ARTICLE

Methylation and Inactivation of Estrogen, Progesterone, and Androgen Receptors in Prostate Cancer

Masahiro Sasaki, Yuichiro Tanaka, Geetha Perinchery, Abhipsa Dharia, Ioulia Kotcherguina, Sei ichiro Fujimoto, Rajvir Dahiya

Affiliations of authors: M. Sasaki, Y. Tanaka, G. Perinchery, A. Dharia, I. Kotcherguina, R. Dahiya, Department of Urology, University of California, San Francisco, and Veterans Affairs Medical Center, San Francisco; S. Fujimoto, Department of Obstetrics and Gynecology, School of Medicine, Hokkaido University, Kitaku, Sapporo, Japan.

Correspondence to: Rajvir Dahiya, Ph.D., Urology Research Center (112F), University of California, San Francisco, and Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121 (e-mail: Urologylab{at}aol.com).


    ABSTRACT
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Prostate cancer development is initially steroid hormone dependent. Estrogen receptors (ERs), androgen receptors (ARs), and progesterone receptors (PRs) have been identified in normal and cancerous prostate tissues. We investigated whether the promoter regions of these steroid receptor genes are methylated and inactivated in prostate cancer cells and tissues. Methods: The expression and promoter methylation status of three ER{alpha} isoforms (ER{alpha}-A, ER{alpha}-B, and ER{alpha}-C), ER{beta}, two PR isoforms (PR-A and PR-B), and AR were investigated in five prostate cancer cell lines (ND1, DU145, PC3, LNCaP, and DUPro) and in pairs of normal and cancerous prostate tissues from 38 patients with prostate cancer. Methylation-specific polymerase chain reaction, reverse transcription–polymerase chain reaction, and 5` rapid amplification of complementary DNA ends were used. All statistical tests were two-sided. Results: ER{alpha}-C was expressed in all cell lines, but ER{alpha}-A and ER{alpha}-B were not expressed in any cell line. ER{alpha}-A and ER{alpha}-B promoters were methylated, but ER{alpha}-C was unmethylated. Promoters for ER{beta}, AR, PR-A, and PR-B were methylated and thus inactivated in some cell lines but not in others. Treating cells with the demethylating reagent 5-aza-2`-deoxycytidine restored expression of all steroid receptor genes with previously methylated promoters. All 38 pairs of cancer and normal tissues had unmethylated ER{alpha}-C promoters. Thirty-six (95%) of 38 cancers had methylated ER{alpha}-A, 35 (92%) of 38 cancers had methylated ER{alpha}-B, but all normal tissues had unmethylated ER{alpha}-A and ER{alpha}-B (both P<.001). ER{beta} was methylated in 30 (79%) of 38 cancers but unmethylated in all normal tissues. AR was methylated in three (8%) of 38 cancers but unmethylated in all normal tissues. PR-A and PR-B were unmethylated in all tissues. Conclusion: Certain steroid receptor genes appear to be inactivated by CpG methylation in prostate cancer tissue and cell lines.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Various steroid hormones act via intracellular steroid receptors, which are potent hormonal effectors implicated in the control of proliferation, differentiation, and development of prostate cells (1). Multiple promoters and differential splicing of 5` upstream exons are often found in nuclear receptor genes including steroid receptors (2–6). The prostate gland is under endocrine control during its growth and development (1). Estrogen receptors (ERs) {alpha} and {beta}, androgen receptor (AR), and progesterone receptors (PRs) have been identified in normal and cancerous prostate tissue (1). Three promoters control the expression of human ER{alpha} isoforms ER{alpha}-A, ER{alpha}-B, and ER{alpha}-C (3), and two promoters control the expression of human PR isoforms PR-A and PR-B (7,8). Hypermethylation of cytosine-rich areas in promoters has been associated with the transcriptional inactivation of genes and viewed as functionally equivalent to an inactivating mutation (9). In addition, abnormal methylation in cytosine-rich areas has been detected frequently in cancer (9). The 5` upstream promoter regions of ER{alpha}, PR, and AR genes also contain cytosine-rich areas (2), but their methylation status and corresponding gene expression activity in the prostate have not been completely characterized. However, we have detected the selective methylation of the ER{alpha}-C (10) and PR-B (11) promoters during the development of endometrial cancer, suggesting that selective methylation may be involved in regulating the expression of steroid receptors (10,11).

We hypothesize that the selective expression or inactivation of steroid receptor isoforms is involved in the pathogenesis of prostate cancer, which is initially dependent on steroid hormones. Consequently, we investigated gene expression and methylation status of promoters for three ER{alpha} isoforms (ER{alpha}-A, ER{alpha}-B, and ER{alpha}-C), ER{beta}, two PR isoforms (PR-A and PR-B), and AR in five prostate cancer cell lines and in pairs of cancerous and normal prostate tissues from 38 patients. We also investigated the effect of demethylation on steroid receptor expression by treating cells with the demethylating reagent 5-aza-2`-deoxycytidine.


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tissues and Cell Lines

All five human prostatic cancer cell lines available from cell banks, including the American Type Culture Collection (Manassas, VA)—LNCaP, PC3, ND1, DUPro, and DU145—were used for these experiments and cultured as described previously (12). Cells were cultured with fresh medium containing 5-aza-2`-deoxycytidine (2 µg/mL) on days 1, 3, and 5 and harvested on day 6. Pairs of cancerous and normal prostate tissues from 38 patients were obtained from the Veterans Affairs Medical Center, San Francisco, CA, and the University of California, San Francisco. All cancer tissues were primary adenocarcinomas, not metastatic tumors. From all specimens of cancerous tissue, 5-µm paraffin-embedded sections were cut and stained with hematoxylin–eosin. Normal and cancerous regions in these sections were then identified by light microscopy at a magnification of x100, marked, and microdissected as described previously (12). From each specimen, a pool of normal tissue and a pool of cancerous tissue were prepared. Immunohistochemical analysis was not carried out because, to our knowledge, there are no antibodies specific for ER{alpha} isoforms ER{alpha}-A, ER{alpha}-B, and ER{alpha}-C.

RNA Isolation, Reverse Transcription–Polymerase Chain Reaction, and 5` Rapid Amplification of Complementary DNA Ends

Total RNA was extracted and complementary DNA (cDNA) was synthesized as described by Sasaki et al. (13). For reverse transcription–polymerase chain reaction (RT–PCR), regions specific for all three ER{alpha} isoforms, ER{beta}, PR-A, PR-B, and AR were amplified from the cDNA with specific primers (see Table 1Go). A {beta}-actin primer that contained one intron of the {beta}-actin gene was used as a positive control; in the presence of contaminating genomic DNA, additional larger actin bands would be amplified and, in its absence, the larger actin bands were not amplified. Negative control reactions without RNA and without reverse transcriptase were also performed. A modified 5` rapid amplification of cDNA ends (5` RACE) method was used to detect the expression of each PR messenger RNA (mRNA) as described previously (10).


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Table 1. Summary of the primer sets and polymerase chain reaction (PCR)* conditions for various steroid receptor isoforms
 
DNA Extraction and Sodium Bisulfite Treatment

DNA was isolated from paraffin-embedded sections. Microdissection of normal and cancerous prostate tissues was performed as described previously (13). DNA (approximately 100 ng) was denatured with NaOH and treated with sodium bisulfite for 16 hours (Invitrogen Corp., Purchase, NY) as described previously (13). Before sequencing, all cytosine residues were deaminated and converted to thymine residues by sodium bisulfite modification. 5-Methylcytosine residues were not altered by this treatment.

Methylation-Specific PCR

Fig. 1Go shows the schematic diagram of the ER{alpha}, ER{beta}, AR, and PR genes. Primers and PCR conditions are summarized in Table 1Go. The fragment of DNA to be amplified was intentionally small because DNA fragments isolated from paraffin sections are generally less than 300 base pairs long (10). For methylation-specific PCR (MS-PCR), we used one primer set (U) that anneals to unmethylated DNA and another primer set (M) that anneals to methylated DNA. Unmodified DNA was amplified with the W primer set (wild-type), which serves as a positive control for PCR. PCR was performed with approximately 10 ng of cDNA in 20 µL containing 1.5 mM MgCl2, all four deoxyribonucleoside triphosphates (each at 0.8 mM), and 0.5 U of Taq polymerase (Applied Biosystems, Inc., Foster City, CA). The PCR containing 40 cycles of denaturation (94 °C for 30 seconds), annealing (Table 1Go), and extension (72 °C for 45 seconds) was followed by a final incubation at 72 °C for 8 minutes. Eight microliters of each PCR product was mixed with 1 µL of 10x loading dye, and the mixture was subjected to electrophoresis on 3% agarose gels at 180 V and ambient temperature. The bands on the gels were visualized by ethidium bromide staining. Only gels with clear bands were used in this study.



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Fig. 1. Structure of steroid receptor genes. Positions and orientation of methylation-specific polymerase chain reaction (PCR) primers (MSP) and reverse transcription–PCR (RT–PCR) primers are indicated by black arrows. CpG sites are shown by lollipop signs. The common region of estrogen receptor {alpha} (ER{alpha}) and progesterone receptor (PR) isoforms is in exon 1, indicated by a solid box. ER{alpha} has three promoters for three distinct exon 1 regions (exon 1, exon 1`, and exon 2`) that are activated and translated independently. Exon 1, exon 1`, and exon 2` give rise to isoforms ER{alpha}-A, ER{alpha}-B, and ER{alpha}-C, respectively. PR-A and PR-B originate from translational initiation at AUG-2 and AUG-1, respectively, in exon 1 of the PR gene. Primer sequences are given in Table 1Go. A representation of the modified 5` rapid amplification of complementary DNA (cDNA) ends method to distinguish PR-A, and PR-B messenger RNA (mRNA) expression is shown at the bottom. Rev = reverse.

 
DNA Sequencing

For confirmation of MS-PCR, PCR products were purified by QIAquick PCR Purification Kit (Qiagen, Valencia, CA), and 30 ng of the product was used as a template for sequencing (10). Double-strand sequence analysis was performed with each primer set by use of an ABI 377 Sequencer and Dye Terminator Cycle sequencing kit (Applied Biosystems, Inc.).

Statistical Analyses

Chi-square analysis with the Yate's correction was used to determine differences in methylation status of these steroid receptor isoforms between endometrial cancerous and normal tissues. All statistical tests were two-sided.


    RESULTS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell Lines

We first determined the expression status of steroid receptor isoforms in five prostate cancer cell lines (LNCaP [Fig. 2Go], PC3, ND1, DU145, and DUPro). In all prostate cancer cell lines, ER{alpha}-C was expressed, but ER{alpha}-A and ER{alpha}-B were not expressed. ER{beta} was not expressed in ND1 and DU145 cells but was weakly expressed in PC3, LNCaP, and DUPro cells. AR was not expressed in ND1, DU145, and DUPro cells but was expressed in PC3 and LNCaP cells. PR-A and PR-B were not expressed in PC3 cells but were expressed in LNCaP, ND1, DU145, and DUPro cells.



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Fig. 2. Messenger RNA (mRNA) expression (A), methylation status (B), and 5` rapid amplification of complementary DNA (cDNA) ends (5` RACE) (C) of the prostate cancer cell line LNCaP before and after treatment with the demethylating reagent 5-aza-2`-deoxycytidine. Lane L = 100-base-pair (bp) ladder marker. A) Lanes 110 = before treatment; lanes 1120 = after treatment. Lanes 1 and 11 = estrogen receptor {alpha} isoform A (ER{alpha}-A); lanes 2 and 12 = ER{alpha}-B; lanes 3 and 13 = ER{alpha}-C; lanes 4 and 14 = ER expression common to all ER{alpha} isoforms; lanes 5 and 15 = ER{beta}; lanes 6 and 16 = androgen receptor (AR); lanes 7 and 17 = progesterone receptor (PR) expression common to PR-A and PR-B; lanes 8 and 18 = PR-B; lanes 9 and 19 ={beta}-actin mRNA expression as positive controls and for detecting DNA contamination; and lanes 10 and 20 = negative controls without template RNA. B) U = unmethylated bands; M = methylated bands. Lanes 114 = before treatment; lanes 1528 = after treatment; lanes 1, 2, 15, and 16 = ER{alpha}-A; lanes 3, 4, 17, and 18 = ER{alpha}-B; lanes 5, 6, 19, and 20 = ER{alpha}-C; lanes 7, 8, 21, and 22 = ER{beta}; lanes 9, 10, 23, and 24 = AR; lanes 11, 12, 25, and 26 = PR-A; and lanes 13, 14, 27, and 28 = PR-B. C) Lane 1 = 5` RACE product before the treatment; lane 2 = 5` RACE product after the treatment; and lane 3 = negative control without template RNA. By the 5` RACE method, longer bands were derived from PRB mRNA, and shorter bands were derived from PR-A mRNA.

 
We then determined the methylation status of steroid receptor isoforms in these cell lines (Fig. 2Go). In all cell lines, the ER{alpha}-C promoter was unmethylated, whereas the ER{alpha}-A and ER{alpha}-B promoters were methylated. The ER{beta} promoter was fully methylated in ND1 and DU145 cells but was partially methylated in PC3, LNCaP, and DUPro cells. The AR promoter was methylated in ND1, DU145, and DUPro cells but was unmethylated in PC3 and LNCaP cells. PR-A and PR-B promoters were methylated only in PC3 cells and were not methylated in LNCaP, ND1, DU145, and DUPro cells.

To investigate whether the expression of mRNAs for these steroid isoforms is inactivated by methylation of their promoters, we treated cells with the demethylating agent 5-aza-2`-deoxycytidine and then assessed expression by use of MS-PCR and RT–PCR (Fig. 2Go). Demethylation restored the expression of ER{alpha}-A and ER{alpha}-B in all cell lines, ER{beta} in ND1 and DU145 cells, and PR-A and PR-B in PC3 cells. Thus, the expression of these steroid receptor isoforms was related to the methylation status of their corresponding promoters.

Pairs of Cancer and Normal Tissues From the Same Patient

To determine whether promoters for these steroid receptor isoforms were methylated in prostate cancer tissues, we analyzed pairs of cancerous and normal tissues from 38 patients (Fig. 3Go). The promoter for ER{alpha}-A was methylated in 36 (95%) of 38 cancerous tissues but was unmethylated in all 38 normal tissues (all P<.001). The promoter for ER{alpha}-B was methylated in 35 (92%) of 38 cancerous tissues but was unmethylated in all 38 normal tissues (P<.001). Of interest, the promoter for ER{alpha}-C was unmethylated in all 38 pairs of cancerous and normal tissues. The promoter for ER{beta} was methylated in 30 (79%) of 38 cancerous tissues and unmethylated in all normal tissues. The promoter for AR was methylated in three (8%) of 38 cancerous tissues and unmethylated in all normal tissues. Promoters for PR-A and PR-B were unmethylated in all cancerous and normal tissues. We confirmed the results for methylation status of these steroid receptor isoforms by direct DNA sequencing (Fig. 4Go). All cytosines were deaminated and converted to thymines after sodium bisulfite modification for these steroid receptors in normal tissues, but 5-methylcytosines were not altered in cancer tissues because methylation protected the residues.



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Fig. 3. Methylation status of various steroid receptor isoforms in a pair of prostate cancer and normal prostate tissues from one patient with prostate cancer. U = unmethylated bands; M = methylated bands; N = normal tissues; and C = cancerous tissues. Lanes 14 = estrogen receptor {alpha} isoform A (ER{alpha}-A); lanes 58 = ER{alpha}-B; lanes 912 = ER{alpha}-C; lanes 1316 = androgen receptor (AR); lanes 1720 = progesterone receptor isoform A (PR-A); lanes 2124 = PR-B; and lanes 2528 = ER{beta}.

 


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Fig. 4. Examples of direct DNA sequencing chromatograms for steroid receptors in pairs of cancer and normal prostate cancer tissue. CpG sites are underlined. All cytosines (C) are deaminated and converted to thymines (T) in normal tissue, but 5-methylcytosines (shown as C) remained unaltered in cancer tissue ({blacktriangledown}). ER = estrogen receptor; AR = androgen receptor.

 
Table 2Go shows the association of the methylation status of ER{alpha}-A, ER{alpha}-B, ER{beta}, and AR with the tumor–node–metastasis classification of prostate cancer (12). ER{alpha}-A promoters were unmethylated in two (15%) of 13 T2 samples (early-stage prostate cancer) but methylated in all 20 T3 samples (late-stage prostate cancer). ER{alpha}-B was unmethylated in three (23%) of 13 T2 samples but methylated in all 20 T3 samples. ER{beta} in five (38%) of 13 T2 samples was unmethylated but methylated in all 20 T3 samples. AR was unmethylated in all T2 samples but methylated in three (15%) of 20 T3 samples.


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Table 2. Association with methylation status of ER{alpha}-A, ER{alpha}-B, ER{beta}, and AR and clinical stage in cancer tissues*
 

    DISCUSSION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Steroid receptors are potent hormonal effectors implicated in the control of proliferation, differentiation, and development of prostate cells (1). Many receptor genes, including those for steroid hormones, have multiple transcription units (5,6,14,15). The existence of an equivalent upstream exon 1 in the human vitamin D receptor has been reported previously (16–18). Several promoters have also been described for the chicken PR gene (19) and human and mouse retinoic acid receptor genes (20,21). Two promoter elements may be used by the rat thyroid hormone receptor gene (22). Thus, multiple promoters may be a feature of the steroid receptor gene family.

Estrogens play an important role in female sexual development and regulation of the menstrual cycle. In males, estrogens affect benign prostatic hyperplasia and the development of prostate cancer (1,23). Loss or inhibition of ER expression in prostate cancer has been documented (23). An inverse association was found between ER expression and histologic grade or pathologic stage of prostate cancer. Estrogen has been used for the treatment of prostate cancer, and its inhibitory effects have been widely acknowledged (1). A low level of ER expression has been associated with a poor response to endocrine therapy (23). Unfortunately, the mechanisms regulating the expression of ER{alpha} are poorly defined, and no mutation or other gross structural alteration of the ER{alpha} gene in prostate cancer tissue has been reported that could clarify the mechanisms (11,24).

The three isoforms of ER{alpha} are transcribed from three promoters active in a cell- and tissue-specific manner (25). The proximal promoter A was identified as a result of cloning the ER cDNA, followed later by the identification of distal promoters B and C (17). These promoters, which regulate the synthesis of specific transcripts for ER{alpha}-A, ER{alpha}-B, and ER{alpha}-C isoforms, are regulated independently. Although the specific roles of the three ER{alpha} isoforms are unclear, the mechanisms regulating their production suggest that the relative levels of ER{alpha}-A, ER{alpha}-B, and ER{alpha}-C in cells are critical for appropriate cellular responses to estrogen.

In this study, we detected the expression of ER{alpha}-C but not that of ER{alpha}-A and ER{alpha}-B in all prostate cancer cell lines and tissues tested. In these cell lines, the promoter for ER{alpha}-C was unmethylated, whereas those for ER{alpha}-A and ER{alpha}-B were methylated. In pairs of cancerous and normal prostate tissues, all cancerous tissues had unmethylated ER{alpha}-C promoters, but 36 of 38 tumors had methylated ER{alpha}-A promoters, and 35 of 38 had methylated ER{alpha}-B promoters. Thus, in prostate cancer, the activation status of each ER{alpha} isoform appears to be specifically regulated through promoter methylation.

The PR also has two distinct isoforms, PR-A and PR-B, which arise from differential transcription from different promoters within the same gene (4). Because both PR isoforms are detected consistently in both normal and cancerous prostate cells, these receptors should have some role in these cells (26,27). Progesterones have been used to treat certain stages of prostate cancer, although the mechanism of action is unresolved (28). In prostate cancer cell lines, we found that both PR-A and PR-B promoters were methylated and inactivated in PC3 cells but were unmethylated and activated in other cell lines. We found that, in pairs of normal and cancerous prostate tissues, all tissues had unmethylated PR-A and PR-B promoters. Our data suggest that PR-A and PR-B methylation may be a late event in prostate carcinogenesis because PR methylation was observed in only one prostate cancer cell line but not in cancer tissues.

Because the AR protein is a key mediator of growth in the prostate, much research has focused on the role of the AR gene in the development of prostate cancer (29). Although several groups (30) have attempted to find mutations of the AR gene in prostate cancers, only a few mutations have been found, especially in primary prostate cancer. However, in a previous study (13), we reported that the AR promoter was methylated in endometrial cancer during carcinogenesis. When we examined pairs of normal and cancerous prostate tissues, the three prostate cancer tissues that had methylated AR promoters were at a relatively late stage, T3a or T3. Methylation of the AR promoter thus may be a late event in prostate carcinogenesis.

To our knowledge, this is the first report in which the selective methylation and inactivation of promoters for ER{alpha}-A and ER{alpha}-B have been detected in prostate cancers. ER{alpha}-C was unmethylated and activated, and ER{alpha}-A and ER{alpha}-B were methylated and inactivated in all prostate cancer cell lines and in almost all prostate cancer tissues. Our data suggest that each ER{alpha} isoform has specific characteristics in prostate carcinogenesis through its methylation and activation status.


    NOTES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Supported by Public Health Service grants DK47517 (National Institute of Diabetes and Digestive and Kidney Diseases) and CA64872 (National Cancer Institute), National Institutes of Health, Department of Health and Human Services; and by Veterans Affairs Medical Center Research Enhancement Award Program and a Department of Veterans Affairs Merit Review.


    REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received June 21, 2001; revised December 19, 2001; accepted December 31, 2001.


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