Japanese Journal of Clinical Oncology 32:79-84 (2002)
© 2002 Foundation for Promotion of Cancer Research
Promoter Hypermethylation and Post-transcriptional Mechanisms for Reduced BRCA1 Immunoreactivity in Sporadic Human Breast Cancers
1Carcinogenesis and 4Pathology Division, National Cancer Center Research Institute, Tokyo, 2Second Department of Surgery, Hiroshima University School of Medicine, Hiroshima and 3Department of Surgery, National Cancer Center Hospital, Tokyo, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Background: Germline mutation of BRCA1 is well known to cause familial breast cancer. Although somatic mutations of the BRCA1 gene are rare in sporadic breast cancers, a high incidence of reduced BRCA1 immunoreactivity has been demonstrated. As one of the mechanisms for this, gene silencing by hypermethylation of the BRCA1 promoter region has been reported. Here, we show the presence of a post-transcriptional mechanism by examining promoter hypermethylation, mRNA expression levels and immunoreactivity of BRCA1 in sporadic human breast cancers
Methods: Paired samples of 20 invasive ductal carcinomas and one invasive lobular carcinoma were obtained from sporadic breast cancer cases. The BRCA1 protein expression levels were determined by immunohistochemistry using a well-characterized antibody. The methylation status of the BRCA1 promoter region was determined by sequencing after bisulfite modification. The mRNA expression levels were determined by semi-quantitative reverse transcription polymerase chain reaction (PCR). Mutations in the entire BRCA1 coding region were analyzed by PCR single-strand conformation polymorphism analysis.
Results: Reduced immunoreactivity was observed in 13 of the 21 cancers. Hypermethylation was observed in five of the 13 cancers with reduced immunoreactivity and mRNA expression was almost absent in these five cancers. In the remaining eight cancers, mRNA expression was not decreased. None of the 21 cancers examined harbored BRCA1 mutations.
Conclusion: These findings showed that post-transcriptional mechanisms, such as low efficiency of translation or reduced stability of BRCA1 protein, are also involved in reduced BRCA1 immunoreactivity in sporadic breast cancers.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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The BRCA1 gene, located on chromosome 17q21, was originally cloned as the gene responsible for familial breast cancers (1). Its germline mutations are observed in 16–61% of familial breast cancers of Caucasians (2–5), 43–47% of those in Ashkenazi Jews (6–8) and 4–14% of those in the Japanese (9–11). It is known that somatic mutations of BRCA1 are rare in sporadic breast cancers (12). However, the minimal common region for loss of heterozygosity (LOH) in 17q21 in sporadic breast cancers falls into the BRCA1 region (13) and decreased mRNA expression (14,15) and reduced immunoreactivity (16,17) are observed in sporadic breast cancers. From these findings, it is now considered that reduction of BRCA1 protein is involved in sporadic breast carcinogenesis, not only in familial breast cancers.
Reduced BRCA1 immunoreactivity is observed in 82% of histologically high-grade sporadic breast carcinomas (16). As for a mechanism for the reduced immunoreactivity, aberrant hypermethylation of the BRCA1 promoter region has been implicated. The BRCA1 gene has two alternative exons, 1A and 1B (18), and each of them has its own promoter, 
and ß (19). In adult mammary cells, it is known that exon 1A is mainly transcribed from the promoter (18). Hypermethylation of the promoter has been observed in 0–33% of sporadic breast cancers by Southern blot analysis using a methylation-sensitive restriction enzyme (20,21) or by sequencing after bisulfite modification (22,23). However, the reported incidences of hypermethylation in the range 0–33% are lower than those reported for reduced BRCA1 immunoreactivity. In a report where both the methylation status of the BRCA1 promoter region and the immunoreactivity of the BRCA1 protein were examined (24), the reduction of BRCA1 immunoreactivity was observed in seven of 22 breast cancers that did not have promoter hypermethylation.
Although mRNA expression levels will be key information to identify the mechanism of reduced immunoreactivity without promoter hypermethylation, there are no previous reports in which the methylation status of the promoter region, mRNA levels and immunoreactivity were examined in the same breast cancer samples. In this study, we analyzed 21 sporadic breast cancers for hypermethylation of BRCA1 promoter by sequencing after bisulfite modification, mRNA expression levels by reverse transcription polymerase chain reaction (RT-PCR) and immunoreactivity of BRCA1 protein by immunohistochemistry using a well-established antibody. In addition, somatic and germline mutations of BRCA1 were analyzed by PCR single-strand conformation polymorphism (SSCP) analysis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Tumor Samples and DNA/RNA Extraction
Twenty-one pairs of breast cancer and surrounding non-cancerous tissue were obtained with informed consent from patients undergoing mastectomy due to breast cancers at the National Cancer Center Hospital. As a tumor specimen, 3–5 mm3 of macroscopic tumor was isolated and frozen in liquid nitrogen. Adjacent tumor tissue underwent microscopic examination, was classified and graded according to the criteria by Elston and Ellis (25) and presence of tumor cells at 70% or more was confirmed. The 21 cancers comprised 20 invasive ductal carcinomas and one invasive lobular carcinoma (Table 1). As a non-cancerous specimen, 5–10 mm3 of macroscopic non-cancerous specimens were isolated and frozen in liquid nitrogen. Adjacent tissue underwent microscopic examination and absence of cancer cells was confirmed. DNA was extracted by serial extraction with phenol and chloroform and ethanol precipitation (26) and total RNA was isolated using Isogen (Nippon Gene, Toyama, Japan).
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Immunohistochemistry
BRCA-1 Ab-1 antibody, a mouse monoclonal antibody to the N-terminus of BRCA1 protein (Oncogene Research Products, Cambridge, MA), was used to examine BRCA1 immunoreactivity. Formalin-fixed, paraffin-embedded sections were sliced at 5 µm thickness, deparaffinized and heated in 10 mM citrate buffer (pH 6.0) for 25 min in a microwave oven. After blocking endogenous peroxidase activity and non-specific binding, the sections were incubated with the antibody at a dilution of 150-fold at 4°C overnight. The binding of the first antibody was detected by a specific second antibody and the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Slides were counterstained with Mayer’s hematoxylin.
Positive staining of normal breast epithelial cells that co-existed on the same section was confirmed. The intensities of the staining in the cancer tissues were diagnosed in three grades. When the immunoreactivity was comparable to that of the normal breast epithelium or nuclear staining was observed in >50% of cancer cells, it was diagnosed as ‘positive’. When the staining was clearly weaker than normal surrounding cells or nuclear staining occurred in 10–50% of cancer cells, it was diagnosed as ‘moderately reduced’. When there was no staining or nuclear staining occurred in <10% of cancer cells, it was diagnosed as ‘markedly reduced’.
Sodium Bisulfite Modification and Sequencing
Sodium bisulfite modification was performed according to previous reports (27,28). Briefly, 3 µg of genomic DNA were digested with EcoRI or BamHI restriction enzyme. After purification by phenol extraction and ethanol precipitation, 1 µg of DNA was denatured in 0.6 M NaOH at 37°C for 15 min. To the denatured DNA, NaHSO3 (pH 5.3) and hydroquinone were added at final concentrations of 3.1 and 0.5 mM, respectively, and the sample was incubated at 55°C for 16 h. The sample was desalted with the Wizard DNA cleanup system (Promega, Madison, WI) and desulfonated by treatment with NaOH at a final concentration of 1 M. The DNA sample was ethanol-precipitated with ammonium acetate and dissolved in 10 µl of TE buffer.
The promoter, from nt. –79 to nt. 72 (GenBank accession U37574), was amplified using 2 µl of the sodium-bisulfite-treated DNA by PCR with primers, 5'-GTATTTTGAGAGGTTGTTGTTTAG-3' and 5'-ATCTAAAAAACCCCACAACC-3'. The PCR conditions were annealing at 56.5°C and Mg2+ concentration 1.5 mM. The PCR product was cloned into pGEM-T-Easy TA (Promega). Ten insert-positive clones were sequenced by the cycle sequencing method using the primers used for the initial PCR and run in an Applied Biosystems 310 sequencer. Complete conversion of cytosines in non-CpG sites to thymines was confirmed.
RT-PCR Analysis
A 3 µg amount of total RNA was treated with DNase I (Life Technologies, Rockville, MD) and reverse transcribed with oligo-dT primer (Promega) and Superscript II reverse transcriptase (Life Technologies). The primer sequences used were as follows: (BRCA1)-F, 5'-TCAACTGGAATGGATGGTACAG-3' (sense); (BRCA1)-R, 5'-ACAGGTGCCTCACACATCTG-3' (antisense); glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-F, 5'-CGG AGT CAA CGG ATT TGG TCG TAT-3' (sense); (GAPDH)-R, 5'-AGC CTT CTC CAT GGT GGT GAA GAC-3' (antisense). PCR conditions were annealing at 58°C, Mg2+ concentration 1.5 mM and 31 cycles for BRCA1, and annealing at 58°C, Mg2+ concentration 1.5 mM and 23 cycles for GAPDH. The amounts of the first-strand cDNA (~50 ng) that should give equal amounts of GAPDH products were adjusted by the intensities of bands in an ethidium bromide-stained 2% NuSieve (BMA, Rockland, ME) gel. PCR for BRCA1 was performed using the cycle numbers that gave a linear increase of the products. All reactions were performed with RT-free controls.
Analysis of BRCA1 Mutation
Exons 2–24 of BRCA1 were analyzed using 26 pairs of primers which covered the BRCA1 coding regions and exon–intron splicing boundaries and PCR-SSCP analysis was performed as described (29).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Reduced Immunoreactivity of BRCA1 in Sporadic Breast Cancers
Immunohistochemical analysis was performed on 21 sporadic breast cancers using the antibody, Ab-1, which had been demonstrated to give the strongest signal with the least amount of background signal in formalin-fixed, paraffin-embedded samples (16). Marked reduction of BRCA1 immunoreactivity was observed in 13 cancers and moderate reduction was observed in six cancers (Fig. 1, Table 1). When the cancers were classified by the histological grades, marked reduction of immunoreactivity was observed in 11 of 14 cancers with grade 3 and in one of six cancers with grade 2 (Table 2).
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Hypermethylation of BRCA1 Promoter Region
The methylation status of the promoter
was analyzed in the 21 pairs of breast cancer and surrounding non-cancerous tissue. In four cases (cases 1, 6, 8 and 14), 70–80% of DNA molecules were methylated in most of the CpG sites in the region and in case 7, 20–40% of DNA molecules were methylated (Fig. 2). Considering that normal cells were contaminated in cancer samples at populations ranging from 20 to 40%, these methylation statuses might indicate methylation of both alleles or one allele in cancer cells. In the other 16 cases, methylation of the promoter region was almost absent (Table 1).
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Analysis of BRCA1 mRNA Expression
mRNA expression levels were analyzed in the 21 pairs by the semi-quantitative RT-PCR method. In the five carcinomas with promoter hypermethylation (cases 1, 6, 7, 8 and 14), mRNA expression was almost absent, while mRNA was expressed in the other carcinomas without hypermethylation (Fig. 3, Table 1).
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Analysis of BRCA1 Mutation
No mutations in the BRCA1 coding region or in the splice acceptor and donor sites of introns 2–23 were observed in the sporadic breast cancers and normal surrounding tissues.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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In this study, reduced immunoreactivity of BRCA1 protein was observed in sporadic breast cancers, using the Ab-1 antibody that had been demonstrated as most reliable after an extensive analysis of 19 kinds of antibody (16). The immunoreactivity of BRCA1 was markedly reduced in 13 of the 21 cases (62%) and was moderately reduced in six of the 21 cases (29%). Of the 13 cases with marked reduction of BRCA1 immunoreactivity, five cases were with the hypermethylation of the
promoter. As expected from previous studies (20–23), mRNA expression was almost absent in these cases. The remaining eight cases were without promoter hypermethylation and mRNA expression levels were not decreased. None of the cancers analyzed in this study harbored BRCA1 mutations. These findings indicated that some post-transcriptional mechanism(s), such as reduced efficiency of translation or reduced stability of BRCA1 protein, are involved in the reduction of BRCA1 immunoreactivity in these sporadic cases. Recently, degradation by a cathepsin-like protease, in fine balance with mRNA transcription, was shown to be responsible for maintaining a steady level of BRCA1 protein (30). There is a possibility that an increase in protease activity is involved in the reduction of BRCA1 immunoreactivity in sporadic breast cancers. The reduction of BRCA1 immunoreactivity was more prominent in high-grade invasive breast cancers, which was consistent with a previous report (16). Hypermethylation was also observed more frequently in high-grade breast cancers, which was also consistent with previous reports (24,31). Although in this study we did not use quantitative RT-PCR methods, such as Taq-Man or real-time PCR, the numbers of PCR cycles were kept to a minimum for both GAPDH and BRCA1 and the results can be regarded as semi-quantitative.
These results showed that reduction of BRCA1 immunoreactivity is observed in a majority of sporadic human breast cancers and suggested that the reduction is caused by a post-transcriptional mechanism(s), in addition to promoter hypermethylation.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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We thank Dr Eriko Okochi for critical discussions and Ms Shoko Oishi for technical assistance. This study was supported by a Grant-in-Aid for the 2nd term 10-year Comprehensive Strategy for Cancer Control from the Ministry of Health, Labor and Welfare and a Grant-in-Aid from the Ministry of Education, Science, Culture and Sports.
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FOOTNOTES |
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+ For reprints and all correspondence: Toshikazu Ushijima, Carcinogenesis Division, National Cancer Center Research Institute, 1–1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. E-mail: tushijim@ncc.go.jp
Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; SSCP, single-strand conformation polymorphism; LOH, loss of heterozygosity; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma
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Received September 13, 2001; accepted December 11, 2001.
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