Tumour-specific distribution of BRCA1 promoter region methylation supports a pathogenetic role in breast and ovarian cancer

Tina Bianco1,2, Georgia Chenevix-Trench5, David C.A. Walsh3, John E. Cooper4 and Alexander Dobrovic1,2,6

1 Department of Haematology–Oncology,
2 University of Adelaide Department of Medicine,
3 University of Adelaide Department of Surgery and
4 Department of Histopathology, The Queen Elizabeth Hospital, Woodville, SA 5011 and
5 The Queensland Institute of Medical Research and The University of Queensland, Royal Brisbane Hospital, Herston, QLD 4029, Australia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The role of BRCA1 in sporadic breast and ovarian cancers remains elusive. Direct involvement of BRCA1 in the development of breast and ovarian cancer is suggested by the finding that the BRCA1 promoter region CpG island is methylated in a proportion of breast and ovarian cancers. The aim of this study was to compare the incidence of BRCA1 promoter region methylation in tumours in which loss of BRCA1 has been shown to play a role in pathogenesis (breast and ovarian carcinomas) with the incidence in tumours in which BRCA1 is unlikely to play a role in pathogenesis. Promoter region hypermethylation was significantly more common (P < 0.008) in breast and ovarian cancer (6/38 tumours methylated) than in colon cancer (0/35 tumours methylated) or in leukaemias (0/19 samples methylated). The restriction of BRCA1 promoter region hypermethylation to breast and ovarian cancer is consistent with a pathogenetic role of BRCA1 promoter methylation in these tumours. We suggest that the rarity of observed BRCA1 mutations in sporadic breast and ovarian cancer is due to the greater likelihood of BRCA1 inactivation by non-mutational mechanisms such as methylation.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In normal somatic cells, most CpG dinucleotides are methylated but those in CpG islands associated with promoter regions are generally unmethylated. CpG islands are associated with the promoter regions of many genes (1,2) and their methylation results in the gene becoming silenced (35). In tumours, there is a shift in the distribution of methylation with an overall decrease in methylation (6) but with de novo methylation of some CpG islands (79).

De novo promoter region methylation is a mechanism that can lead to the inactivation of tumour suppressor genes. It was first reported for the RB gene in which hypermethylation of the promoter region CpG island was found in some sporadic retinoblastomas (1013). Hypermethylation markedly reduced expression of the RB gene and abolished binding of transcription factors (14). Methylation of the promoter region CpG island has subsequently been implicated in the inactivation of other tumour suppressor genes, including the VHL gene in renal carcinomas (15), p15 in haematological malignancy and gliomas (16,17) and p16 in numerous cancers (1820).

Germline mutations in the BRCA1 gene lead to a very high probability of a female developing breast or ovarian cancer (21) and the normal allele is often removed by loss of heterozygosity (22). The BRCA1 region also displays loss of heterozygosity in 40–80% of sporadic breast carcinomas and 30–60% of sporadic ovarian carcinomas (23). However, there have been no reported BRCA1 mutations in sporadic breast tumours (23) and only a handful of sporadic ovarian tumours have been reported to have BRCA1 mutations (2426).

While the lack of mutation may indicate that BRCA1 does not normally have a role in sporadic cancer, BRCA1 inactivation might be achieved by means other than coding region mutation, such as epigenetic silencing or regulatory changes. Significantly, recent reports have found that BRCA1 is expressed at substantially lower levels in breast cancer samples and cell lines compared with normal breast tissue (2729). This is reflected in the levels of BRCA1 protein, which is reduced or absent in high grade ductal carcinoma (30,31).

Another observation that indicates that the reduction in BRCA1 activity in sporadic breast and ovarian cancers plays a definite role in pathogenesis is that the introduction of a normal BRCA1 gene inhibited the growth of breast and ovarian cancer cell lines in vitro and inhibited the tumorigenicity of the MCF-7 breast cancer cell line in nude mice (32). No growth inhibition was observed in colon and lung cancer cell lines, reinforcing the idea that loss of BRCA1 activity is an important stage in the development of breast and ovarian tumours but not other tumours.

Hypermethylation of the promoter region CpG island of the BRCA1 gene was initially reported in two of seven breast tumours (33) and subsequently in two out of six breast tumours and two out of five ovarian tumours (34). Catteau et al. (35) recently reported a larger study of 96 breast carcinomas and 43 ovarian carcinomas in which methylation was observed in 11 and 5% of tumours, respectively.

As hypermethylation does not account for the decrease in BRCA1 activity in most cases of breast cancer, it is possible that BRCA1 promoter region methylation arises as a non-specific change following alteration of methylation patterns in cancer. The aim of this study was to determine the specificity of methylation changes of the BRCA1 gene by comparing methylation rates in cancers where BRCA1 may play a role in their aetiology, such as sporadic breast and ovarian cancers, with cancers in which BRCA1 is unlikely to play a role, such as colon cancers and leukaemia.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Southern blotting
Ten micrograms of DNA was digested with 100 U of TaqI (New England Biolabs, Beverley, MA), precipitated and digested with 100 U of the methylation-sensitive enzyme CfoI (Roche, Mannheim, Germany) or HpaII (New England Biolabs). After digestion, the samples were precipitated, washed in 70% ethanol and run on a 20 cm 1.5% agarose gel at 100 V in 0.5x TBE buffer for 5 h. The gel was blotted overnight without prior depurination onto Hybond N+ membrane (Amersham, Little Chalfont, UK) in 0.4 M sodium hydroxide. After transfer, the membrane was washed with 2x SSC before crosslinking the DNA to the membrane with 120 000 µJ/cm2 UV irradiation using a Stratalinker (Stratagene, La Jolla, CA). Membranes were prehybridized for at least 2 h at 42°C in a 20 ml solution with 50% deionized formamide, 1M NaCl, 1% SDS, 10% dextran sulphate (Pharmacia, Uppsala, Sweden) and 0.2 mg/ml sonicated and denatured salmon sperm DNA. The probe (25 ng) was labelled using the GIGAprime DNA labelling kit and [{alpha}-32P]dCTP (Geneworks, Adelaide, Australia). After hybridization at 42°C overnight, membranes were washed to a final stringency of 0.1x SSC, 0.1% SDS at 65°C and autoradiographed (Hyperfilm; Amersham) using Cronex Hi-Plus intensifying screens at –70°C.

The 217 bp probe (33) detects a 2686 bp TaqI fragment in the BRCA1 promoter CpG island (Figure 1Go) which contains BRCA1 exons 1A, 1B and 2 and exon 1A of the adjacent NBR2 gene (36,37). When the BRCA1 promoter CpG island is unmethylated, the probe detects a 217 bp fragment (89 bp of homology) and a 72 bp (72 bp homology) CfoI fragment or a 299 bp (175 bp of homology) HpaII fragment. The probe also detects a 1514 bp TaqI fragment containing the promoter region and exon 1A of NBR1 and exons 1A and 1B of a partial duplication of the BRCA1 gene (here referred to as the duplicated BRCA1 region) (38). The last 174 bp of the probe are homologous (with 17 mismatched bases) to the duplicated BRCA1 exon 1A. When the 1514bp TaqI fragment is unmethylated, the probe detects a 282 bp (174bp of close homology) CfoI fragment or a 364 bp (132 bp of close homology) HpaII fragment.



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Fig. 1. Genomic organization of regions recognized by the BRCA1 probe. The BRCA1 probe detects a 2686 bp TaqI fragment from the BRCA1 promoter region and a 1514 bp TaqI fragment from the duplicated BRCA1 region. The numerals indicate the HpaII and CfoI fragments seen in the absence of methylation. The T refers to the TaqI restriction sites.

 

    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Colon tumours and leukaemias
No BRCA1 promoter region methylation was found for any of the 35 colon adenocarcinomas (Dukes stage A, 3/35; B, 16/35; C, 9/35; N/A, 7/35) (Figure 2Go) or 19 leukaemias (11 AML; one CML; five CLL; two ALL) examined. Three of the leukaemias had an extra band of ~600 bp in TaqI/HpaII digests (results not shown) which resulted from the 3'-terminal HpaII site of the duplicated BRCA1 region being methylated. Peripheral blood lymphocytes showed only unmethylated bands (data not shown), consistent with previously reported results (33,34).



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Fig. 2. A Southern blot showing five colon tumours. Lane 1 is a TaqI alone control. Lanes 2, 4, 6, 8 and 10 are tumour DNA cut with TaqI and CfoI. Lanes 3, 5, 7, 9 and 11 are tumour DNA cut with TaqI and HpaII. Lanes 2 and 3, lanes 4 and 5, lanes 6 and 7, lanes 8 and 9 and lanes 10 and 11 are from five separate tumour samples.

 
Breast tumours
Eleven sporadic breast cancers (Table IGo) were analysed for BRCA1 promoter methylation. Two tumours showed higher molecular weight bands (in addition to unmethylated bands derived from the normal cells in the biopsy). In the tumour from patient 5, the 2686 bp TaqI fragment (Figure 3Go, lane 10) did not cut with CfoI, indicating that the CfoI sites were all methylated. In the tumour from patient 6 (Figure 4Go, lane 2) the band was slightly less than 2686 bp in the TaqI/CfoI digest, indicating that all the CfoI sites with the exception of one or more of the 5'-terminal sites were methylated. The CfoI sites in the NBR1 region were unmethylated for both patients.


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Table I. BRCA1 methylation in breast tumours
 


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Fig. 3. A Southern blot showing five breast tumours. Lane 1 is a TaqI alone control. Lanes 2, 4, 6, 8 and 10 are tumour DNA cut with TaqI and CfoI. Lanes 3, 5, 7, 9 and 11 are tumour DNA cut with TaqI and HpaII. Lanes 2 and 3 are from patient 1, lanes 4 and 5 are from patient 2, lanes 6 and 7 are from patient 3, lanes 8 and 9 are from patient 4 and lanes 10 and 11 are from patient 5.

 


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Fig. 4. A Southern blot showing five further breast tumours. Lane 1 is a TaqI alone control. Lanes 2, 4, 6, 8 and 10 are tumour DNA cut with TaqI and CfoI. Lanes 3, 5, 7, 9 and 11 are tumour DNA cut with TaqI and HpaII. Lanes 2 and 3 are from a patient 6 tumour, lanes 4 and 5 are from patient 7, lanes 6 and 7 are from patient 8, lanes 8 and 9 are from patient 9 and lanes 10 and 11 are from patient 10.

 
The tumour from patient 5 was also methylated at the BRCA1 region HpaII sites as shown by the intact 2686 bp TaqI fragment (Figure 3Go, lane 11). Additional bands were seen at a slightly lower molecular weight than 2686 bp, indicating that subpopulations of cells were unmethylated at terminal HpaII sites. Similarly, in patient 6 (Figure 4Go, lane 3) the HpaII sites were all methylated within the BRCA1 region except for a 3'-terminal HpaII site, which accounts for the downward band shift. All the HpaII sites were methylated in the duplicated region of both patients as the 1514 bp fragment produced with TaqI alone was visible in the TaqI/HpaII digest. The lack of digestion by HpaII was not due to partial digestion by the restriction enzyme as other probes gave expected fragment sizes (results not shown).

Ovarian tumours
Two of the 20 ovarian cancers (Table IIGo) examined for BRCA1 methylation were found to be methylated. For tumour 91.007, the BRCA1 promoter region was hypermethylated, except for the 5'-terminal CfoI sites, producing a downward shift of the 2686 bp band (Figure 5Go, lane 4). The second novel band in the TaqI/CfoI lane (Figure 5Go, lane 4) comes from the duplicated BRCA1 region with all the CfoI sites methylated except for a 5'-terminal CfoI site. There appeared to be methylation of all HpaII sites in the 2686 and 1514 bp TaqI fragments except for terminal sites, producing slight downward band shifts (Figure 5Go, lane 5). Tumour 90.076 (data not shown) was also methylated at the CfoI and HpaII sites in both the BRCA1 promoter and duplicated BRCA1 regions.


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Table II. BRCA1 methylation in ovarian tumours
 


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Fig. 5. A Southern blot showing five ovarian tumours. Lane 1 is a TaqI alone control. Lanes 2, 4, 6, 8 and 10 are tumour DNA cut with TaqI and CfoI. Lanes 3, 5, 7, 9 and 11 are tumour DNA cut with TaqI and HpaII. Lanes 2 and 3 are from tumour 90.075, lanes 4 and 5 are from tumour 91.007, lanes 6 and 7 are from tumour 91.033, lanes 8 and 9 are from tumour 93.018 and lanes 10 and 11 are from tumour 93.035.

 
Breast and ovarian cancer cell lines
No BRCA1 methylation was found in eight breast cancer cell lines (MDA-MB231, MDA-MB453, MDA-MB468, Hs578t, T47-D, ZR75-1, MCF-7 and PMC42). We previously reported MDA-MB453, MDA-MB468 and T47-D to be unmethylated in both regions using AvaII as the flanking enzyme (33). We also reported that MCF-7 was methylated at CfoI sites in the duplicated region. AvaII detects a fragment that contains only the duplicated BRCA1 exons 1A, 1B and 2 and none of the NBR1 promoter region. No methylation of MCF-7 was seen here (data not shown). This indicates that the NBR1 promoter region is unmethylated whereas the BRCA1 homologous region is partially methylated.

The seven ovarian cancer cell lines examined (JAM, PEO4, PEO14, OAW42, OAW28+53, 27/87 and C1-80-135) showed no BRCA1 promoter methylation although several showed a small amount of methylation in the duplicated region (data not shown). A band of ~600 bp was seen in the TaqI/CfoI digests of 27/87 and was explained by the CfoI site within the duplicated BRCA1 exon 1A being methylated. The primary tumour from which 27/87 was derived was not methylated at this site. C1-80-135 and OAW28+54 both showed an extra band of ~600 bp in the TaqI/HpaII digests. This was the same band that had been seen in some of the leukaemia samples.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We observed methylation of the BRCA1 promoter region CpG island in 4/18 (22%) sporadic breast tumours and in 2/20 (10%) sporadic ovarian tumours examined by Southern blotting (Table IGo). Mancini et al. (34), using genomic sequencing, found 2/6 breast cancers and 2/5 ovarian cancers to be methylated at the BRCA1 promoter region, while Catteau et al. (35), using a different Southern blotting protocol from ours (PstI/SmaI digests), found 10/96 breast cancers and 2/43 ovarian cancers to be methylated. If these studies are combined, methylation has been observed in 16/120 (13%) breast tumours and 6/68 (9%) ovarian tumours. It is difficult to reconcile the above results with the report of Magdinier et al. (29) who found no methylation in 37 sporadic breast tumours using Southern blotting.

If BRCA1 methylation was not specific and not directly related to the loss of BRCA1 activity that has been reported for breast cancer (27,29,31), one might expect to find BRCA1 methylation in other tumour types. None of the 35 sporadic colon tumours and 19 leukaemias that we examined showed BRCA1 methylation. A two-tailed Fisher exact test shows that the incidence of BRCA1 methylation in BRCA1-associated tumours (breast and ovarian) is significantly different from the incidence in tumours (colorectal and leukaemia) that are not associated with BRCA1 (P < 0.008). Thus, the absence of BRCA1 methylation in colon cancers and leukaemias suggests that BRCA1 methylation is selected for in breast and ovarian cancers, where it plays a role in pathogenesis.

All four breast tumours that were methylated at the BRCA1 promoter region were also ER-negative (Table IGo). An association between BRCA1 promoter region methylation and ER status was first noted by us (33) and subsequently confirmed by Catteau et al. (35). We are currently assessing whether there is an elevated hypermethylation of other sites in BRCA1-methylated tumours. The restriction of BRCA1 methylation to breast and ovarian cancers contrasts with the ER gene, whose promoter region is also hypermethylated in a fraction of breast tumours (39), in nearly all colon tumours (40) and in leukaemias (41).

We found no BRCA1 promoter region methylation in eight breast cancer cell lines and seven ovarian cell lines. Only one breast or ovarian cell line with BRCA1 promoter region methylation (28) has been reported so far of the 21 investigated. This shows that the BRCA1 region is resistant to non-specific methylation, as cancer cell lines often acquire de novo methylation at CpG islands (42).

In our previous study we used AvaII as a flanking enzyme (33). Whereas the probe detects a fairly similar region of the BRCA1 promoter with both AvaII and TaqI, it detects the NBR1 promoter region in the duplicated BRCA1 region only if TaqI is used. The use of TaqI in this study allowed us to determine both BRCA1 and NBR1 promoter region methylation. The two ovarian tumours with methylation of the BRCA1 promoter region were the only two tumours to show extensive methylation of the NBR1 promoter region island at both CfoI and HpaII sites. It would be of interest to see if hypermethylation of the NBR1 promoter occurs in other ovarian cancers with BRCA1 methylation, especially as this gene may encode the CA125 tumour marker (43).

Both breast tumours with methylation of the BRCA1 promoter region showed partial methylation of the duplicated BRCA1 region, with higher bands in the HpaII but not the CfoI digests. The difference in CfoI and HpaII cutting in the breast tumours may be explained by the different distribution of the sites in the duplicated region (Figure 1Go). The HpaII sites are clustered within the ARPP1 pseudogene insert and the BRCA1 duplicated exons, whereas the CfoI sites are distributed throughout the region. Although the ARPP1 pseudogene region is normally unmethylated, it showed a tendency to become methylated in the tumours we studied. We also detected some methylation of the duplicated BRCA1 region HpaII sites in three leukaemia patients and two ovarian cancer cell lines.

Until recently, the virtual absence of mutation in sporadic breast and ovarian cancers had been difficult to understand. However, the loss of BRCA1 activity in most sporadic breast cancers and in a substantial proportion of ovarian cancers (27,29,31) and the growth inhibitory effects of BRCA1 on breast and ovarian cancer cell lines (32) indicate that the loss of BRCA1 activity is important in the pathogenesis of sporadic breast and ovarian cancer. There are multiple pathways that may lead to this loss of activity. Mutations are presumably rarely observed only because other events leading to a down-regulation or loss of activity are more common. Methylation of the BRCA1 promoter region is a more common event leading to loss of activity. The other pathways remain to be determined.

The pattern of BRCA1 lesions is reminiscent of the VHL and RB genes in which the methylation in sporadic tumours is restricted to the tumour types that occur when there is a germline mutation of these genes. Thus, the restriction of BRCA1 methylation to those tumour types to which germline mutations of BRCA1 predispose reunites the pathogenetic mechanisms of sporadic and inherited cancers. The results presented here point to BRCA1 methylation having a specific role in sporadic breast and ovarian tumours.


    Acknowledgments
 
We thank Damian Hussey for his technical support, Lisa Butler and Prue Cowled for some of the colon cancer DNAs, Drs B.Ward, A.Wilson, I.Hayward, T.Bradley, I.Bertoncello and R.Whitehead for some of the cell lines used, Jean-Pierre Issa and Lor Wai Tan for helpful comments on the manuscript and Ed Sage for his support.


    Notes
 
6 To whom correspondence should be addressedEmail: adobrovic{at}medicine.adelaide.edu.au

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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 10, 1999; revised October 8, 1999; accepted October 8, 1999.