Epigenetic-mediated upregulation of progesterone receptor B gene in endometrial cancer cell lines

Gynecologic Oncology 99 (2005) 135 – 141 www.elsevier.com/locate/ygyno

Epigenetic-mediated upregulation of progesterone receptor B gene in endometrial cancer cell lines Yuning Xiong a, Sean C. Dowdy a, Jesus Gonzalez Bosquet a, Ying Zhao a, Norman L. Eberhardt b,c, Karl C. Podratz a, Shi-Wen Jiang a,b,* a

Department of Obstetrics and Gynecology, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, MN 55905, USA b Department of Medicine, Division of Endocrinology, Mayo Clinic and Foundation, Rochester, MN 55905, USA c Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905, USA Received 6 January 2005 Available online 15 July 2005

Abstract Objectives. To determine if epigenetic interference can restore progesterone receptor-B (PR-B) expression in PR-B negative endometrial adenocarcinoma cell lines, and to characterize the kinetics of PR-B induction mediated by DNA methyltransferase and histone deacetylase inhibitors. Methods. The PR-B negative endometrioid cancer cell lines KLE and HEC-1B were used as study models. PR-B mRNA and protein expression levels were measured using real-time PCR and Western blot analysis, respectively. DNA methylation levels of the PR-B promoter were determined by methylation-specific PCR. Dose – response correlations and the duration of response to aza-deoxycytidine (ADC) and trichostatin A (TSA) were characterized. Cell responses to prolonged and repeated drug treatment were also examined. Results. Relatively low concentrations of ADC and TSA over a 24-h period induced PR-B expression. Furthermore, ADC and TSA acted synergistically to reactivate PR-B expression. Depending on the cell line used, PR-B mRNA was induced 10 – 110 fold. This elevated PR-B expression continued for 48 h after drug withdrawal. Sustained upregulation of PR-B mRNA and protein was observed during prolonged and repeated drug treatment. Conclusion. The epigenetically silenced PR-B gene remains sensitive to changes in DNA demethylation and histone acetylation in uterine adenocarcinoma cell lines. Treatment with ADC and/or TSA results in a robust and sustainable PR-B upregulation. These small molecule epigenetic modifying agents may be used to sensitize poorly differentiated, PR-B negative endometrial cancers to progestational therapy. D 2005 Elsevier Inc. All rights reserved. Keywords: Progesterone receptor; DNA methylation; Epigenetics; Endometrial cancer

Introduction The availability of endogenous progesterone is important to protect the endometrium from the hyperplastic effects of estradiol [1]. In the case of exogenous estrogen administration, progesterone is routinely given in combination with estrogen to prevent the development of endometrial malig* Corresponding author. Department of Obstetrics and Gynecology, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, MN 55905, USA. Fax: +1 507 255 4828. E-mail address: [email protected] (S.-W. Jiang). 0090-8258/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2005.05.035

nancies [2]. Progestins have also been used effectively to treat early stage endometrial cancers in women wishing to preserve fertility [1,3]. Progesterone exerts its regulatory effects by binding to the progesterone receptor (PR). PR is a member of a closely related subgroup of nuclear receptors that includes the androgen, mineralocorticoid, and glucocorticoid receptors. Two isoforms of the progesterone receptor, PR-A and PR-B, are expressed in normal endometrium. These isoforms are transcribed from alternative promoters located in proximity to a single gene, and both may be required to limit the proliferation and promote differentiation of the endometrial glandular cells [4].

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Previous studies have indicated that the loss of one or both PR isoforms may provide certain advantages for endometrial cancer progression [4]. A deceased ratio of PRB to total PR was found to be associated with compromised survival [5,6], suggesting an important PR-B specific action in the development of endometrial cancers. Many of the PR-negative tumors present in advanced stages, associated with deep invasion, high recurrence rates, and poor prognosis [5,6]. In addition to its role in tumorigenesis, PR negativity is also considered to be responsible for resistance to progestin treatment [7]. However, the presence of an intact PR gene in PR negative cancers raises the possibility for resensitizing these tumors to hormonal treatment following PR induction. An accumulating body of evidence suggests that epigenetic events, including changes in DNA methylation and/or histone acetylation, regulate gene transcription by modulating chromatin conformation [8,9]. Hypermethylated DNA and deacetylated histones in the promoter region are usually associated with downregulated or silenced gene expression. Two lines of reagents, affecting DNA methylation and histone acetylation, respectively, have been used to reactivate epigenetically silenced genes. Aza-deoxycytidine (ADC) is a modified nucleoside homolog that becomes incorporated into nascent DNA strands during cell replication [10,11]. After incorporation, ADC covalently binds to and arrests DNA methyltransferase (DNMT), leading to genome-wide demethylation [11]. Trichostatin A (TSA) inhibits histone deacetylase (HDAC), leading to the accumulation of acetylated histone [12,13]. While it was reported that PR-B is silenced by a DNA methylation mechanism and PR-B expression was upregulated by ADC treatment [14], it is not clear how HDAC inhibitors alone, or in combination with DNMT inhibitors, may affect PR-B expression. Furthermore, important features of PR-B induction by epigenetic interference, such as the time and dose response patterns, have not been determined. The aim of this investigation was to determine if PR-B expression can be restored by epigenetic interference, and if so, to characterize the kinetics of PR-B expression induced by ADC and TSA in PR-negative endometrial cancer cell lines.

Materials and methods

facturer’s recommendation. Aza-deoxycytidine and trichostatin A were purchased from Sigma (St. Louis, MO). Cell treatment, RNA isolation, cDNA synthesis, and quantitative real-time PCR Cells were plated in 10-cm dishes at 20% confluence. When cultures grew to approximately 50% density, ADC or TSA was added in various concentrations as indicated in the figure legends. Except for the time point study, cells were treated for 48 h prior to harvesting for RNA and DNA isolation. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized with 1 Ag RNA using the SuperScripti kit (Invitrogen, Carlsbad, CA). The 20 Al reverse transcription products were diluted to 100 Al and 2 Al used for each real-time PCR. Reactions were performed in a volume of 25 Al containing 140 ng primers and 12.5 Al SYBR green Master Mix (Stratagene, Cedar Creek, TX). The primer sequences are: PRB-F, 5VACTGAGCTGAAGGCAAAGGGT and PRB-R, 5VGTCCTGTCCCTGGCAGGGC. As an input control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA levels were measured using primers: GAPDH-F, 5VGAAGGTGAAGGTCGGAGTC and GAPDH-R, 5V-GAAGATGGTGATGGGATTTC. PCR conditions were: initial denaturing, 95-C for 5 min; 40 cycles of repeated denaturing at 95-C for 15 s; annealing at 56-C for 30 s; and extension at 72-C for 30 s. Methylation-specific PCR Genomic DNA was isolated using Wizard Genomic DNA Isolation Kit (Promega) following the manufacturer’s instructions. 2 Ag DNA was subject to sodium bisulfite conversion using an EZ DNA methylation Kiti (ZYMO Research). The converted DNA was eluted from DNA affinity columns and 2 Al was used for PCR. PCR was performed using the following primers: PRB-MF, 5VGATTGTCGTTCGTAGTACG, and PRB-MR, 5V-CGACAATTTAATAACACGCG for methylated DNA, and primers PRB-UF, 5V-TGATTGTTGTTTGTAGTATG and PRB-UR, 5V-CAACAATTTAATAACACACA for unmethylated DNA. PCR products were resolved in 2% agarose gels and DNA bands visualized by ethidium bromide staining.

Cell lines and reagents Western blot analysis Human endometrioid cancer cell lines KLE and HEC-1B were purchased from American Type Culture Collection (ATCC, Rockville, MD). The cells were grown in DMEM/ F12 containing 10% fetal bovine serum (BioWhittaker, Walkersville, MD), 100 Ag/ml streptomycin, 100 units/ml penicillin, and 2 mM l-glutamine. Cells were maintained at 37-C in an atmosphere containing 5% CO2 and 100% humidity. The rabbit PR-B antibody was purchased from Upstate (Waltham, MA) and applied following the manu-

Cell cultures were rinsed three times with cold phosphate buffered saline (PBS) and harvested by scraping with a plastic policeman in lysis buffer containing 20 mM Hepes, pH 7.2, 25% Glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. To prevent protein degradation, the solution was supplemented with 1 protease inhibitor cocktail (Sigma, St. Louis, MO). Cell lysates were centrifuged at

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14,000 rpm for 15 min at 4-C and the insoluble debris discarded. Protein concentrations were determined using Coomassie Protein Assay Reagent (Pierce, Rockford, IL). Cell extracts (20 Ag) were mixed with 5 Al gel-loading buffer (250 mM Tris –HCl, pH8.0, 20% h-mercaptoethanol, 40% glycerol, 8% SDS, 1.2 mg/ml bromophenol blue), heated for 5 min at 95-C, and resolved in SDS polyacrylamide gels (Ready Gel, 4 – 15% gradient, BIO-RAD Laboratories, Hercules, CA). Proteins were electro-transferred onto Immun-Bloti PVDF membranes (Bio-Rad). The membranes were blocked for 2 h in PBS containing 0.1% Tween-20 and 10% nonfat dry milk. Primary antibody binding was performed at 4-C overnight with constant shaking. The secondary anti-rabbit or anti-mouse antibodies labeled with horseradish peroxidase (Amersham Corp, Arlington Heights, IL) were applied at 1:5,000 dilutions. Secondary antibody binding was carried out at room temperature for 1 h. Chemiluminescence detection was performed with the ECL plus Western Blotting Detection System (Amersham Corp, Arlington Heights, IL) following the manufacturer’s protocols. The blots were re-probed with h-actin antibody and the results provided controls for protein loading. Data analysis The threshold cycle number (CT) measured by real-time PCR was used to calculate relative PR-B mRNA levels. The CT reading for PR-B (CTPR-B) was normalized against GAPDH using the formula DCT = CTPR-B CTGAPDH. The difference between PR-B and GAPDH was further converted to relative fold (F = 2DCT). For the convenience of data presentation, relative PR-B mRNA levels were shown in the figures, with the control group arbitrarily set at 1. Standard errors were proportioned to each mean and indicated. All data groups were analyzed using multivariate ANOVA to determine if there were significant differences among the data. For all experimental groups that satisfied the initial ANOVA criterion, individual comparisons were performed with the use of post hoc Bonferroni t tests with the assumption of two-tail distribution and two samples with equal variance. Statistically significant differences (P < 0.05) are indicated by an asterisk in each figure.

Results To identify appropriate cell lines as study models, we examined six human endometrioid cancer cell lines for PRB gene expression and found HEC-1B and KLE cells to have diminished levels of PRB mRNA and protein (data not shown). Using methylation-specific PCR, we confirmed that the CpG islands with the PR-B promoters in these cells are highly methylated (Fig. 1A). Treatment with the DNMT inhibitor ADC led to demethylation of the PR-B promoter and a concomitant increase in PR-B mRNA in both cell

Fig. 1. PR-B gene methylation and mRNA expression in KLE and HEC-1B cells. (A) Methylation-specific PCR was performed with sodium bisulfite converted genomic DNA. In both HEC-IB and KLE cells, a band representing methylated PR-B promoter is detected. Following ADC treatment, it is evident that the PR-B promoter is demethylated in both cell lines. (B and C) ADC (2.5 AM) and TSA (7 nM) treatment for 48 h significantly increased the PR-B mRNA levels in HEC-1B (B) and KLE cells (C). Standard error was shown for each group and statistical significance (P  0.05) of the difference between treatment and control groups is indicated by asterisk on the top of standard error bars.

lines (Figs. 1B, C), confirming PR-B gene upregulation by epigenetic modification. HEC-1B and KLE cells were subsequently used for kinetic studies on PR-B induction. HEC-1B and KLE cells responded well to low AM and nM ranges of ADC and TSA, respectively. Treatment with 2.5 AM ADC alone induced a modest 2- to 4-fold increase in PR-B mRNA levels (Figs. 1B, C). In contrast, TSA (7 nM) treatment led to a 4-fold increase in PR-B mRNA levels in HEC-1B cells and a 50-fold induction of PR-B mRNA in KLE cells, indicating that promoter demethylation is not required for PR-B induction. The highest level of PR-B expression, however, was observed when cells were simultaneously treated with both agents. Inductions as high as 10- and 110-fold were achieved in HEC-1B and KLE cells, respectively, indicating a strong synergism between

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DNMT and HDAC inhibitors, confirming that epigenetic regulation plays a dominant role in PR-B silencing in endometrial cancer. To determine PR-B protein expression, we performed Western blot analysis on cells treated with ADC or TSA alone, or with a combination of the two reagents. PR-B protein was not detected in these cell lines under normal conditions, but significantly increased following ADC and TSA treatment (Fig. 2). Similar effects on PR-B expression were observed in HEC-1B (Fig. 1, left side) and KLE (right side) cells. Consistent with the changes observed in mRNA levels, a synergistic effect on PR-B protein levels was noted with combined administration of DNMT and HDAC inhibitors. To characterize the dose – response relationship, we treated KLE cells with increasing concentrations of ADC or TSA. ADC was able to significantly induce PR-B mRNA transcription at concentrations as low as 0.05 AM, but the greatest effects were observed using 2.5 AM (Fig. 2A). Further increases in ADC concentrations led to cytotoxic effects evidenced by massive cell death, dramatic morphological changes (data not shown), and decreased PR-B production (Fig. 3A, 25 AM). With TSA, a significant induction was observed using a concentration of only 0.14 nM (Fig. 3B). A progressive enhancement in PR-B expression was seen with increasing concentrations of TSA until 7 nM, at which time further increases were counterproductive for PR-B expression. Similar to the use of high concentrations of ADC, 70 nM of TSA caused significant cell death, a phenomenon well documented for HDAC inhibitors in cancer cells [15]. Thus, administration of both reagents induces robust PR-B gene expression over a relatively broad range of concentrations. We next performed time course studies on PR-B upregulation. A significant induction in PR-B mRNA was seen at 12 h with ADC, followed by a steady increase up to a maximum level of induction at 48 h (Fig. 4A). TSA appears to require a shorter treatment time than ADC to induce PR-B mRNA expression (Fig. 4B). A significant induction was readily observed after 6 h with a maximal induction at 24 h. Longer treatments did not further increase

Fig. 2. PR-B protein expression in KLE (left side) and HEC-1B (right side). Western blot analysis shows that ADC (2.5 AM) and TSA (7 nM) treatment for 48 h increased the PR-B expression. A synergistic effect was observed when the two drugs were used in combination. The blot was stripped and re-detected with h-actin antibody. Comparable density of h-actin bands indicates nearly equal protein loading for each sample.

Fig. 3. Dose – response studies for ADC (A) and TSA (B). KLE cells were treated with increasing concentrations of ADC and TSA for 48 h before mRNA was extracted and PR-B expression measured. The results were compared to the correspondent controls and statistical significance (P < 0.05) is indicated by asterisks.

Fig. 4. Time course studies for ADC (A) and TSA (B). KLE cells were treated with 2.5 AM ADC or 7 nM TSA for various times (h) as indicated. The results were compared to the untreated controls as described under Materials and methods. Statistical significance (P < 0.05) is indicated by asterisks.

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PR-B expression levels. Thus, the two reagents exhibited distinct time response patterns, reflecting their different actions on chromatin modification. Both reagents, however, were able to support elevated PR-B expression lasting for at least 3 (TSA) or 4 (ADC) days. As part of the kinetic study, we also examined how long the PR-B upregulation could last in the absence of DNMT and HDAC inhibitors. Following a 2-day exposure to ADC or TSA, cell cultures were changed to regular media containing no inhibitor and the PR-B mRNA levels were

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monitored at different time points thereafter. The results showed that ADC effects on PR-B expression gradually decrease following drug withdrawal, with a 2-fold induction remaining after 48 h (Fig. 5A). Following treatment with TSA, however, PR-B mRNA levels decreased sharply 12 h after the drug was withdrawn (Fig. 5B), but remained many times higher than the baseline level for at least 48 h. These results demonstrate the persistent nature of epigenetic modifications on gene expression in that reversal of changes in DNA methylation and histone acetylation patterns may take days to complete. To determine the cellular response to repeated drug exposure, we treated KLE cells with ADC or TSA for 48 h and then changed them to drug-free media. After 7 days of recovery, drugs were again added for a second round of treatment and PR-B mRNA levels were monitored at each stage of the experiment. Repeated exposure of KLE cells to ADC or TSA induced PR-B expression to the same extent as the original treatment (Fig. 5C). These data suggest that no significant adaptation or resistance to these agents was developed by these cells, at least in the short term.

Discussion

Fig. 5. Effects of drug withdrawal and repeated treatment with DNMT and HDAC inhibitors on PR-B expression. KLE cells were treated with 2.5 AM ADC (A) or 7 nM TSA (B) for 48 h. Cultures were then continued in the absence of inhibitors and PR-B mRNA levels were measured at different time point. The asterisks indicate statistical significance (P < 0.05) of each treatment group compared to the untreated control. For repeated treatment, KLE cells treated with ADC (C, left side, 2.5 AM for 48 h) or TSA (C, right side, 7 nM for 48 h) were maintained in normal media for 1 week. The cells were again treated with ADC (2.5 AM) and TSA (7 nM) for 48 h. PR-B mRNA levels were compared before and after drug treatment and statistical significance is marked by asterisks.

The biological effects of progesterone are mediated by its specific interaction with PR, resulting in the transport of PR from the cytosol to the nucleus and activation of PRmediated transcription. This leads to changes in a variety of cellular functions, including enhancement of cell differentiation, inhibition of mitosis, and reduction of protein synthesis, the basis for its therapeutic use [16 – 18]. Administration of exogenous progesterone is well known to cause significant endometrial cell apoptosis both in vitro and in vivo [19,20]. Amezcua et al., studied hyperplastic endometrial tissue from patients treated with progestins and found that apoptosis is an early result of progestin therapy [19]. Furthermore, a direct correlation between PR positivity and response to progestin treatment was observed [21]. Unfortunately, the most advanced endometrial cancers are often PR-negative and recalcitrant to progestational effects [21]. One subtype of progesterone receptor, PR-B, may play a dominant role in the progestin-mediated inhibition of endometrial cell growth and invasiveness [4]. Using adenovirus vectors, Dai et al. overexpressed PR-B in PRnegative endometrial cancer cells and found that cell adhesion molecules were significantly downregulated by PR-B. Our results indicate that relatively low concentrations of ADC and TSA induce significant PR-B expression following a relatively short treatment duration. As little as 0.05 AM ADC and 0.14 nM TSA readily increase PR-B mRNA levels. These concentrations represent a fraction of the dose previously used for the induction of other genes in different cell lines representing diversified cancers [22 –24], suggesting that the key components of the transcription/translation

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machineries required for PR-B expression are not impaired in endometrial cancer cells and that the silenced PR-B gene remains exquisitely sensitive to DNA demethylation and histone acetylation. From a pharmacological standpoint, PRB expression appears to be sustainable with repeated treatments and may be applicable in a clinical setting (Fig. 5). Our data also demonstrate that both DNA methylation and histone deacetylation are involved in the PR-B downregulation, and simultaneous interference with both mechanisms is required for a maximal level of PR-B expression. It is important to point out that while ADC and TSA may directly upregulate PR-B expression by modifying the structure of chromatins surrounding this gene’s promoter, both drugs are capable of affecting the expression of many other genes. Therefore, indirect actions of these drugs, such as through affecting upstream genes, may also contribute to the induction of PR-B expression. Further studies are required for a thorough understanding of the mechanisms involved in PR-B upregulation. Both DNMT and HDAC inhibitors are currently being examined for treating hematological as well as solid tumors [25 –27]. These drugs are small hydrophobic molecules that can easily penetrate cytoplasmic membrane to reach their nuclear targets. ADC is used in Phase II clinical trials for the treatment of nonsmall cell lung cancers, chronic myeloid leukemia, and prostate cancers [28]. Phase I trials utilizing HDAC inhibitors in patients with advanced solid tumors have shown these agents to be efficacious and well-tolerated [29,30]. Endometrial cancer appears to be an attractive target for epigenetic modification drugs, because the potential benefits of PR reexpression may be augmented by the generic effects of ADC and TSA on genes involved in DNA repair, cell cycle control, and apoptosis. Simultaneous silencing of key tumor suppressor genes may be a crucial step in the malignant transformation of normal endometrium [31]. DNA hypermethylation and histone deacetylation are also responsible for the silencing of PTEN and the MLH1 DNA repair gene, both of which have been shown to be reactivated by epigenetic interference [32,33]. Although we have not yet examined whether epigenetic induction of PR-B could restore sensitivity to progesterone, previous studies in other genes suggest this is highly possible. It has been reported that treatment with ADC and TSA not only reactivated ER-a expression, but also restored the normal transcriptional regulation by estrogen responsive elements in ER-negative breast cancer cells [4]. Using a similar epigenetic approach, Plumb et al., have successfully reestablished MLH1 expression, restored the DNA repair function, and hence reversed the resistance to genotoxic drugs in a xenograft model [27]. These results, together with our current findings, suggest the potential development of a novel chemotherapy strategy for PR-negative endometrial cancers using epigenetic modification drugs in combination with progestins. Further pre-clinical studies, including pharmacological studies in animal models, are required to

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