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Clinical Cancer Research Vol. 12, 2404-2413, April 15, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Transcriptional and Posttranscriptional Down-Regulation of the Imprinted Tumor Suppressor Gene ARHI (DRAS3) in Ovarian Cancer

Zhen Lu1, Robert Z. Luo1, Hongqi Peng1, Daniel G. Rosen3, E. Neely Atkinson4, Carla Warneke4, Maosheng Huang1, Arata Nishmoto1, Jinsong Liu3, Warren S-L. Liao2, Yinhua Yu1 and Robert C. Bast, Jr.1

Authors' Affiliations: Departments of 1 Experimental Therapeutics, 2 Biochemistry and Molecular Biology, 3 Pathology, and 4 Biostatistics and Applied Mathematics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Robert C. Bast, Jr., Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 355, Houston, TX 77030. Phone: 713-792-7743; Fax: 713-745-2107; E-mail: rbast{at}mdanderson.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: ARHI expression is lost or markedly down-regulated in the majority of ovarian cancers. The mechanism by which ARHI is down-regulated in ovarian cancers is still not clear. Our previous reports indicated that ARHI promoter activity was reduced in ovarian cancer cells, due in part to the effects of negative regulatory transcription factor(s).

Experimental Design and Results: We now show that E2F1 and E2F4, but not E2F2, E2F3, or E2F5, bind to the ARHI promoter and repress its activity in ovarian cancer cells. Consistent with this observation, immunochemical staining of cell lines and of 364 samples of ovarian cancer tissue show that the expression of E2F1 and E2F4 proteins is much higher in ovarian cancer cells than in normal ovarian epithelial cells, and that increased expression of E2Fs was negatively correlated with ARHI expression (P < 0.05). Mutation of the putative E2F binding site in the ARHI promoter reversed this inhibitory effect and significantly increased ARHI promoter activity. In addition to the effects of transcriptional regulation, ARHI mRNA also exhibited a significantly reduced half-life in ovarian cancer cells when compared with that in normal ovarian epithelial cells (P < 0.01), suggesting posttranscriptional regulation of ARHI expression. ARHI mRNA contains AU-rich elements (ARE) in the 3'-untranslated region. We have found that these AREs interact with HuR, an ARE-binding protein that stabilizes bound mRNAs, possibly contributing to the rapid turnover of ARHI mRNA. Finally, reduced HuR ARE binding activity was observed in ovarian cancer cells when compared with normal ovarian surface epithelium.

Conclusions: Taken together, our data suggest that ARHI expression is regulated at both the transcriptional and the posttranscriptional levels, contributing to the dramatic decrease in ARHI expression in ovarian cancers.

Our group recently identified ARHI (Ras homologue member I, DRAS3), a novel maternally imprinted tumor suppressor gene that encodes a 26 kDa small GTPase protein with 60% homology to Ras and Rap (1). ARHI is expressed in normal ovarian epithelial cells but its expression is lost or markedly down-regulated in the majority of ovarian cancers (1, 2). Using histochemical analysis of 407 ovarian cancers, we showed that ARHI expression was down-regulated in 63% of invasive ovarian cancer specimens and could not be detected in 47% (2). The introduction of this gene into cancer cells that lack ARHI expression truncates signaling through Ras/mitogen-activated protein kinase, activates c-Jun-NH2-kinase, induces p21WAF1/CIP1, down-regulates cyclin D1, inhibits motility, and triggers apoptosis (1, 3). Loss of any of these functions may contribute to oncogenesis. Expression of human ARHI in mice is associated with small size, infertility, and inhibition of lactation (4). These data suggest that ARHI functions as a negative regulator of growth and a suppressor of tumorigenesis.

Most Ras family members cycle between an active GTP-bound state, and an inactive GDP-bound state, to regulate many cellular processes, such as vesicular trafficking, nuclear-cytoplasmic trafficking, spindle microtubule assembly events, and cytoskeletal rearrangements. Constitutive activation of many Ras family members has been shown to induce cellular transformation and oncogenesis (5). ARHI, however, differs from other Ras proteins in residues critical for GTPase activity (6). Due to these sequence differences, ARHI is constitutively activated in the GTP-bound form and thus lacks the molecular switch by which other GTPases regulate their cellular activities. As a result, ARHI cannot depend primarily on the regulation of the GTP/GDP cycle to regulate its activity. Rather, ARHI activity may be modulated by regulating the levels of its protein expression.

Several different mechanisms may contribute to the down-regulation of ARHI protein expression. ARHI is one of 40 genes from the entire human genome that is known to be imprinted (7). The maternal copy is silenced and ARHI is expressed only from the paternal allele in all normal cells. Loss of the paternal allele can occur through a genetic event, such as the loss of heterozygosity observed in 40% of ovarian cancers (8). Aberrant methylation in the ARHI promoter of the nonimprinted allele or reduced histone H3 acetylation and increased histone H3 methylation have been associated with reduced ARHI expression in breast cancers (9, 10). Other mechanisms, including transcriptional and posttranscriptional regulation, have not been explored in ovarian cancers.

Specific transcriptional repressors expressed in cancer cells, but not in normal epithelial cells, may also contribute to the lower levels of ARHI expression. A number of potent transcriptional regulators, such as PEA3 and E1A, have been shown to repress HER2/neu gene expression. Expression of these transcriptional regulators resulted in the down-regulation of HER2/neu promoter activity and reversed the transformed phenotype of the cancer cells in vitro (11, 12).

Posttranscriptional gene regulation also plays an important role in regulating translation efficiency and mRNA stability. mRNA stability is one of the central factors in the control of gene expression. The balance between mRNA synthesis and its degradation determines the steady-state levels of individual mRNA in different cells. In vertebrates, mRNA half-lives vary from 10 minutes to >24 hours. Consequently, up to 1,000-fold differences in the cellular abundance of various mRNAs can result from seemingly minor differences in half-lives (13). These changes are ultimately reflected in the amount of protein produced. In contrast, the steady-state levels of tumor suppressor p53 mRNA were dramatically lowered in immortal compared with primary chicken embryo fibroblast cells. The half-life of p53 mRNA in primary cells was found to be as long as 23 hours, compared with only 3 hours in immortal cells (14). Insight into the molecular events controlling gene expression preceded the discovery that adenylate- and uridylate-rich (AU-rich) elements (ARE) offer posttranscriptional control of expression by affecting mRNA stability or translation (15–17). Several trans-acting factors can bind to these AU-rich sequences to either stabilize or destabilize bound mRNAs.

To further understand the molecular mechanisms that control the expression of ARHI, we examined the transcriptional regulation of ARHI and the stability of ARHI mRNA in normal and malignant ovarian epithelial cells. We found that increased levels of transcription factors, E2F1 and E2F4, in cancer cells were associated with decreased ARHI expression. We also showed that reduced levels of HuR in cancer cells may contribute to faster decay of ARHI mRNA.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. Three human primary ovarian epithelial cell cultures (OSE98, OSE113, and OSE115) were grown in 1:1 MCDB 105 and medium 199 with 10% fetal bovine serum and 10 ng/mL epidermal growth factor (Sigma-Aldrich, St. Louis, MO). Ovarian cancer cell lines, SKOv3 and DOV13, were maintained in McCoy's medium with 10% fetal bovine serum.

Patients and tissues. Subjects with primary epithelial ovarian cancer who had undergone initial surgery at The University of Texas M.D. Anderson Cancer Center between 1990 and 2004 were included in this study. A total of 482 patients were collected and 364 valid cases were used in this study after applying selection criteria. Histopathologic diagnosis was based on WHO criteria. The samples were assigned a grade based on Gynecologic Oncology Group criteria and staged according to the International Federation of Gynecology and Obstetrics system. Studies of tissue blocks and patient information were approved by the Institutional Review Board at The University of Texas M.D. Anderson Cancer Center.

DNA/RNA extraction. Genomic DNA was extracted from cell lines using QIAamp DNA Mini Kits (Qiagen, Valencia, CA) and total RNA was extracted with TRIzol Reagent, purchased from Invitrogen (Carlsbad, CA), using procedures recommended by the manufacturer. All samples were dissolved in nuclease-free water and quantified by reading absorbance at 260/280 nm.

Reverse transcription-PCR. The reverse transcription (RT) reaction for the first-strand cDNA synthesis was carried out with reverse transcriptase (Invitrogen) using 2 µg total RNA. Semiquantitative RT-PCR of ARHI mRNA was done using specific sense and antisense PCR primers for amplification across the intron from the first exon to the second exon of the ARHI gene, yielding a PCR product of ~250 bp. The sequence of the paired primers was as follows: the sense primer was 5'-TCTCTCCGAGCAGCGCA-3' and the antisense primer was 5'-ATCTTCCTGTGGGGCTTGAAGG-3'.

Real-time quantitative RT-PCR. Quantitative RT-PCR was used to measure the expression levels of ARHI in cells treated with 5 µg/mL actinomycin D. The cDNA used for real-time PCR was the same as that used for the RT-PCR reaction. Total RNA from different cell lines was purified and the cDNA synthesized using 2 µg of total RNA. The reverse transcriptase reaction was done according to the manufacturer's instructions using oligo(dT)16 and SuperScript II reverse transcriptase (Invitrogen). Real-time quantitative PCR was done in a reaction mixture with two gene-specific primers (NY2P1, 5'-TCTCTCCGAGCAGCGCA-3'; and NY2P2, 5'-TGGCAGCAGGAGACCCC-3'), a labeled probe 5'-TGTCTTCTAGGCTGCTTGGTTCGTGCC-3' (5'-fluorescent label, 6-FAM; 3'-label, 6-carboxytetramethylrhodamine), 2 µL of reverse transcriptase reaction mixture, and 12.5 µL of Master Mix on an ABI PRISM 7700 Sequence Detection System (Perkin-Elmer, Wellesley, MA), following the manufacturer's protocol.

Measurement of mRNA instability. To determine the half-life of ARHI mRNA in normal or malignant ovarian cells, primary human ovarian epithelial cells, and ovarian cancer cell lines were treated with actinomycin D for increasing time periods. When cells reached 80% confluence, cultures were treated with 5 µg/mL of actinomycin D for the indicated times. The expression levels of ARHI were determined by real-time PCR. The calculated cycle threshold (Ct) provided an arbitrary cutoff point at which a Ct value was assigned for each sample. Copy numbers of each sample were calculated based on the standard curve. The half-life of mRNA was calculated from the time required for ARHI mRNA to decay by 50%.

Cytoplasmic and nuclear extract preparation. Normal ovarian epithelial cells and cancer cells were grown as monolayers and trypsinized, washed, and pelleted. Nuclear and cytoplasmic extracts were prepared following the manufacturer's instructions with Nuclear Extraction Reagents and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL).

Electrophoretic mobility shift assay and supershift assays. Electrophoretic mobility shift assay (EMSA) was done as described previously (18). For oligonucleotide competition experiments, the protein samples were incubated with 32P-labeled probe in the presence of an excess of wild-type or mutant oligonucleotides. For antibody supershift assays, 1 µg of antibody was added to the nuclear extract mix at 4°C for 1 hour before addition of the radiolabeled probe. All E2F antibodies and normal rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

The oligonucleotides used were:

A2 (wild-type): 5'-TTATTTTTGGCGGGGGGGAATCTATAG-3'
A2-M1 (mutant 1): 5'-TTACTGTTAGCGGGGGGGAATCTATAG-3'
A2-M2 (mutant 2): 5'-TTATTTTTGGAGTGGTGGAATCTATAG-3'
A2-M3 (mutant 3): 5'-TTATTTTTGGCGGGGGGTAAGCTGTAG-3'
A2-ME (mutant E2F): 5'-TTATTTTTGGATGGGGGGAATCTATAG-3'
E2F1 (wild-type): 5'-ATTTAAGTTTCGCGCCCTTTCTCA-3'
E2F1-M (mutant): 5'-ATTTAAGTTTCGATCCCTTTCTCA-3'

Chromatin immunoprecipitation assays. Chromatin immunoprecipitation assays were done as previously described (9) with minor modifications. Briefly, the immunoprecipitated DNA-protein cross-links in the chromatin complexes were reversed by heating at 65°C overnight and DNA was eluted. The ARHI promoter DNA in the immunoprecipitates was detected by PCR using gene-specific primers as described below. PCR products were analyzed by electrophoresis on a 2% agarose gel. The semiquantitative PCR was also done using the Bioanalyzer 2100 (Agilent, Germantown, MD) and the DNA 500 LabChip Kit (Agilent) to determine the quantity of PCR product.

ARHI promoter mutation assays. Site-directed mutation of the ARHI-promoter/luciferase reporter vectors was carried out using a QuickChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). Substitutions of nucleotides in primers were designed to match the mutations in the primers (A2-M1, A2-M2, A2-M3, and A2-ME) used in EMSA and supershift assays. The mutated ARHI-promoter/luciferase reporter constructs were sequenced to confirm the proper mutations.

Transient transfection and luciferase assays. SKOv3 ovarian cancer cells were transfected with E2F1-E2F6 expression vectors (kindly provided by Dr. Kristian Helin, the FIRC Institute for Molecular Oncology, Milan, Italy) and the luciferase activities measured by using a dual-luciferase report assay system in an Analytical Luminometer Monolight 2010 (BD PharMingen, San Diego, CA) as described previously (19). All samples in experiments were assayed in triplicate.

Immunostaining. For immunochemical staining, SKOv3 ovarian cancer cells and normal ovarian epithelial cells were grown on collagen-coated chamber slides (BD Bioscience) in appropriate medium supplemented with 10% fetal bovine serum and immunostained as described previously (2). Anti-E2F1 (Santa Cruz Biotechnology) or anti-E2F4 (GeneTex, San Antonio, TX) antibody was used as the primary antibody. A diaminobenzidine tetrachloride supersensitive substrate kit (Biogenex, San Ramon, CA) was used to visualize the antibody-antigen complex and a light counterstaining was done with hematoxylin. A negative control with IgG was carried out in all experiments.

Tissue microarray staining was similar as above except for the initial deparaffinization. Deparaffinized sections were steamed in 10 mmol/L citrate buffer (pH 6.0) to restore latent epitopes. Anti-ARHI, E2F1 (1:50, Santa Cruz Biotechnology) or E2F4 (1:200, GeneTex) antibody was used as the primary antibody.

Analysis of protein-RNA interactions. The cytoplasmic and nuclear extracts from normal and cancer cells were prepared as previously described (20), except that all reagents were prepared with 0.1% diethyl pyrocarbonate–treated water and all experiments were conducted under RNase-free conditions. The ARHI mRNA ARE fragment 5'-GUAGUUUCUCAUGUCAUUUAAAAAUGUUUGAGUAUUCUGCAUAGCAGUUUGUA-3' was synthesized and end-labeled with {gamma}-32P ATP. 32P-labeled RNA probes (~50 fmol) were incubated for 20 minutes on ice with 5 µg of cytoplasmic or nuclear extract in 10 µL of bandshift buffer [10 mmol/L Hepes (pH 7.6), 3 mmol/L MgCl2, 20 mmol/L KCl, 1 mmol/L DTT, 5% glycerol] and heparin sulfate (Sigma-Aldrich) were added to 5 mg/mL, followed by incubation on ice for a further 5 minutes. Then, 5 µL of loading buffer was added and RNA-protein complexes were resolved by electrophoresis on a 6% native Tris-borate EDTA acrylamide gel. Supershift and competition experiments were conducted with similar protocols, except that protein was preincubated with antibody, control IgG, or unlabeled competitor RNA prior to the addition of probe. For UV cross-linking, binding reaction mixtures were UV-irradiated in 96-well trays in a Stratalinker 2400 (Stratagene) for 5 minutes and then incubated with 10 µg of RNase A and 5 units of RNase T1 (Ambion) for 30 minutes at room temperature. UV cross-linked RNA-protein complexes were also immunoprecipitated with a monoclonal antibody against HuR or with control IgG. The 32P-labeled protein-RNA complexes on the dried SDS gel were visualized by autoradiography.

Construction of tissue microarrays. Sample preparation for tissue microarrays was the same as described previously (2). Five-micron sections were obtained and stained with H&E to confirm the presence of tumor and to assess tumor histology. Each core was scored individually and the results were presented as the mean of the two replicate core samples. Cases in which no tumor was found, no cores were available, or had <20% of tumor were excluded from the final data analysis.

Statistical analysis. The data are expressed as the mean ± SE. Differences in proportions were evaluated by the {chi}2, Fisher's exact test of the Spearman correlation, as appropriate. Student's t test was used to evaluate differences in different groups. P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
ARHI expression is down-regulated by transcriptional regulation in ovarian cancer cells. Our previous studies have shown markedly reduced ARHI promoter activity and mRNA levels in ovarian cancer cells when compared with normal cells (1, 19). We showed that the regulatory region in the ARHI promoter that contributes to its reduced activity was centered on a region covered by our P2 construct (nucleotides –420 to +68; ref. 19). To more precisely identify the putative transcription factor(s) that negatively regulate ARHI promoter activity in cancer cells, we have screened this P2 region with EMSA. The EMSAs were done using labeled oligonucleotides that matched this region with nuclear extracts from ovarian cancer cell line, SKOv3, which exhibited markedly down-regulated expression of ARHI and compared with those of normal ovarian surface epithelial cells OSE035 that expressed ARHI. A sequence-specific protein-DNA complex with oligonucleotide A2 (–385 to –409) was observed with nuclear extracts from cancer cells but not from normal cells (Fig. 1A ), suggesting that this A2-matched fragment might bind to a transcriptional repressor(s) that negatively regulates ARHI expression in cancer cells. Similar EMSA analysis confirmed that each of three ovarian cancer cell lines (SKOv3, DOV13, and Hey), but not three normal ovarian epithelial cell cultures (OSE113, OSE115, and OSE120), exhibited A2-DNA binding activity (Fig. 1B).


Figure 1
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  		Fig. 1. E2F transcription factor(s) bind to the A2 element in the ARHI promoter region. A schematic representation of the A2 ARHI promoter construct (A). An oligonucleotide A2 (nucleotides –385 to –409) binds to putative transcription factor(s) in the ovarian cancer line SKOv3, but not to factor(s) in normal ovarian epithelial cells (OSE035). Binding could be inhibited with specific oligonucleotides (A). Similar results were obtained with two additional ovarian cancer cells lines and three additional cultures of normal ovarian epithelial cells (B). TESS search suggested that the E2F, Sp1, and AP2 transcription factors might bind to the elements in oligonucleotide A2. Mutant oligonucleotide constructs were developed (A2-M1, A2-M2, A2-M3, and A2ME) to inhibit binding of Sp1, E2F, or AP2, respectively (C). Nuclear extracts prepared from SKOv3 ovarian cancer cells were incubated with 32P-radiolabeled A2 oligonucleotide, with or without nonlabeled A2 or A2 mutant oligonucleotides in EMSA. Mutants with alterations in the E2F binding elements failed to block the binding of labeled A2 (D). A2 failed to block the binding of transcription factors to 32P-radiolabeled Sp1 (E) or AP2 (F) oligonucleotides, whereas nonlabeled Sp1 or AP2 oligonucleotides blocked binding.

 
A2 fragment contains a putative E2F binding site. Analysis of the A2 fragment sequence by a TESS search (http://www.cbil.upenn.edu/tess) suggested three putative transcription factor binding sites (Sp1, E2F, and AP2) in this region. To clarify which transcription factors could bind this fragment, three mutant oligonucleotides (A2-M1, A2-M2, and A2-M3; Fig. 1C) were synthesized that covered the entire A2 fragment and that could potentially affect the putative consensus elements for binding of Sp1, E2F, or AP2. EMSA analysis with A2 and different mutant constructs showed that both A2-M1 and A2-M2 failed to compete with the A2 element for specific binding (Fig. 1D), consistent with the importance of the E2F or Sp1 binding sites. On the other hand, A2-M3, which contains a mutation distant to the E2F binding site, completely blocked the binding activity (Fig. 1C and D). An oligonucleotide mutant, A2-ME, that was localized to the E2F binding site, failed to block A2-DNA binding, consistent with the possibility that E2F was involved in the A2-DNA binding activity. To rule out the possibility that the A2 element may interact with Sp1 or AP2, radiolabeled consensus Sp1 or AP2-binding oligonucleotides were incubated with nuclear extracts from SKOv3 ovarian cancer cells and competed with unlabeled Sp1 and AP2 binding oligonucleotides as well as A2 element. Although unlabeled Sp1 and AP2 oligonucleotides effectively competed the protein-DNA complexes formed with their respective probes, neither the wild-type A2 nor the mutated A2 (A2-M1) was able to block the specific complex formation (Fig. 1E and F).

To further confirm the ability of E2F to bind in the A2 region of the ARHI promoter, we synthesized wild-type and mutant E2F1-binding oligonucleotides and used them as probes in E2F-specific EMSA. The mutant E2F1 probe was identical to the wild-type E2F1 probe, except that it contained a CG to AT substitution of the second CG pairs of the E2F binding site. 32P-labeled E2F oligonucleotide was incubated with nuclear extracts from the SKOv3 ovarian cancer cells. A distinct migration pattern of protein-E2F DNA complexes was observed. The complexes found could be blocked by both unlabeled E2F1 and A2 oligonucleotides, but not by unlabeled E2F1 mutant or A2-ME oligonucleotides (Fig. 2A ). Conversely, when the same nuclear extracts were incubated with 32P-labeled A2 oligonucleotide, unlabeled E2F1, but not E2F1 mutant oligonucleotides, could also block protein-A2 DNA complex formation (Fig. 2B). Taken together, our results strongly suggest that the transcription factor in SKOv3 cancer cells that binds the A2 element is E2F.


Figure 2
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  		Fig. 2. E2F specifically binds to the A2 fragment of the ARHI promoter. Nuclear extracts prepared from SKOv3 ovarian cancer cells were incubated with 32P-labeled E2F1 oligonucleotide and analyzed by EMSA with or without a 200-fold excess of nonlabeled oligonucleotides (A). Nonlabeled E2F1 or A2 inhibited binding, whereas mutant constructs with alterations in the E2F-binding element did not (A). The same nuclear extract was incubated with 32P-labeled A2 oligonucleotide with or without nonlabeled A2, A2-ME, E2F1, or E2F1 mutant oligonucleotides (B). Nonlabeled E2F1 inhibited binding of transcription factor(s) in a dose-dependent manner, whereas E2F1 mutant oligonucleotides did not (B). To identify the specific E2F family members that could bind the A2 fragment, nuclear extracts prepared from SKOv3 ovarian cancer cells were incubated at 4°C with 1 µg of the indicated antibodies against different E2F family members for 1 hour before adding 32P-labeled A2 and EMSA assay. Preimmune IgG was incubated as a control and the specificity of A2 binding was confirmed by incubation with nonlabeled A2. Arrows, shifted bands (C). Supershifts were observed with anti-E2F1, E2F4 and E2F5 antibodies (C). To confirm that the anti-E2F2 and anti-E2F6 antibodies were functional, E2F2, E2F3, and E2F6 constructs were transfected into SKOv3 cells and cell lysates were analyzed in the same supershift assays (D). Supershifts were observed with each of the antibodies (D). To show the presence of E2F in ovarian cancer cell lines, nuclear extracts prepared from ovarian cancer cells (SKOv3, Dov13, and Hey) and normal ovarian epithelial cells (OSE115, OSE120, and OSE113) were incubated with 32P-labeled E2F oligonucleotide and subjected to EMSA (E). Binding was observed with extracts from the cancer cells, but not from normal cells.

 
E2F consists of a small family member known to participate in the transcriptional regulation of target genes. To determine which E2F family members are involved in regulating ARHI expression, antibody supershift assays were done using specific antibodies against each E2F members (anti-E2F1 to anti-E2F6; Santa Cruz Biotechnology). As shown in Fig. 2C, the addition of anti-E2F1, anti-E2F4, and anti-E2F5 antibodies to the EMSA reaction mixture produced clear supershifts of the protein-A2 DNA complexes, whereas anti-E2F2, anti-E2F3, and anti-E2F6 antibodies had no effect. To confirm that anti-E2F2, E2F3, and anti-E2F6 antibodies were functional in supershift assays, E2F2, E2F3, and E2F6 expression constructs (from Dr. K. Helin) were transiently transfected into SKOv3 cells and the cell lysates were used for the supershift assay. The results showed that all antibodies tested recognized the specific E2Fs (Fig. 2D).

E2F DNA-binding activity is increased in ovarian cancer cells. To examine E2F-binding activities in cancer and normal cells, nuclear extracts prepared from ovarian cancer cells (SKOv3, Dov13, and Hey) and normal ovarian epithelial cells (OSE115, OSE120, and OSE113) were incubated with 32P-labeled E2F1 oligonucleotide. The EMSA results showed markedly increased E2F binding activity in ovarian cancer cells when compared with the normal ovarian epithelial cells (Fig. 2E).

Both E2F1 and E2F4 bind to the ARHI promoter. To further verify that E2F transcription factors could bind to A2 fragment, chromatin immunoprecipitation assays were done with SKOv3 cells. When chromatin-DNA fragments were immunoprecipitated with antibodies against E2F family members (confirmed to recognize E2F-DNA complexes in the supershift EMSA assays) and used as a template. PCR amplification with primers for the ARHI promoter region produced DNA of the expected size from the anti-E2F1 and anti-E2F4 immunoprecipitates; whereas no amplified products were detected from the anti-E2F2 immunocomplex (Fig. 3A ). Additionally, the ratio of immunoprecipitates to input confirmed that E2F1 and E2F4, but not E2F2 bound to the A2 fragment of the ARHI promoter (P < 0.001; Fig. 3B). These results show that in SKOv3 cells, the P2 region of the ARHI promoter is bound by both E2F1 and E2F4 transcription factors.


Figure 3
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  		Fig. 3. E2F1 and E2F4 bind to the ARHI promoter in SKOv3 cells. To confirm that E2F transcription factors could bind to the A2 fragment of the ARHI promoter in intact cells, chromatin immunoprecipitation assays were done with SKOv3 cells using antibodies against E2F1 and E2F4. PCR was done with primers around the A2-E2F binding site in the ARHI promoter (A). Immunoprecipitated promoter fragments (IP) were measured by densitometry using ImageQuant software (Molecular Dynamics) and presented as a ratio of IP to Input (*, P < 0.001; B). To test whether E2F1 and E2F4 inhibited ARHI promoter activity, SKOv3 cells were transiently cotransfected with an ARHI promoter-luciferase reporter construct and expression vectors for E2F family member, including three E2Fs shown to produce a supershift in the EMSA assay (Fig. 2D). Cell extracts were prepared and luciferase activity measured. Expression of excess E2F1 and E2F4, but not E2F3 and E2F5 proteins significantly inhibited (*, P < 0.01) ARHI promoter activity (C). Mutation of the putative E2F binding site in the A2 region of the ARHI promoter (the same mutation as in the A2-ME oligonucleotide) significantly increased (P < 0.01) ARHI promoter activity (D). Columns, mean from three independent experiments; bars, ±SE. Conversely, E2F siRNAs were used to knock-down E2F expression to determine the effect on subsequent ARHI expression (E). E2F expression was reduced as shown by Western blots (E, bottom) and ARHI expression was increased significantly (*, P < 0.05; **, P < 0.01) by semiquantitative PCR (E, top), with additive effects observed when both E2F1 and E2F4 expression were reduced.

 
E2F1 and E2F4 negatively regulate ARHI promoter activity. To confirm the effect of individual E2F family members on ARHI promoter activity, six E2F family expression vectors, generously provided by Dr. K. Helin, were transiently cotransfected with an ARHI/luciferase reporter into SKOv3 ovarian cancer cells. Figure 3C shows that the expression of E2F1 and E2F4 reduced luciferase activity by >3-fold (*, P < 0.01), whereas expression of E2F2 and E2F5 did not alter the luciferase activity. To examine the importance of the E2F-binding site in the ARHI promoter, we generated a mutant ARHI/luciferase reporter in which two bases in the E2F-binding site have been mutated (the same mutation as the A2-ME oligonucleotide). Consistent with its repressive function, an ARHI/luciferase reporter with a mutated E2F-binding site resulted in significantly increased ARHI promoter activity (*, P < 0.05; Fig. 3D). Because E2Fs are often overexpressed in cancer cells, we sought to examine whether elevated levels of E2F1 and E2F4 contribute directly to the low ARHI expression. Short interfering RNA (siRNA) specific for E2F1 and E2F4 were prepared and used to knock-down E2F expression in SKOv3 cells. E2F2 siRNA was included as a negative control. As shown by Western blots, E2F siRNA (E2F1, E2F2, E2F4, and combined E2F1 and E2F4) significantly and specifically decreased the expression of their respective E2F family proteins in SKOv3 cells (Fig. 3E). However, ARHI expression was significantly increased only in cells treated with E2F1 and E2F4 siRNA (*, P < 0.05; **, P < 0.01; Fig. 3E). This result supports the notion that overexpression of E2F1 and E2F4 in SKOv3 cells contributes to the down-regulated ARHI expression.

Expression of E2F1 and E2F4 increased in ovarian cancer cells. The observation that E2F1 and E2F4 seemed to down-regulate ARHI promoter activity in ovarian cancers prompted us to examine the levels of E2F protein in ovarian cancer cells. Expression of E2F1 and E2F4 proteins were markedly increased in ovarian cancer cell lines (SKOv3, DOV13, and Hey) compared with normal ovarian epithelial cells (OSE115, OSE120, and OSE113; Fig. 4B ). Consistent with Western blot analysis, immunochemical staining showed that the E2F proteins, localized mainly in the nucleus, were significantly higher in SKOv3 ovarian cancer cells (Fig. 4A, b and d), whereas very small amounts of E2F1 and E2F4 were detected in normal ovarian epithelial cells (Fig. 4A, a and c).


Figure 4
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  		Fig. 4. Expression of E2F1 and E2F4 is increased in ovarian cancer cells, associated with decreased expression of ARHI. SKOv3 ovarian cancer cells (b and d) and OSE115 normal ovarian epithelial cells (a and c) were cultured in chamber slides for 2 days and stained with anti-E2F1 (a and b) or anti-E2F4 (c and d) antibody and immunoperoxidase (A). E2F proteins were detected in the nuclei of SKOV3 ovarian cancer cells, but not in the OSE115 cells (A). Lysates from SKOv3, DOV13, and Hey ovarian cancer cells as well as from OSE115, OSE120 normal ovarian epithelial cells were subjected to Western blot analysis with anti-E2F1 or anti-E2F4 antibody to verify protein expression (B). Greater expression of E2F1 and E2F4 was observed in the ovarian cancer cell lines when compared with normal ovarian epithelial cells (B). Immunohistochemical staining was used to measure E2F1 (a-d) or E2F4 (e-h) in normal ovarian epithelial cells (a and e) and in ovarian cancer cells (b, c, d, f, g, h) taken directly from patients (C). Low E2F expression (b and f) and increased E2F expression (c, d, g, and h) detected in ovarian cancers. Significantly increased (P < 0.01) expression of E2F1 and E2F4 was detected in ovarian cancers (D). E2F1 was found predominantly in serous and endometrioid histotypes of invasive cancers, whereas E2F4 was overexpressed by all histotypes in borderline and invasive lesions (D). Overexpression of E2F4 or of both E2F1 and E2F4 correlated with decreased expression of ARHI (P < 0.05; E).

 
To confirm the observations from cultured cells in clinical samples, 364 surgically confirmed ovarian cancer specimens in tissue microarrays were subjected to immunochemical staining with ARHI, E2F1, and E2F4 antibodies. Results showed that a majority of ovarian samples exhibited increased E2F1 (66%) and E2F4 expression (78%), whereas ARHI expression was down-regulated in 64% of ovarian cancers (Fig. 4C and D). Increased expression of E2F1 correlated with increased expression of E2F4 (P < 0.001). Increased expression of E2F4 or a combination of E2F1 and E2F4 correlated with decreased expression of ARHI in ovarian cancers (P < 0.05; Fig. 4E).

ARHI mRNA has a significantly reduced half-life in ovarian cancer cells. In general, normal ovarian epithelial cells showed much higher steady-state levels of the ARHI mRNA than ovarian cancer cells, although the differences were small in some cell lines, such as OSE113 and Dov13 (Fig. 5A ). To examine the stability of ARHI mRNA, ovarian cancer cells and normal ovarian epithelial cells were treated with actinomycin D (5 µg/mL), an RNA synthesis inhibitor. ARHI mRNA levels were measured in normal and cancer cells at different times after the addition of actinomycin D using both RT-PCR (Fig. 5B) and quantitative real-time PCR (Fig. 5C). The mRNA levels of ARHI from three independent experiments were quantified using the Beckman Du640 Spectrophotometer. Figure 5B shows that, before adding actinomycin D, tumor cells already contained significantly lower levels (up to 1,000-fold lower for SKOv3 ovarian cancer cells) of ARHI mRNA than did normal cells. During the actinomycin D chase, ARHI mRNA in normal cells exhibited half-lives of 17 to 19 hours. By contrast, ARHI mRNA had a significantly reduced half-life of ~8 to 9 hours in ovarian cancer cell lines (P < 0.01; Fig. 5C), suggesting a much faster decay of ARHI mRNA in ovarian cancer cells. Given the shortened half-life of mRNA in ovarian cancer cell lines, proteins were sought that might stabilize or destabilize ARHI mRNA.


Figure 5
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  		Fig. 5. The half-life of ARHI mRNA is reduced in ovarian cancer cells. Ovarian cancer cells (SKOv3 and DOV13) and normal ovarian epithelial cells (OSE98, OSE113, and OSE115) were cultured in 60 mm plates and treated with 5 ng/mL actinomycin-D for the indicated times (2, 4, 8, 16, 24, 32, and 48 hours) to block endogenous mRNA synthesis. Total mRNA from cultured cells was extracted and subjected to semiquantitative RT-PCR analysis (A). Real-time quantitative RT-PCR amplification, done as described in the text, of the ARHI mRNA was conducted (B) and the half-life of ARHI mRNA was calculated (C). B, points, mean from three independent experiments; bars, ± SE; C, columns, mean from three independent experiments; bars, ± SE (P < 0.01).

 
HuR binds to the 3'-untranslated region ARE of ARHI mRNA. The 3'-untranslated region (UTR) of ARHI mRNA contained four AREs or U-rich motifs (AUUUA/GUUUG; Fig. 6A ). To identify proteins interacting with 3'-UTR ARE elements, an ARHI ARE probe was used in EMSAs with the cell fraction extracts from normal ovarian epithelial cells or cancer cell lines. Both cytoplasmic and nuclear extracts showed specific ARE probe-protein complexes. Higher levels of nuclear binding protein were observed in the normal OSE cells than in the cancer cells (Fig. 6B). Adding unlabeled ARE RNA probe in increasing concentrations specifically depleted the radiolabeled RNA-protein complexes in the extracts from both cytoplasmic and nuclear fractions (Fig. 6C). A novel supershifted complex was observed in both nuclear and cytoplasmic extracts (Fig. 6D) in the presence of a monoclonal antibody against the RNA-binding protein HuR, but not in the presence of an antibody against the RNA-binding protein TTP or control IgG. This observation is consistent with reports that HuR is a protein that shuttles between the nucleus and the cytoplasm (19). RNA-protein binding reactions were set up using nuclear extracts and cytoplasmic extracts with the same probe. After treatment with UV to cross-link RNA-protein, complexes were treated with RNase T1 or a combination of RNase T1 and RNase A, separated by SDS-PAGE, and detected by autoradiography. Under these conditions, two cross-linked RNA-protein complexes were detected (Fig. 6E). UV cross-linked RNA-protein complexes could be immunoprecipitated using an anti-HuR antibody, but not by normal IgG. The masses of the digested cross-linked complexes were ~48 and 70 kDa (Fig. 6E, b). However, after more effective RNA digestion with combined RNases (RNase T1 + A), a single protein band was detected, which was similar to the 34 kDa HuR protein (Fig. 6E, c). The data are consistent with the possibility that the cross-linked complexes contain one molecule of HuR with different numbers of RNA molecules to which the protein is cross-linked.


Figure 6
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  		Fig. 6. ARE-binding protein HuR binds to ARHI AREs. ARHI mRNA contains four AU-rich sequence or U-rich motifs (AUUUA/GUUG) in the 53-nucleotide sequence of the ARHI ARE (A). 32P radio end-labeled ARE probe was incubated with cytoplasmic or nuclear lysate (10 µg) from normal or cancer ovarian epithelial cells at room temperature prior to the addition of heparin and electrophoresis on native strength polyacrylamide gels. Arrows, protein-RNA complexes (B). The specificity of ARHI ARE binding was determined by adding increasing amounts (25- to 200-fold) of unlabeled ARHI ARE RNA to 32P-radiolabeled ARHI ARE prior to the addition of 10 µg of ovarian cells extracts (C). EMSA was done with 32P-radiolabeled ARHI ARE probe and 10 µg of cytoplasmic or nuclear extracts from normal ovarian epithelial cells. An antibody against HuR, an antibody against TTR, or control IgG was incubated with the protein extracts in binding buffer for 10 minutes prior to the addition of probe. Arrow, supershifted complexes for HuR (D). To determine the molecular weight of the protein in the protein-ARE complex, binding reactions were set up as above except that 20 minutes later, reaction mixtures were UV cross-linked and treated with RNase T1 (E, b) or RNase T1 plus RNase A (E, c). RNA-protein complexes were also immunoprecipitated with an anti-HuR antibody, or control IgG. Immunoprecipitated complexes were subjected to SDS-PAGE on a 12% polyacrylamide gel as the starting materials before RNase digestion (E, a). Cross-linked complexes were detected by autoradiography. The results presented are representative of experiments done in triplicate.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Several mechanisms have been shown to regulate ARHI expression. As an imprinted gene, only the paternal allele is expressed. ARHI expression from the paternal allele can be lost through a genetic event, such as loss of heterozygosity, observed in 40% of ovarian and breast cancers (8). Epigenetic factors can also regulate ARHI expression. Aberrant methylation of the CpG islands of the promoter, as well as reduced histone H3 acetylation of this imprinted gene can contribute to decreased expression of ARHI protein in breast cancers (9, 10). In this report, we show that regulation of ARHI gene expression occurs at both transcriptional and posttranscriptional levels in ovarian cancers.

Our data suggest that ARHI expression can be down-regulated by E2F1 and E2F4. E2F has a critical role in cell growth regulation, DNA damage repair, as well as tumorigenesis. In quiescent cells, E2F is complexed with retinoblastoma. After phosphorylation of retinoblastoma, E2F is released and mediates the activation of genes important for DNA replication and cell proliferation. Two distinct subgroups have been identified within the E2F family: E2F4 and E2F5 generally act as repressors, whereas E2F1, E2F2, and E2F3 function as both activators and repressors (21). Recent studies have highlighted that in certain settings, E2F1 and E2F4 can exhibit similar functions in transcriptional regulation (22). By chromatin immunoprecipitation and gel supershift assays, Blais et al. showed that E2F1 and E2F4 associate with the regulatory elements of the p18INK4c promoter in vivo as well as in vitro (23). Yoshida et al. studied the regulation of E2F on Geminin and Cdt1 expression and found that both E2F1 and E2F4 could activate these genes (24). In the case of ARHI, we have found that both E2F1 and F2F4 can bind to the ARHI promoter and negatively regulate its activity (Figs. 2 and 3). E2F3 and E2F5 failed to affect ARHI promoter activity, although they could bind to A2 fragment (Figs. 2 and 3). Supershift by an E2F3 antibody may have resulted from a lack of specificity for the antibody, or corepressors might exert different effects on different E2F family members. Consistent with the findings observed in cultured cells, protein microarray analyses of 364 cases of ovarian cancer samples showed that the increased expression of E2F1 and E2F4 in ovarian cancer samples was negatively correlated with decreased ARHI expression. Thus, increased E2F1 and E2F4 expression in ovarian cancer cells may, at least partially, suppress the transcriptional activity of the ARHI promoter and lead to reduced mRNA transcription of ARHI in ovarian cancer cells.

An additional level of regulation relates to the stability of ARHI mRNA in ovarian cancer cells. The half-life of the ARHI mRNA in ovarian cancer cells averages only 7.8 hours compared with >18 hours in normal ovarian epithelial cells. The quick decay of ARHI mRNA in ovarian cancers could dramatically attenuate the expression of ARHI. mRNA stability is one of the important factors in the control of gene expression.

Posttranscriptional regulation is considered to be mediated by the 3'-UTR of the ARHI mRNA through a conserved U- or AU-rich sequence element contained within the 3'-UTR. AREs are potent cis-acting determinants of rapid cytoplasmic mRNA turnover in mammalian cells (25). They generally consist of one or more overlapping AUUUA pentamers contained within or near a U-rich tract. The context of these AUUUA/GUUUG motifs within the 3'-UTR implicates the involvement of this region in rapid decay of mRNA (26). In this study, we showed that nuclear/cytoplasmic proteins specifically bind to the ARHI ARE, and we postulate that this interaction regulates the stability of ARHI mRNA. This finding provides evidence that decreased expression of specific ARE-binding protein(s) may down-regulate the stability of ARHI mRNA and subsequently the levels of ARHI protein expressed in ovarian cancer cells.

Several investigators have shown that proto-oncogene mRNA and cytokine mRNAs are rapidly degraded, and that this is mediated by AREs. Here, we have shown that ARHI mRNA is rapidly degraded, and that the ARE-containing 3'-UTR of ARHI could mediate this destabilization. Along these same lines, alterations in ARE binding by actinomycin D may directly inhibit mRNA translation because ARE-dependent mRNA turnover is proposed to be coupled to translation (27). Although a number of RNA-binding proteins that recognize AREs have been reported, the mechanism by which these factors mediate mRNA degradation or translational inhibition is unknown. Insight into the molecular events involved in posttranscriptional control has been facilitated through the identification of these proteins. Several ARE-binding proteins have been identified and show a wide variety of activities ranging from pre-mRNA processing, developmental control, and metabolic catalysis (27). More importantly, the AUBF, HuR, TTP, and AUF1 ARE-binding proteins have been shown to directly affect ARE-mediated mRNA decay (17, 25, 28). In this study, we showed that ARE-binding protein HuR binds to the 3'-UTR ARE element of the ARHI mRNA, and observed a reduced HuR-ARE binding activity in ovarian cancer cells. HuR is ubiquitously expressed in all cells and is one of the best-characterized ARE-binding proteins that stabilize mRNA and enhance translation (29). It has a high binding affinity for several AREs, and although predominantly nuclear, it shuttles between the nucleus and the cytoplasm, regulating mRNA stability and translation (25). HuR may bind the ARE-containing mRNAs in the nucleus and transport them to the cytoplasm, where they access the translational apparatus or are released for rapid degradation (30). Lower HuR-ARE binding activity in ovarian cancers will reduce its ability to stabilize the ARHI mRNA and contribute to faster mRNA turnover in ovarian cancer cells.

Taken together, the expression of ARHI can be lost in ovarian and breast cancer cells through several mechanisms that involve imprinting, loss of heterozygosity, CpG methylation, decreased histone H3 acetylation, transcriptional repression by E2F1 and E2F4, and destabilization of mRNA by down-regulation of HuR binding. Different mechanisms are likely to be found in cancers from different individuals. Loss of ARHI protein enhances the growth and activity of cancer cells. The variety of mechanism underlying this loss suggests that ARHI may play an important role in ovarian and breast oncogenesis.

ARHI expression correlates inversely with progression-free survival in ovarian cancer (2). At a clinical level, loss of ARHI expression might have an affect on chemoresistance or on the biological aggressiveness of ovarian cancer. Preliminary data suggests that ARHI reexpression increases the sensitivity of ovarian cancer cells to cisplatin and paclitaxel.5 In addition, the reexpression of ARHI can inhibit proliferation (1), motility, and invasiveness (31). Overexpression of ARHI using dual adenoviral vectors induces calpain-dependent, caspase-independent apoptosis in ovarian and breast cancer cells, and inhibits the growth of xenografts in immunodeficient mice (3). Recognition that E2F1 and E2F4 regulate ARHI in cell culture and correlate inversely with ARHI expression in ovarian cancer specimens suggests another approach to therapy. Expression of E2F4 is increased in 78% of ovarian cancers. Treatment with siRNA against E2F4 can enhance drug-induced apoptosis produced by cisplatin and other agents in human cancer cells (32). Further studies will be required to determine the potential therapeutic activity of siRNA directed against E2F4. In these studies, up-regulation of ARHI might contribute to enhanced sensitivity to drug-induced apoptosis and serve as a biomarker for E2F4 regulation in at least a fraction of ovarian cancers.


   Footnotes
 
Grant support: NIH grants CA 64602 (R.C. Bast), National Foundation for Cancer Research grant LF2004-00009224HM (R.C. Bast), and CA 80957 (Y. Yu).

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.

Note: Z. Lu and R.Z. Luo contributed equally to this report.

5 Lu, Luo, Bast, unpublished data. Back

Received 5/11/05; revised 1/25/06; accepted 2/ 3/06.


   References
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 Abstract
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 Discussion
 References
 

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