BRCA1 dysfunction in sporadic basal-like breast cancer

Oncogene (2007) 26, 2126–2132 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc SHORT COMMUNICATION BRCA1 dysfunction in sporadic basal-like breast cancer NC Turner1, JS Reis-Filho1,2, AM Russell1, RJ Springall3, K Ryder3, D Steele1, K Savage1, CE Gillett3, FC Schmitt2, A Ashworth1 and AN Tutt1,3 1 Chester Beatty Laboratories, The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London, UK; Medical Faculty and IPATIMUP Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal and 3 Breast Pathology Laboratory, Guy’s Hospital, London, UK 2 Basal-like breast cancers form a distinct subtype of breast cancer characterized by the expression of markers expressed in normal basal/myoepithelial cells. Breast cancers arising in carriers of germline BRCA1 mutations are predominately of basal-like type, suggesting that BRCA1 dysfunction may play a role in the pathogenesis of sporadic basal-like cancers. We analysed 37 sporadic breast cancers expressing the basal marker cytokeratin 5/6, and age- and grade-matched controls, for downregulation of BRCA1. Although BRCA1 promoter methylation was no more common in basal-like cancers (basal 14% vs controls 11%, P ¼ 0.72), BRCA1 messenger RNA expression was twofold lower in basal-like breast cancers compared to matched controls (P ¼ 0.008). ID4, a negative regulator of BRCA1, was expressed at 9.1-fold higher levels in basal-like breast cancer (Po0.0001), suggesting a potential mechanism of BRCA1 downregulation. BRCA1 downregulation correlated with the presence of multiple basal markers, revealing heterogeneity in the basal-like phenotype. Finally, we found that 63% of metaplastic breast cancers, a rare type of basal-like cancers, had BRCA1 methylation, in comparison to 12% of controls (Po0.0001). The high prevalence of BRCA1 dysfunction identified in this study could be exploited in the development of novel approaches to targeted treatment of basal-like breast cancer. Oncogene (2007) 26, 2126–2132. doi:10.1038/sj.onc.1210014; published online 2 October 2006 Keywords: BRCA1; basal-like; breast cancer; ID4; metaplastic Basal-like breast cancers form a distinct subtype of breast cancer that can be identified using gene expression microarrays (Perou et al., 2000), and are characterized by the expression of markers expressed in normal basal/myoepithelial cells (Perou et al., 2000; Nielsen et al., 2004). Basal-like cancers only infrequently express Correspondence: Dr AN Tutt, Chester Beatty Laboratories, The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, 237 Fulham Road, Chelsea, London SW3 6JB, UK. E-mail: Received 24 April 2006; revised 3 August 2006; accepted 17 August 2006; published online 2 October 2006 the oestrogen or progesterone receptor and rarely overexpress HER2 (Nielsen et al., 2004). Understanding the pathogenesis of this form of breast cancer, and the identification of potential therapeutic targets, is of major importance if the poor prognosis of this subtype is to be improved. Unlike sporadic cancers, where basal-like cancer represent 15% of invasive cancers (Nielsen et al., 2004; Abd El-Rehim et al., 2005), 80–90% of cancers arising in BRCA1 germline mutation carriers are of the basallike subtype (Foulkes et al., 2003; Lakhani et al., 2005). Expression microarray analyses have also indicated strong similarities in gene expression between BRCA1 familial cancers and sporadic basal-like cancers (Sorlie et al., 2003). This phenotypic overlap has raised the possibility that inactivation of BRCA1 function may also underlie sporadic basal-like cancers, and that loss of BRCA1 function may have an aetiological role in the development of the basal-like phenotype. A number of previous studies have examined BRCA1 in unselected sporadic breast cancer, identifying both methylation of the BRCA1 promoter in 10–15% of cancers and loss of BRCA1 expression in a number of breast cancers (Catteau et al., 1999; Esteller et al., 2000; Turner et al., 2004). The incidence of BRCA1 methylation has appeared to be more common in cancers with a high histological grade, and with a triple negative phenotype, suggesting that BRCA1 methylation could underlie basal-like cancers (Magdinier et al., 1998; Catteau et al., 1999; Esteller et al., 2000). However, recent studies examining the basal-like subtype specifically have generated conflicting results and it is presently unclear whether BRCA1 is dysfunctional in these cancers (Abd El-Rehim et al., 2005; Matros et al., 2005; Richardson et al., 2006). Here, we examine BRCA1 silencing and downregulation in a large cohort of basal-like cancers with a control non-basal-like population matched for age and grade to eliminate potential confounding factors (Lambie et al., 2003). In addition, we examined a cohort of metaplastic breast cancers, rare tumours that form a distinct group within the spectrum of basal-like tumours (Reis-Filho et al., 2006). The Guy’s Hospital Tissue Microarray (Gillett et al., 2000) representing 1455 breast cancers was stained by immunohistochemistry for basal cytokeratin 5/6 (Ck5/6) BRCA1 downregulation in basal breast cancer NC Turner et al 2127 Figure 1 BRCA1 promoter methylation and BRCA1 expression in basal-like cancers and controls. (a) Cores from tissue microarray stained for the basal Ck5/6 (brown) with D516B4 (Chemicon, Temecula, CA, USA) and counterstained with light haematoxylin. Example cores demonstrating no Ck5/6 expression and strong Ck5/6 expression (original magnification  10, with inset  40). Crosssection of normal breast duct with positive staining of outer myoepithelial/basal layer, and negative staining of inner – luminal layer (original magnification  40). (b) BRCA1 promoter methylation analysis with MSP. Microdissected DNA, prepared as described previously (Simpson et al., 2004), was modified with sodium bisulphite as described by Herman et al. (1996) and using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA). Primers that do not discriminate between methylated and unmethylated sequence were used to nest PCR amplify bisulphite-treated DNA; forward, 50 -TATTTTGAGAGGTTGCTGTTTAG-30 and reverse 50 -CTAAAAAACCCCACAACCTATCCC-30 . The nest-amplified product was diluted 1:50 before subsequent MSP using primers published previously (Esteller et al., 2000). Controls for all reactions were a negative control with no DNA, unmethylated normal lymphocyte DNA (U Control) and lymphocyte DNA methylated in vitro with the Sss-I methylase (M Control) (NEB, Ipswich, CA, USA). Example PCR run with primers specific for unmethylated (U) and methylated DNA (M). The first four tumours are unmethylated for BRCA1, and last methylated. (c) Incidence of BRCA1 methylation in control cancers (4/37 (11%); 95% confidence interval (CI) 4–25%) and Ck5/6-positive cancers (4/29 cases (14%); 95% CI 4–32%). P ¼ 0.72, Fisher’s exact test. Error bars represent binomial 95% CIs. (d) (...truncated)