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Haplo-insufficiency of BRCA1 in Sporadic Breast Cancer¹

Laboratory of Cancer Biology, Institute of Medical Technology [S. S., J. I., M. T.], and Department of Clinical Oncology, University Hospital of Tampere [M. T.], University Hospital of Tampere, Tampere, Finland

	ABSTRACT

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Copy number deletions and loss of heterozygosity of the BRCA1 gene have been frequently reported in sporadic breast cancer. We studied their relationship with BRCA1 gene expression (the haplo-insufficiency hypothesis) with real-time quantitative reverse transcription-PCR. Expression levels of both full-length and BRCA1-11b splice variant mRNA were studied, and they showed strong correlation (Pearson r = 0.89). Copy number deletion of BRCA1, found in 45% (27 of 60) of the sporadic breast tumors, was associated with ErbB2 oncogene amplification (P = 0.001) and DNA aneuploidy (P = 0.037), but not with stage, grade, or hormone receptor status. The presence of BRCA1 copy number deletion associated significantly with low levels of full-length BRCA1 mRNA (P < 0.0001). The BRCA1 promoter hypermethylation, found in 6 of 53 tumors (11%) by methylation-specific PCR, was also correlated with low BRCA1 expression (P = 0.005). In statistical multiple regression analysis, decreased expression of BRCA1 mRNA showed strongest association with BRCA1 copy number deletion (P < 0.0001) but was also significantly linked to negative progesterone receptor status (P = 0.02) and BRCA1 promoter hypermethylation (P = 0.041). These findings demonstrate that deletion of the BRCA1 gene copies results in haplo-insufficiency, i.e., decreased BRCA1 mRNA expression. This, in turn, suggests that the BRCA1 gene might have a tumor suppressor function also in sporadic breast cancer.

	INTRODUCTION

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Individuals carrying a germ-line mutation of the BRCA1 gene (1) are predisposed to early onset breast and ovarian cancer syndrome. A multitude of different germ-line mutations have been identified, a majority of which leads to a premature stop codon in the transcript. BRCA1 gene encodes a large multifunctional protein, which is likely to be involved in genomic integrity of the cells (2) . This indicates a tumor suppressor function, the loss of which is explained to cause BRCA1-linked hereditary breast cancer. Hereditary BRCA1 breast tumors have high rate of LOH³ at the BRCA1 locus accounting mostly for biallelic inactivation of BRCA1 (3 , 4) . In contrast, promoter hypermethylation seems to be a rare cause of inactivation of the wild-type BRCA1 in familial BRCA1 breast tumors (4) .

The tumor suppressor function of BRCA1 has remained unclear in the development of sporadic breast cancer, because almost no somatic mutations have been found (5) . Nevertheless, somatic genetic "hits," such as physical deletions and LOH, occur frequently (in 30–50%) in sporadic breast tumors (6, 7, 8, 9, 10, 11) . Decreased BRCA1 mRNA and protein levels have also been described in tumors versus normal breast epithelium (12, 13, 14, 15) . Decreased expression of BRCA1 has recently been associated with epigenetic regulation via its promoter hypermethylation in a small proportion of sporadic breast tumors (16, 17, 18, 19) .

The frequent allelic loss of BRCA1 in the absence of somatic mutations suggests that LOH might promote tumorigenesis as such via decreased BRCA1 gene expression. This concept, denoted as the haplo-insufficiency theory (20) , has empirical support from recent studies of the tumor suppressor genes APC and NF1 (21 , 22) . In the present study, we tested the haplo-insufficiency hypothesis with regard to BRCA1 gene in sporadic breast cancer. We analyzed primary sporadic breast cancers for BRCA1 deletion by FISH and quantified the expression of full-length BRCA1 mRNA as well as BRCA1-11b-isoform mRNA (23) using quantitative real-time RT-PCR system (Light Cycler). The impact of epigenetic regulation, the BRCA1 promoter hypermethylation, was also studied.

	MATERIALS AND METHODS

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Tumor Samples.
Freshly frozen tumor specimens were available from altogether 62 primary sporadic breast carcinomas and obtained from the Tampere University Hospital (Tampere, Finland) and Tampere City Hospital (Tampere, Finland). The mean age at diagnosis of these breast cancer patients was 62 years, and the median age was 65 years. All but two of the tumors were invasive ductal carcinomas. Seventeen percent of the invasive ductal carcinomas were grade I, 45% were grade II, and 38% were grade III. Forty-eight percent of the tumors had sent nodal or distant metastasis at the time of diagnosis. The frequency of BRCA1 mutations among unselected Finnish breast cancer patients has been reported to be very low, 0.39% (24) . Because none of the patients of our study had strong familial history for breast and ovarian cancer, it is likely that there are no carriers of a germ-line BRCA1 mutation in our material. A hereditary breast cancer control was derived from a mouse xenograft model of BRCA1 mutation-associated breast cancer⁴ (25) .

FISH.
A PAC probe specific for the BRCA1 gene (PAC 103014) and pericentromeric probe for the chromosome 17 (p17H8) was labeled with digoxigenin-dUTP (Roche Diagnostics) and FITC-dUTP (NEN, Boston, MA), respectively, by nick translation (26) . Two-color FISH procedure has been described previously (26) . Hybridization signals from 50 to 100 nuclei were scored to assess the copy number of BRCA1. Deletion was defined as an average ratio 0.8 of BRCA1 signals relative to chromosome 17 centromere signals or as monosomy of chromosome 17.

The same set of sporadic tumors was also analyzed for the ErbB2 oncogene amplification using the same method but commercially available ErbB2 probe (27) .

The ploidy status was determined by DNA flow cytometry (28) and the hormone receptors and histopathology using standard procedures.

DNA Methylation.
DNA methylation in the CpG islands of the BRCA1 promoter region was determined by methylation-specific PCR (29) . The primer sequences have been described previously (18) . The product of the unmethylated PCR reaction is 86-bp long, and the product of the methylated reaction is 75 bp. Universally methylated human male DNA (Intergen Co.) was used as a positive control, and DNA from normal lymphocytes was used as a negative control for methylation. Bisulfite modification of DNA was performed using CpG Genome Modification Kit (Intergen Co.), according to the manufacturer’s instructions. The PCR conditions consisted of 5 min of initial denaturation at 95°C, 35 cycles of denaturation at 95°C for 30 s, annealing for 30 s at 62°C (unmethylated reaction), 40 s at 65°C (methylated reaction), and elongation at 72°C for 30–45 s. Ten microliters of each PCR reaction product were loaded onto NuSieve GTG 4% agarose gels (BMA, Rockland, ME) and visualized under UV illumination.

Quantitative Real-time RT-PCR.
Total RNA from the frozen sporadic primary tumors and the xenograft tumor was isolated using the Sigma GenElute Mammalian Total RNA Kit (Sigma-Genosys, United Kingdom) according to the manufacturer’s instructions. The RNA samples were used for the first-strand cDNA synthesis with Superscript II reverse transcriptase and random hexamer primer (Invitrogen, Life Technologies, Inc.). To prepare the standard curve for the real-time semiquantitative RT-PCR analyses (Light Cycler), RNA from the breast cancer cell line HBL-100 was extracted, reverse transcribed, and used in serial dilutions corresponding to the cDNA transcribed from 750, 150, 30, 6, and 1.2 ng of the total RNA.

Primers were designed for both the full-length BRCA1 (mRNA with intact exon 11) and BRCA1-11b splice variant isoform. To avoid amplification of genomic DNA, primers were designed to amplify several exons or cover exon boundaries. In addition, primers were tested negative on samples of the first strand cDNA synthesis without adding reverse transcriptase. Because the expression of BRCA1 gene is known to be cell cycle dependent (30) , we used the similarly cell cycle-dependent cyclin B1 mRNA (31) to adjust for the variation in the tumor proliferation rate. We preferred cyclin B1 instead of the housekeeping gene TBP as the reference, because the former could be used to adjust both for the differences in the sample RNA concentration and differences in the tumor proliferation rate. The primer and probe sequences are shown in Table 1 . For the two different BRCA1 isoforms (full-length, i.e. with intact exon 11 and 11b), the exonic location of the primers is indicated in parentheses after the primer sequences in Table 1 , and the schematic presentation of the reactions is shown in Fig. 1 .

View this table: [in this window] [in a new window]   Table 1 The real-time RT-PCR primer and probe sequences for the BRCA1, BRCA1-11b, and Cyclin B1 genes The location of BRCA1 primers within the BRCA1 gene sequence (the number of exon) is shown in parentheses.

View larger version (10K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. The structure of the detected BRCA1 mRNAs and location of the cDNA primers used to amplify them. The primers are shown by arrows. The hybridization probes used to detect the variants in the real-time quantitative RT-PCR assay are shown as gray and open boxes.

Thermocycling for each reaction was done in a final volume of 20 µl containing 4 µl of cDNA sample (diluted 1:4 from the original first-strand synthesis reaction) or standard, 2.5 mM MgCl₂, 0.5 µM each primer, 0.2 µM fluorescein probe, and 0.4 µM LC Red 640- or 705-labeled probes, and finally 1x ready-to-use reaction mix containing TaqDNA polymerase, reaction buffer, and deoxynucleotide triphosphate mix. After 10 min of initial denaturation at 95°C, the cycling conditions of 45 cycles consisted of denaturation at 95°C for 10 s, annealing for 5 s at 55°C (BRCA1 11b-isoform), 56°C (full-length BRCA1), and 58°C (Cyclin B1) and elongation at 72°C for 9 s (Cyclin B1 and BRCA1 11b-isoform) and 12 s (full-length BRCA1).

The Light Cycler measured the fluorescence of each sample in every cycle at the end of the annealing step. After proportional background adjustment, the fit point method was used to determine the cycle in which the log-linear signal was distinguished from the background, and that cycle number was used as the crossing point value. The software produced the standard curve by measuring the crossing point of each standard and plotting them against the logarithmic values of concentrations.

Statistical Analyses.
The association of BRCA1 gene copy number status or promoter hypermethylation with BRCA1 expression was studied using t test. Multiple linear regression analysis was used to study the relation of covariates to BRCA1 mRNA expression levels. Expression variables with grossly asymmetric distribution were log transformed before statistical analyses. The association of BRCA1 deletion with ErbB2 gene copy number status, BRCA1 promoter hypermethylation, or clinicopathological characteristics was analyzed using Fisher’s exact test.

	RESULTS

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Deletion of BRCA1 in Sporadic Tumors.
Sixty primary sporadic breast cancer tumors were studied for BRCA1 gene copy number by FISH. Twenty-seven of the 60 tumors analyzed (45%) showed physical deletion of the BRCA1 gene (Fig. 2, A–C) . Deletion was present in 13 chromosomally aneuploid tumors as a copy number ratio of 4:2 (four copies of 17 centromere; 2 copies of BRCA1) and in 10 diploid tumors as the presence of one BRCA1 gene allele or in 4 tumors as monosomy of chromosome 17. Thirty-three tumors (55%) didn’t show any relative BRCA1 gene copy number change (equal number of BRCA1 and chromosome 17 centromere signals; Fig. 2, D–F ). Tumors with no relative change in BRCA1 gene copy number were diploid, triploid, or tetraploid.

View larger version (104K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Two-color FISH analysis of BRCA1 (red fluorescent signals) and chromosome 17 centromere (green fluorescent signals) in sporadic breast cancer tumors. Tumor nuclei were counterstained with 4',6-diamidino-2-phenylindole (blue). Physical deletion of BRCA1 gene (loss of BRCA1) was detected by FISH as 4:2 BRCA1 deletions (A), as diploid tumors with only one BRCA1 gene allele (B) or as monosomy of chromosome 17 (C). Sporadic breast cancer tumors with no relative BRCA1 gene copy number change (equal number of BRCA1 and chromosome 17 centromere signals) were diploid (D), triploid (E), or tetraploid (F).

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Example of methylation-specific PCR (MSP) analysis of promoter region of the BRCA1 gene in primary sporadic breast cancer tumors. The presence of a PCR product in the lanes marked by U indicates the presence of unmethylated BRCA1 gene alleles, and the presence of a PCR product in the M lanes indicates the presence of methylated BRCA1 gene alleles. Universally methylated human male DNA was used as a positive control for methylation, and normal human lymphocyte DNA was used as a negative control for methylation. Sporadic tumor sample 1 (Lanes 5 and 6) provides an example of hypomethylated BRCA1 promoter region, whereas sporadic tumor sample 2 (Lanes 7 and 8) is an example of a tumor with BRCA1 promoter hypermethylation.

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. A, relative expression levels of full-length BRCA1 mRNA analyzed by real-time RT-PCR in primary sporadic breast cancer tumors with (deletion +) or without deletion (deletion -) of the BRCA1 gene and in a control sample of a xenograft model of BRCA1 mutation-induced breast cancer (four parallel samples assayed in the same run). In deletion + group, tumors with only one copy of BRCA1 are shown by black dots (), and tumors with two BRCA1 copies are shown by open dots (). B, relative expression levels of full-length BRCA1 mRNA analyzed by real-time RT-PCR in primary sporadic breast cancer tumors with (methylation +) or without (methylation -) BRCA1 promoter hypermethylation. The median value of expression is shown by a horizontal line. In methylation + group, tumors of double BRCA1 inactivation (deletion with hypermethylation) are marked as black dots (). Two of these tumors showed 4:2 and one 2:1 BRCA1 deletion.

A statistical multivariate analysis was done to study whether BRCA1 deletion affects mRNA expression independently or via its association with other variables. In multiple regression analysis, decreased levels of BRCA1 expression showed strongest association with BRCA1 deletion (P < 0.0001), followed by negative PgR status (P = 0.020) and BRCA1 promoter hypermethylation (P = 0.041; Table 3 ). The variation in BRCA1 expression was not dependent on histological grade or ER status (Table 3) . R square value was 0.455, indicating that the regression model explains 45% of the variation in expression levels of BRCA1 mRNA (Table 3) .

Expression of BRCA1-11b Splice Variant mRNA.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Correlation of relative expression levels of full-length BRCA1 and its 11b splice variant mRNA in primary sporadic breast cancer tumors (Pearson’s correlation coefficient r = 0.89). Tumors with both deletion and hypermethylation are shown by black dots (), tumors with deletion alone by spotted dots ( ), tumors with hypermethylation alone by dots with stripes ( ), and tumors with no genetic or epigenetic changes by open dots ().

	DISCUSSION

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Here, we found a statistically highly significant association between BRCA1 deletion (loss of gene copies) and low levels of BRCA1 mRNA. We have shown previously that in sporadic breast tumors, LOH of BRCA1 results from physical deletion of the BRCA1 gene, which can be detected by FISH on clinical tumor samples (26) . BRCA1 deletions were mostly of gene copy ratio 4:2 (four copies of chromosome 17 centromere and two copies of BRCA1) and less frequently of ratio 2:1 (two copies of chromosome 17 and one copy of BRCA1; Ref. 26 ). The 4:2 deletion most likely results from endoreduplication of the entire genome, which is known to occur at late stage during breast cancer development (35) . In the present study, tumors with 2:1 or 1:1 deletion of the BRCA1 gene showed similar levels of full-length BRCA1 mRNA when compared with tumors with BRCA1 deletion of copy number ratio 4:2. Thus, the relative allelic loss as such seems to be more important than the copy number in the regulation of BRCA1 expression. In statistical multivariate analysis, the association between BRCA1 deletion and low levels of BRCA1 mRNA was independent of the impact of histological grade, hormone receptor status, or BRCA1 promoter hypermethylation. Thus, these findings provide direct empirical evidence for haplo-insufficiency of BRCA1 gene in sporadic breast cancer. Our results are in line with those made using conventional RT-PCR (14 , 33) , which, however, do not provide fully quantitative evidence of decreased BRCA1 expression. Studies of BRCA1 protein expression have failed to report an association with LOH (10 , 36) . This might reflect the lack of true association at protein level but also the reported technical difficulties of the BRCA1 immunohistochemistry (37) . Here, we used quantitative real-time RT-PCR, which allows quantifying the amount of mRNA based on determining the PCR cycle during which the reaction enters the exponential phase. Specificity of the method is improved by the use of two labeled detector probes in addition to two primers. Therefore, to produce a specific signal, four different oligonucleotides have to anneal to the target sequence.

Quantitative real-time RT-PCR also made it possible to detect BRCA1 splice variants (Fig. 1) , the expression and significance of which has remained unclear. According to our results, the expression levels of full-length mRNA (i.e., with intact exon 11) and 11b splice variant were strongly correlated, suggesting that they might be similarly regulated. The protein product of 11b splice variant has recently been shown to contain an NH₂-terminal nuclear localization sequence that enables it to enter the cell nucleus and form DNA damage-inducible foci almost identical to full-length BRCA1 (38 , 39) . The strong correlation between full-length and 11b isoforms suggests that both forms might be functionally important in sporadic breast cancer.

As shown previously (18 , 19) , BRCA1 promoter hypermethylation was also associated with low BRCA1 mRNA expression. The association was statistically highly significant, despite the fact that methylation was found only in a minority of tumors (11%). Thus, epigenetic regulation (promoter hypermethylation) is likely to play a role in the regulation of BRCA1 expression too, although only in a small proportion of tumors. There was no association between BRCA1 deletion and promoter hypermethylation, indicating that these two mechanisms are independent of each other. However, double inactivation (both deletion and hypermethylation in the tumor) associated significantly with lower levels of full-length BRCA1 mRNA expression compared with tumors with either deletion or hypermethylation. In multiple regression analysis, BRCA1 deletion was the strongest determinant of low BRCA1 expression, followed by negative PgR status and promoter hypermethylation. Thus, genetic and epigenetic mechanisms were found not only to take place in different tumors but also to complement each other in the down-regulation of BRCA1 expression. In biostatistical terms, the multiple regression model, including BRCA1 deletion, promoter hypermethylation, and PgR as significant determinants, explained 45% of the variation in BRCA1 mRNA expression. Thus, it is likely that other mechanisms, such as upstream regulatory proteins, could affect the transcription of the BRCA1 gene in sporadic breast cancers where deletions or promoter hypermethylation are absent and do not explain the variation in BRCA1 expression.

The functional consequences of allelic loss of BRCA1 have also been studied indirectly by correlating the phenomenon with clinical parameters. Allelic loss has been associated previously with high histological grade and hormone receptor negativity (6 , 7) , both recognized as markers of aggressive tumor type. We found that BRCA1 deletions associated with amplification of the ErbB2 oncogene and aneuploidy, which too are markers of an aggressive breast cancer. An association between high ErbB2 and low BRCA1 expression has been reported previously (34) , and, based on our findings, it seems to be attributable to genetic rearrangements of these genes.

In conclusion, our results indicate that loss of BRCA1 gene is common and associates with decreased expression of both full-length BRCA1 and 11b mRNA in sporadic breast cancer. Our results support the haplo-insufficiency of BRCA1 in sporadic breast cancer. The haplo-insufficiency, in turn, suggests that BRCA1 gene might have a tumor suppressor function in sporadic breast cancer.

	ACKNOWLEDGMENTS

 
We thank Päivi Järvinen and Sari Toivola for their excellent technical assistance.

	FOOTNOTES

 
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.

¹ Supported by the Finnish Cancer Society, Finnish Breast Cancer Group, Satakunta Cultural Foundation, Finnish Medical Association, and Medical Research Fund of Tampere University Hospital.

² To whom requests for reprints should be addressed, at Laboratory of Cancer Biology, Institute of Medical Technology, Lenkkeilijänkatu 6, 33014 University of Tampere, Finland. Phone: 358-3-215 6729; Fax: 358-3-215 8923; E-mail: jorma.isola{at}uta.fi

³ The abbreviations used are: ER, estrogen receptor; LOH, loss of heterozygosity; FISH, fluorescence in situ hybridization; PgR progesterone receptor; RT-PCR, reverse transcription-PCR.

⁴ The xenograft specimen was a gift from Professor Åke Borg, University of Lund, Lund, Sweden.

Received 1/ 7/03. Revised 5/ 4/03. Accepted 6/10/03.

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