Abstract 4727: Breast cancer prevention and selective estrogen response modulators (SERMs): Pharmacogenomics and differential estrogen and SERM regulation of BRCA1 and BRCA2 expression

Abstract

Abstract Introduction: The NSABP P-1 and P-2 breast cancer prevention trials have demonstrated that the SERMs tamoxifen and raloxifene can reduce the risk of breast cancer in high risk women. Methods: We performed a nested matched case-control GWAS utilizing DNA from participants enrolled in P-1 and P-2. 592 participants developed invasive breast cancer or ductal carcinoma in situ, and were matched with 1171 controls who did not. Genome-wide genotyping with the Illumina 610-Quad chip and CYP2D6 genotyping for pertinent alleles were performed. Functional genomic studies involved the use of cultured cells, with siRNA knockdown, followed by qRT-PCR and Western blot analyses. Results: Eleven SNPs with p-values < 3E-05 were identified. Twenty one additional SNPs with p < 4E-05 were identified by imputation around the genotyped SNPs on chromosomes (Chrs) 4, 8, 9, 13, 16 and 22, which were then genotyped with the Invader platform. Initial functional genomic studies focused on 6 SNPs on Chr16 (p-values 1.81-9.55E-06), all of which were in ZNF423, a gene encoding a putative zinc-finger protein. The Chr16 minor variant SNP sequences had less risk than the common variant (OR=0.7). Our functional studies showed that incubation of Hs578T cells stably transfected with estrogen receptor (ER) α with 0.1 nM estradiol (E2) induced expression of ZNF423, BRCA1/2 for WT but not variant SNP sequences. There was also a striking difference between E2-induced ZNF423, BRCA1/2 expression for variant and WT SNPs in lymphoblastoid cell lines (LCLs) stably transfected with ERα, with only WT showing induction. ChIP assay showed that ZNF423 could bind to the 5’-flanking region of BRCA1, and reporter gene assays showed that ZNF423 could regulate BRCA1/2 transcription. In LCLs stably transfected with ERα and genotyped for ZNF423 SNPs, estrogen-dependent expression of all 3 genes occurred only in the presence of the WT, but not variant ZNF423 SNP sequences. Blockade of ERα with 4-OH tamoxifen prevented induction of expression in cells with WT sequences, but resulted in gene dose-dependent increases in BRCA1/2 expression in cells with variant SNP sequences-suggested that the minor variant has a “protective” effect during clinical SERM therapy and verified by ChIP assays. Conclusions: The SNPs in ZNF423 identified during GWAS were associated with differential E2-dependent induction of ZNF423, BRCA1 and BRCA2-implying that ZNF423 is “upstream” for the estrogen-dependent induction of BRCA1/2 expression. However, LCLs with variant SNP sequences showed greatly enhanced expression of ZNF423, BRCA1/2 during 4-OH tamoxifen exposure, while cells with WT sequences did not. These results may reveal a novel mechanism for SERM-dependent regulation of BRCA1/2 expression and may have implications for patient selection for SERM therapy. Funded by U10-CA37377, U10-CA69974 and U14-GM61388. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4727. doi:10.1158/1538-7445.AM2011-4727