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Molecular Biology, Pathobiology and Genetics |
1 Center for Clinical Cancer Genetics, Department of Medicine and Departments of 2 Health Studies, 3 Pathology, and 4 Human Genetics, University of Chicago, Chicago, Illinois
Requests for reprints: Olufunmilayo I. Olopade, Center for Clinical Cancer Genetics, Department of Medicine, University of Chicago, Chicago, IL 60637-1463. Phone: 773-702-1632; Fax: 773-702-0963; E-mail: folopade{at}medicine.bsd.uchicago.edu.
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Abstract |
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Introduction |
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Hypermethylation of the BRCA1 promoter has been proposed as one mechanism for functionally inactivating BRCA1 and has been reported to occur in 7% to 31% of sporadic breast and ovarian cancers (3). BRCA1-methylated sporadic tumors have been described in young women and display pathologic features similar to those of BRCA1-mutated hereditary breast cancers (3–5). Interestingly, sporadic tumors with aberrant methylation of the BRCA1 promoter can be clustered with tumors derived from women with inherited BRCA1 germ line mutations because of similarities in their global gene expression profiles (5, 6).
Thus, an important role of functional inactivation of BRCA1 by promoter methylation in sporadic breast cancer seems plausible but the mechanisms of tumor initiation and progression in these tumors are largely unknown. It has not been shown if the silencing of BRCA1 in sporadic tumor cells is associated with the deletion of the BRCA1 locus and/or the loss of chromosome 17, as is often the case in BRCA1-mutated cells. In this study, we have done an extensive analysis of BRCA1 promoter methylation in relation to BRCA1 gene copy number to gain a better understanding of the mechanisms involved in epigenetic regulation of the gene.
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Materials and Methods |
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Patient materials. The study was conducted under research protocols approved by the University of Chicago Institutional Review Board. Sporadic breast tumors were randomly selected from the pathology archives and tumor bank at the University of Chicago Hospitals as previously described (7). Of the 131 breast tumors analyzed in this study, 64 were paraffin-embedded specimens and 67 were fresh frozen specimens. Samples containing >50% tumor area were selected after microscopic examination of representative sections from each case. Frozen breast tissue specimens containing normal breast epithelia from three patients were used as controls.
DNA isolation. Genomic DNA was extracted using either QIAamp kit (Qiagen, Inc., Valencia, CA) for paraffin-embedded tissue or DNAzol isolation protocol (Invitrogen) for frozen tissue. The integrity of DNA extracted from paraffin-embedded tissue was confirmed by PCR with primers for exon 11A of the BRCA1 gene (8, 9). Microdissection was done on 8-µm cryosections from 18 frozen specimens using Leica Laser Microdissection technology (Leica, Bensheim, Germany). From microdissected breast tumor cells, the DNA was extracted using the QIAamp kit (Qiagen).
DNA bisulfite modification. Sodium bisulfite conversion was done on 1 µg of tumor DNA per sample using previously described methods (10). Alkali-denatured DNA was incubated in 3 mol/L NaHSO3 and 0.5 mmol/L hydroquinone for 16 hours at 54°C. Modified DNA was purified using the Wizard DNA Clean-up System (Promega, Madison, WI) and eluted into 50 µL of sterile water. DNA was precipitated with 0.5 mol/L ammonium acetate (pH 4.6), 1.5 µL of 20 mg/mL glycogen, and ethanol and then resuspended in Tris-EDTA.
BRCA1 methylation. Methylation analysis was done using a methylation-specific PCR–based approach as previously described (11). Primer sequences of BRCA1 for the methylated reaction were 5'-TCGTGGTAACGGAAAAGCGC-3' (sense) and 5'-AACGAACTCACGCCGCGCAA-3' (antisense) and for the unmethylated reaction, 5'-TTGAGAGGTTGTTGTTTAGTGG-3' (sense) and 5'-AACAAACTCACACCACACAA-3' (antisense). The sense primer of the methylated reaction begins at 1,543 bp and the sense primer of the unmethylated reaction begins at 1,507 bp. The primers were predicted to give two PCR products with a 36-bp difference in size, each containing the transcription start site at 1,581 bp (GenBank BRCA1 sequence U37574).
RNA isolation and reverse transcription-PCR. Total cellular RNA was isolated from microdissected frozen tumor cells using the RNAeasy Midi kit (Qiagen) and from breast cancer cells lines using Trizol reagents (Invitrogen). Reverse transcriptase reactions were done with 2 µg of DNase-treated RNA and 1 µL of oligo(dT)12-18 as the reverse transcription primer using the SuperScript One-Step reverse transcription-PCR (RT-PCR) System and Platinum Taq DNA Polymerase kit (Invitrogen). cDNA primers were designed to amplify exon 20 of the BRCA1 gene: 5'-TGCTGAGTTTGTGTGTGAACGG-3' (sense) and 5'-CTGTGCCAAGGGTGAATGATG-3' (antisense). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a normalization control.
Fluorescence in situ hybridization. A BAC probe containing DNA sequences specific for the human BRCA1 gene on 17q12-q21 (283G2, P1 library dmpc-HFF-1; ref. 12) was indirectly labeled by nick translation with biotin-conjugated dUTP (Boehringer Mannheim, Indianapolis, IN) and was detected with FITC-conjugated avidin as a green fluorescent signal (13, 14). A commercial CEP17 probe is directly labeled with SpectrumOrange and contains 
-satellite DNA for hybridization to the centromeric region of chromosome 17 (17p11.1-q11.1; Vysis/Abbott Labs, Downers Grove, IL).
Metaphase cell preparations from cell lines and from normal peripheral blood lymphocytes were done according to routine protocols (13). Imprint touch preparations of fresh frozen breast tumors and two-color fluorescence in situ hybridization (FISH) procedure were as described previously (14). In each sample, hybridization signals from 50 to 100 nuclei were scored. The absolute number of signals and the ratio of BRCA1 signals to chromosome 17 centromere signals were recorded. Chromosome 17 aneusomy was estimated as previously described (7). The hybridization efficiency of the BRCA1 probe was confirmed by performing BRCA1/CEP17 FISH on normal lymphocytes as previously determined (7, 14, 15).
Demethylation of UACC3199 cell line. Cells were seeded at a density of 5 x 105 per 100-mm plate. The cells were allowed to attach for 48 hours. They were then exposed to 5-aza-2'-deoxycytidine (5-aza-dC) or trichostatin A (Sigma, St. Louis, MO) at 10, 50, or 100 ng/mL dose levels of each reagent or to the combination of 50 ng/mL 5-aza-dC and 50 ng/mL trichostatin A in triplicates. In parallel, untreated cells in triplicates were used as a control. After 48 hours of continuous exposure, the medium was changed. Cells were harvested and DNA was extracted after an additional 48 hours of incubation.
Sodium bisulfite genomic sequencing of the BRCA1 promoter in UACC3199 cells after demethylation. The functional BRCA1 promoter region of 658 bp, which includes 30 CpG dinucleotides, was sequenced for cytosine methylation using two rounds of PCR with nested primers specific to the bisulfite-modified sequence as previously determined (16). Purified PCR products were cloned into a TA vector according to the instructions of the manufacturer (TOPO TA Cloning kit for Sequencing, Invitrogen). Ten recombinants were isolated using a Qiaprep Spin Plasmid Miniprep kit (Qiagen) and sequenced on an ABI automated DNA sequencer. The methylation status of individual CpG sites was estimated by determining the number of bisulfite-converted cases that differed from corresponding wild-type cases. For each dinucleotide, the proportion of methylated clones to the total number of clones analyzed (n = 10 in each experiment) was calculated.
Statistical analysis. Summary statistics were computed for patient demographic and disease characteristics, expressed on a continuous scale, and compared between BRCA1-methylated and unmethylated tumors using the two-sample t test. For characteristics classified into discrete categories, frequency distributions by methylation status were compared using Fisher's exact test.
Mean numbers of BRCA1 and CEP17 signals per tumor for BRCA1-methylated and unmethylated cases were plotted, and for each tumor, the ratio of mean BRCA1 signals to CEP17 signals was computed. Mean copy numbers and mean ratios were compared between groups using the two-sample t test. For each tumor sample, proportions of cells exhibiting one, two, and three or more copies of BRCA1 and CEP17 signals were determined. Methylated and unmethylated tumor groups were compared for proportions of cells with aneusomy via t tests on arcsine transforms of these values, which are approximately normally distributed. For these tests and those described above, results of the t test and its nonparametric counterpart (Wilcoxon rank-sum test) were similar.
To evaluate the relationship between BRCA1 methylation status and gene copy number while accounting for association with other patient/tumor characteristics, a linear regression model was fit, with BRCA1 mean copy number as the response variable and covariates for methylation status and other patient/tumor characteristics associated with methylation status as the predictors. Similar adjusted comparisons were conducted for CEP17 copy number and mean BRCA1/CEP17 ratio.
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Results |
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BRCA1 methylation in primary breast tumors. Thirty-nine of 131 (29.8%) tumors were BRCA1 methylated. Prevalence of BRCA1 methylation was similar in paraffin-embedded samples (32.8%) and fresh frozen samples (26.9%; P = 0.57). Among methylated tumors, we observed significant heterogeneity because BRCA1 methylation was incomplete in the majority of cases. Both methylated and unmethylated products were present in 32 of 39 (82%) BRCA1-methylated tumors. Therefore, complete BRCA1 promoter methylation (only methylated product present) was detected in only 7 of 39 or in 5.4% (7 of 131) of the primary breast carcinomas by conventional methylation-specific PCR assay.
BRCA1 methylation and mRNA expression in microdissected tumors. To determine if the unmethylated product in cases with incomplete BRCA1 methylation is due to contamination by adjacent normal tissue or to incomplete methylation of one or both BRCA1 alleles, methylation-specific PCR was done on tumor cells isolated by microdissection in 18 randomly selected fresh frozen cases (Table 1; tumors 1-8 from Table 1 are also depicted in Fig. 1B). Four of six BRCA1-methylated cases with additional unmethylated product showed only a methylated product after microdissection of tumor tissue. In two methylated cases, unmethylated band remained even after microdissection (tumors 2 and 11), suggesting that incomplete methylation of one or both BRCA1 alleles is accountable for the methylation heterogeneity in these tumors (2 of 7, 30%). In all 18 cases, regardless of the DNA isolation method used, the methylation status of the BRCA1 promoter remained unchanged.
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Correlation of BRCA1/promoter methylation with clinicopathologic features of breast tumors. BRCA1 methylation was more frequently observed in tumors from premenopausal or perimenopausal women (Table 2). Of the 58 cases from women <55 years old, 40% were methylated whereas only 22% of the 67 cases from women >55 years old were methylated (P = 0.05). BRCA1-methylated tumors were also more likely to be of higher grade, with 70.0% of the methylated cases classified as grade 3, whereas only 33.8% of the unmethylated cases fall in this category (P = 0.001). A strong correlation was also found with estrogen receptor negativity (P = 0.04) and progesterone receptor negativity (P = 0.01), with both being more likely a feature of BRCA1-methylated tumors. Correlation was found between the methylation status of BRCA1 and tumor size, stage, histologic type, number of nodes involved, and patient's ethnicity, but did not reach statistical significance.
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BRCA1 copy number in primary breast tumors in relation to promoter methylation and mRNA expression. Imprint touch preparations for FISH were available from 50 of 67 frozen tissues [or 50 of 131 (38%); 37 unmethylated and 13 methylated] including the subset of 18 microdissected tumors. These cases were randomly selected but their clinical characteristics seemed to be representative of the entire cohort (data not shown). Figure 3A shows a plot of the mean number of BRCA1 copies per cell versus the mean number of CEP17 copies per cell, depicted for BRCA1-methylated and unmethylated tumors. The mean number of BRCA1 copies was smaller in methylated cases as compared with unmethylated cases (1.78 versus 2.30, P = 0.001). Similarly, the CEP17 mean copy number was smaller among methylated cases as compared with unmethylated cases (1.85 versus 2.29, P = 0.005). Because methylation was frequently associated with monosomy for chromosome 17 and loss of one copy of BRCA1, the overall BRCA1/CEP17 ratio did not differ between the groups (mean of 0.97 in methylated cases versus 1.01 in unmethylated cases, P = 0.24).
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Results of BRCA1/CEP17 FISH from 18 microdissected primary breast carcinomas are given in Table 1. The mean number of BRCA1 copies per cell among methylated tumors was significantly smaller that that among unmethylated tumors (1.71 versus 2.29, P = 0.02) and correlated with expression of BRCA1 transcripts. Three of four predominantly methylated tumors with no BRCA1 expression (tumors 8-10) had the smallest number of BRCA1 copies per cell, ranging from 1.15 to 1.60, and the highest proportion of cells with BRCA1 copy number loss, ranging between 43.16% and 84.71% (a representative photomicrograph of the BRCA1-methylated tumor 8 after FISH is shown in Fig. 2C; see also Table 1 and Fig. 1B and C). Conversely, the unmethylated tumors displayed the highest mean number of BRCA1 copies per cell (range of the means, 2.01-3.63) and normal mRNA expression (a representative photomicrograph of the unmethylated tumor after FISH is shown in Fig. 2D). Gene copy numbers from the methylated cases, either with incomplete methylation of BRCA1 promoter or reduced BRCA1 transcripts (tumors 1, 2, and 11), were intermediate between those of tumors that were heavily methylated/undetectable transcripts and unmethylated/normal transcripts. In three unmethylated cancers (tumors 4, 7, and 12) and one methylated case (tumor 6), there was no correlation between methylation-specific PCR, RT-PCR, and FISH results, supporting the suggestion of other investigators that mechanism(s) other than aberrant methylation can be involved in LOH and diminished expression of BRCA1 gene product (1, 21).
A similar pattern was seen with respect to chromosome 17 across all 18 cases in which chromosome 17 aneusomy was frequently associated with BRCA1 copy number alterations. However, in methylated tumor 10 (Table 1), the proportion of cells with reduction of BRCA1 to one copy (43.16%) was much higher than the proportion of cells monosomic for CEP17 (5.26%). The BRCA1/CEP17 ratio of 1:2 was recorded in 38% of tumor cells, suggesting that in this tumor the physical deletion of BRCA1 was submicroscopic and did not involve the loss of whole chromosome 17. Such event has been observed in hereditary BRCA1-associated tumors (15).
Thus, there was good correlation between gene copy number, degree of BRCA1 promoter methylation, and mRNA expression. In a multivariable model including estrogen receptor and progesterone receptor status, tumor grade, and patient age, methylation status remained a significant predictor of reduced BRCA1 and CEP17 copy number (P = 0.01). When patient and tumor characteristics were taken into account via inclusion in a statistical model predicting methylation status with the reduced BRCA1 and CEP17 copy number and other factors associated with methylation, a similar trend was observed.
Demethylation and reexpression of BRCA1 after treatment of UACC3199 cells with 5-aza-2'-deoxycytidine and trichostatin A. Methylation-specific PCR analysis of DNA extracted before and after exposure to 5-aza-dC and/or trichostatin A showed restoration of the unmethylated state of BRCA1 at all applied dose levels (Fig. 4A). The intensity of unmethylated band correlated with drug concentration. However, RT-PCR analysis revealed that only 50 and 100 ng/mL 5-aza-dC and 100 ng/mL trichostatin A or the combination of 50 ng/mL 5-aza-dC and 50 ng/mL trichostatin A was needed to induce the reexpression of BRCA1 transcripts (Fig. 4B). We found that the combination of 5-aza-dC and trichostatin A led to a higher degree of BRCA1 promoter demethylation and higher levels of mRNA reexpression than treatment with either inhibitor alone. Bisulfite DNA sequencing confirmed the partial demethylation of the BRCA1 promoter by these agents (Fig. 4C). After combined 5-aza-dC/trichostatin A drug treatment, the demethylation event of different degrees was observed in 13 of 30 (43%) CpG sites. In five of these sites (or 5 of 30, 17%) demethylation was significant. The percent methylation at CpG dinucleotides, located at positions –533, –355, –173, –21, and +44 from the transcription start site, was 90%, 50%, 90%, 40%, and 100% before treatment and decreased to 20%, 0%, 40%, 0%, and 20% after treatment, respectively.
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Discussion |
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We have previously shown that tumors from both BRCA1-mutated hereditary cancers and sporadic controls displayed chromosome 17 aneusomy and were often monosomic for chromosome 17 (7). In the present study, FISH analysis clearly showed significant differences in chromosome 17 copy number among methylated and unmethylated cases. The significant proportion of cells with chromosome 17 monosomy in the methylated tumors suggests that BRCA1 methylation might have resulted in loss of chromosome 17. The initial reduction in expression of BRCA1 could create subsequent genomic instability. It is likely that chromosome 17 aneusomy was specific and nonrandom because we had previously shown that BRCA1-methylated cells are more likely to have MYC gene copy number gain/amplification and chromosome 8 polysomy compared with unmethylated tumors (11). Thus, our previous and current data suggest that loss of BRCA1 in some sporadic breast cancers through epigenetic mechanism contributes to the development of those tumors and seems to precede and possibly promote specific chromosomal changes, such as chromosome 17 aneusomy and MYC amplification (7, 11).
HCC1937, a breast cancer cell line from a BRCA1 germ line mutation carrier, is polysomic for chromosome 17 and shows concomitant gain of copies of BRCA1 gene and yet has only mutated BRCA1 allele with the 5382insC mutation (17). This suggests that loss of the wild-type BRCA1 allele due to deletion of a whole chromosome 17 was an early somatic genetic event in these cells, followed by gain of the remaining mutant homologue by nondisjunction (15). In addition, our methylation-specific PCR analysis, showing that the retained mutant allele of BRCA1 remained unmethylated in HCC1937 cells, confirmed a previous observation that aberrant methylation of the CpG island rarely occurs when only a mutant BRCA1 allele is present in the tumor (4). However, if two alleles are present and one of them has a germ line mutation, the biallelic inactivation of the gene may be accomplished by promoter methylation of the retained wild-type allele (4, 23). The BRCA1-methylated cell line UACC3199 was disomic for chromosome 17 and yet the cells had a mean BRCA1 copy number of 4.09 and absent mRNA transcripts. Interestingly, a similar paradoxical copy number gain of a mutant BRCA1 allele relative to chromosome 17 has been observed in BRCA1-associated hereditary breast cancers (15). Extrapolating from data on HCC1937 and UACC3199 cells, we suggest that those primary tumors, which were detected in our study as being BRCA1 methylated, yet having two copies or gain of copies of BRCA1 (tumors 1-2, 6, and 11 from Table 1), accomplished biallelic inactivation of BRCA1 by promoter methylation of both alleles and without involvement of genetic deletions/LOH. Alternatively, the possibility of endoreduplication of one methylated allele in conjunction with loss of another allele cannot be ruled out at this time (24).
Esteller et al. (4, 18, 25) previously proposed the functional equivalency between the effect and significance of the epigenetic silencing of BRCA1 in sporadic breast cancer and genetic suppression of the gene in BRCA1 mutation carrier. The results of our methylation-specific PCR, RT-PCR, and FISH analyses indicate that BRCA1 methylation contributes to a subset of sporadic breast cancers with the resulting phenotype similar to that of hereditary BRCA1-mutated breast cancers. These data support a model of breast carcinogenesis in which BRCA1 promoter methylation may serve as a "first hit," much like an inherited germ line mutation, and the "second hit" results in reduced BRCA1 copy number and/or chromosome 17 aneusomy. Our study supports the hypothesis that BRCA1 promoter hypermethylation occurs early and, when complete, causes defects in chromosome structure, cell division, and viability. Thus, a BRCA1-deficient cell must acquire additional alterations that overcome these problems and force tumor progression down the same limited set of molecular pathways, which are characteristic of the progression of hereditary tumors (26, 27).
During the elaboration of our study, Staff et al. (28) reported FISH data on 60 primary tumors in which 45% were found to have a relative loss of BRCA1 copy number that was highly correlated with reduced mRNA expression and DNA aneuploidy. Consistent with our data, they also reported a strong association between reduced BRCA1 expression and progesterone receptor negativity. However, only 11% of their tumors were methylated, and the authors suggested alternative upstream mechanisms for down-regulating BRCA1 as they did not find an association between BRCA1 deletion and methylation. Other groups found that the rate of LOH at the BRCA1 locus may be higher than the percentage of BRCA1-methylated tumors or tumors with low mRNA or protein expression (25). However, loss of BRCA1 expression without LOH and allelic loss has also been reported (29). In our study, we were able to detect the correlation between mean BRCA1 and CEP17 copy numbers and methylation status because we took a different approach by recognizing the significance of methylation heterogeneity and we did not use an arbitrary cutoff point to classify tumors with deletions. Rather, we used increases and decreases in mean gene copy number of BRCA1 as being suggestive of chromosomal gains or losses, along with the calculation of the proportion of tumor cells with a given copy number of the gene and chromosome 17. Whereas Staff et al.'s (28) conclusion that haploinsufficiency at the BRCA1 locus may cause a gene dosage effect is conceivable, it is also possible that the methylation heterogeneity that we observed could explain the differences between our study and that of Staff et al. (28). Of course, both studies had limitations because of the number of tumors available for correlation analysis. Nevertheless, our findings are consistent with a recent FISH study of BRCA1 copy number in ovarian cancer (30) in which 25 of 47 cases (53%) were found to be "BRCA1 deletion positive" (i.e., having a high proportion of tumor cells with BRCA1/CEP17 copy number ratios of 1:2 and 1:1). Sixteen of these cases (34% of the total) actually had a very high proportion of cells with BRCA1 deletions. Whereas the methylation status of the tumors has not been analyzed, the patients with BRCA1 deletion–positive tumors were significantly younger and showed a trend toward poorer survival.
Our study has implications for clinical practice on the use of demethylating agents in cancer prevention or treatment of patients with estrogen receptor– and progesterone receptor–negative BRCA1-methylated sporadic breast cancers. Achieving BRCA1 demethylation and reexpression after treating BRCA1-methylated cells with 5-aza-dC and trichostatin A, we confirmed a reversible mechanism of BRCA1 silencing in tumor cells and a potential role for DNA methylation in the control of BRCA1 expression (31). The combinatorial effect of 5-aza-dC and trichostatin A on BRCA1 reactivation also confirmed the previously published observations on other cancer-related genes that the use of inhibitors of DNA methylation and histone deacetylation in combination may be an effective chemotherapy for breast cancer (32). When we treated BRCA1-methylated cancer cells with 5-aza-dC and trichostatin A for only 48 hours (33), we saw a dramatic induction of BRCA1 demethylation and reexpression levels despite the restricted number of CpGs that were affected. It is conceivable that those CpGs, at positions –533, –355, –173, –21, and +44 from the transcription start site, might be important in the transcriptional regulation of BRCA1. Indeed, a single CpG at position –355 was recently found to be one of four binding sites of BRCA1 DNA for specific protein 1 (31) and is also located within 60 bp of one of the two binding sites of CTCCC binding factor (34). Specific protein 1 and CTCCC binding factor may play a role in maintaining a methylation-free state of the functional BRCA1 promoter region, analyzed in our study, permitting transcription of the BRCA1 gene. Disruption of the binding of specific protein 1 and CTCCC binding factor to DNA may facilitate aberrant DNA methylation of the BRCA1 promoter, leading to genomic instability and development of sporadic breast cancer (34). In addition, methylation at positions –173 and –21 CpG sites may specifically inhibit binding of cAMP-responsive element binding protein and E2F transcription factors to BRCA1 DNA, respectively (24, 35). Thus, the understanding of molecular mechanisms underlying tumor initiation and progression in cells lacking BRCA1 is a critical step toward the development of more effective strategies for prevention, early detection, and treatment of aggressive breast cancer that disproportionately affects young women.
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Acknowledgments |
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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.
We thank Dr. Lucy Godley for critical reading of the manuscript and the Cell Culture Shared Resources for providing us with the UACC1399 breast cancer cell line established at the University of Arizona Cancer Center (Tucson, AZ).
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Footnotes |
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M. Wei is currently at Department of Pharmacology, China Medical University, Shenyang 110001, People's Republic of China.
Received 4/12/05. Revised 8/12/05. Accepted 9/ 1/05.
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genes in breast carcinoma cells. Cancer Chemother Pharmacol 2001;48:71–6.[CrossRef][Medline]This article has been cited by other articles:
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