated 15% to 30% of patients (2). The androgen receptor (AR) is the mediator of the physiologic effects of Activation of E2F transcription factors, through disruption of androgen. It regulates the growth of normal and malignant the retinoblastoma (Rb) tumor-suppressor gene, is a key event prostate epithelial cells. Upon ligand binding, AR translocates to in the development of many human cancers. Previously, we the nucleus, binds to DNA recognition sequences, and activates showed that homozygous deletion of Rb in a prostate tissue transcription of target genes, including genes involved in cell recombination model exhibits increased E2F activity, acti- proliferation, apoptosis, and differentiation (reviewed in ref. 3). vation of E2F-target genes, and increased susceptibility to Androgen ablation therapy is highly successful for the treatment of hormonal carcinogenesis. In this study, we examined the hormone-sensitive prostate cancer; however, hormone resistance expression of E2F1 in 667 prostate tissue cores and compared significantly limits its benefits. Hormonal ablation therapy will it with the expression of the androgen receptor (AR), a marker control metastatic disease for 18 to 24 months (4), but once of prostate epithelial differentiation, using tissue microarray metastatic prostate cancer ceases to respond to hormonal therapy, analysis. We show that E2F1 expression is low in benign and median survival decreases significantly (5). Although the focus of localized prostate cancer, modestly elevated in metastatic rigorous study, the molecular mechanisms underlying the progres- lymph nodes from hormone-naı¨ve patients, and significantly sion of prostate cancer to hormone-independent disease remain elevated in metastatic tissues from hormone-resistant pros- unclear. tate cancer patients (P = 0.0006). In contrast, strong AR The E2F family of transcription factors consists of at least eight expression was detected in benign prostate (83%), localized characterized family members (E2Fs 1–8), which can form prostate cancer (100%), and lymph node metastasis (80%), but heterodimers with differentiating protein family members (DP1, decreased to 40% in metastatic hormone-resistant prostate DP2), giving rise to functional E2F activity (6, 7). E2F controls the cancer (P = 0.004). Semiquantitative reverse transcription- progression through the cell cycle by regulating the transcription of PCR analysis showed elevated E2F1 mRNA levels and genes that are essential for DNA synthesis and cell cycle increased levels of the E2F-target genes dihyrofolate reductase progression (8). Several E2Fs, including E2F1, behave as oncogenes, and proliferating cell nuclear antigen in metastatic hormone– and are able to induce quiescent cells to enter the cell cycle, independent prostate cancer cases compared with benign override various growth-arrest signals, and transform primary cells tissues. To identify a role of E2F1 in hormone-independent (9–11). Elevated E2F1 can induce hyperproliferation, hyperplasia, prostate cancer, we examined whether E2F1 can regulate AR and p53-dependent apoptosis in E2F1 transgenic mice (12). E2Fs expression. We show that exogenous expression of E2F1 also act like tumor suppressors by activating apoptotic pathways significantly inhibited AR mRNA and AR protein levels in (13), suggesting that E2Fs affect multiple downstream targets prostate epithelial cells. E2F1 also inhibited an AR promoter- depending on cell type and environmental context. luciferase construct that was dependent on the transactivation Aberrant expression of E2F1 has been documented in human domain of E2F1. Furthermore, using chromatin immunopre- cancers, including amplification of the E2F1 gene in erythroleuke- cipitation assays, we show that E2F1 and the pocket protein mia cell lines (14), and overexpression of E2F1 protein in breast family members p107 and p130 bind to the AR promoter cancer cell lines (15) and head and neck carcinoma cell lines (16). in vivo. Taken together, these results show that elevated E2F1, Elevated E2F1 expression has been reported in invasive ductal through its ability to repress AR transcription, may contribute breast carcinomas (15) and non–small cell lung carcinomas (17), to the progression of hormone-independent prostate cancer. where high levels of E2F1 were associated with advanced disease (Cancer Res 2006; 66(24): 11897-906) and poor prognosis. E2F3 was found to be elevated in localized prostate cancer and is associated with decreased overall survival Introduction (18). Recently, Rhodes et al. (19) reported that genes with E2F Prostate cancer is the number one diagnosed cancer in males in binding sites are significantly overexpressed in a wide variety of the United States other than skin cancer and is the second leading human cancers, further supporting the view that activation of the cause of cancer-related deaths (1). Locally defined disease is often E2F pathway is a prevalent event in certain human cancers. successfully treated with surgery and/or radiotherapy; however, We have previously shown that disruption of the retinoblastoma (Rb)-E2F complex by homozygous deletion of Rb in a prostate tissue recombination model exhibited increased E2F activity and Requests for reprints: Mark L. Day, Department of Urology, University of activation of E2F-target genes in vitro and predisposed prostatic Michigan, 6131 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0944. epithelium to hormonal carcinogenesis in vivo (20–22). We Phone: 734-763-9968; Fax: 734-647-9271; E-mail: [email protected]. I2006 American Association for Cancer Research. hypothesized that enhanced E2F1 expression may be a common doi:10.1158/0008-5472.CAN-06-2497 event in human prostate cancer and may contribute to prostate www.aacrjournals.org 11897 Cancer Res 2006; 66: (24). December 15, 2006 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research cancer development and progression. In this study, we used tissue Cell lines. LNCaP and 293 cells were purchased from the American Type microarray (TMA) analysis to examine protein expression of E2F1 Culture Collection (Manassas, VA). All cells were maintained in RPMI 1640 and AR during prostate cancer progression. A TMA containing 667 supplemented with 5% fetal bovine serum (FBS), 0.1% penicillin/ tissue cores designed to examine prostate cancer progression to streptomycin, and 0.1% L-glutamine. Semiquantitative reverse transcription-PCR analysis. Total RNA hormone-independent disease was used to assess the relationship from cell lines was isolated using Qiagen RNA Easy kit using the between E2F1 protein expression and prostate cancer progression. manufacturer’s protocol (Qiagen, Valencia, CA). Frozen human prostate We observed that E2F1 was significantly elevated in hormone- tissue samples used for semiquantitative reverse transcription-PCR (RT- resistant prostate cancer. Interestingly, we also observed that PCR) analysis were evaluated by a pathologist to confirm pathologic overall AR levels were significantly down-regulated in hormone- disease. Two to three 10-Am sections were used to isolate total RNA using resistant prostate cancer. To define a role for elevated E2F1 in Qiagen RNA Easy kit using the manufacturer’s protocol. RNA was hormone-resistant prostate cancer, we investigated whether E2F1 quantitated spectrophotometrically and 2 Ag of RNA were used to make could regulate the expression of the AR and found that E2F1 acts as cDNA using Thermoscript RT-PCR Reaction System (Invitrogen, Carlsbad, a transcriptional repressor of the AR gene. This is the first report CA) per manufacturer’s protocol. PCR reaction was done using the ¶ ¶ investigating E2F1 expression during prostate cancer progression following primers: human E2F1, 5 -CATCCAGCTCATTGCCAAGAAG-3 and 5¶-GATCCCACCTACGGTCTCCTCA-3¶; hypoxanthine phosphoribosyltrans- using TMA and the first study to show that E2F1 may be a critical ferase, 5¶-CAGTACAGCCCCAAAATGGT-3¶ and 5¶-TTACTAGGCAGATGGC- component of AR transcriptional regulation. CACA-3¶; proliferating cell nuclear antigen (PCNA), 5¶-GCCACTCCGCCAC- CATGTTCG-3¶ and 5¶-GCCTAAGATCCTTCTTCATCCTC-3¶; dihydrofolate reductase (DHFR), 5¶-GGTTGGTTCGCTAAACTGCATC-3¶ and 5¶-CCTTT- Materials and Methods CTCCTCCTGGACATCAG-3¶; and actin, 5¶-CCTGGACTTCGAGCAAGA- TMA construction. The tissue donation program and rapid autopsy GATG-3¶ and 5¶-GTAACGCAACTAAGTCATAGTCCG-3¶. PCR was done program was approved by the Institutional Review Board of the University using a Thermocycler with an annealing temperature of 59jC. Ten- of Michigan. Benign and localized prostate cancer tissues were constructed microliter aliquots were removed at cycles 25, 28, 31, and 34. Samples were from transurethral resection of the prostate and prostatectomy samples run in triplicate and reaction products were electrophoresed in a 0.8% taken from 28 patients with prostate cancer seen at the University of agarose gel as previously described
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