Vogel, Victor G.

Abstract The selective estrogen receptor modulators (SERM) tamoxifen and raloxifene can reduce the occurrence of breast cancer in high-risk women by 50%, but this U.S. Food and Drug Administration-approved prevention therapy is not often used. We attempted to identify genetic factors that contribute to variation in SERM breast cancer prevention, using DNA from the NSABP P-1 and P-2 breast cancer prevention trials. An initial discovery genome-wide association study identified common single-nucleotide polymorphisms (SNP) in or near the ZNF423 and CTSO genes that were associated with breast cancer risk during SERM therapy. We then showed that both ZNF423 and CTSO participated in the estrogen-dependent induction of BRCA1 expression, in both cases with SNP-dependent variation in induction. ZNF423 appeared to be an estrogen-inducible BRCA1 transcription factor. The OR for differences in breast cancer risk during SERM therapy for subjects homozygous for both protective or both risk alleles for ZNF423 and CTSO was 5.71. Significance: This study identified novel, functionally polymorphic genes involved in the estrogen-dependent regulation of BRCA1 expression, as well as a novel mechanism for genetic variation in SERM therapeutic effect. These observations, and definition of their underlying mechanisms, represent steps toward pharmacogenomically individualized SERM breast cancer prevention. Cancer Discov; 3(7); 812–25. ©2013 AACR. See related commentary by Machiela and Chanock, p. 728 This article is highlighted in the In This Issue feature, p. 705

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

Abstract Background: Controversy exists regarding the association between CYP2D6 enzyme activity and tamoxifen effectiveness in the adjuvant treatment of invasive breast cancer; however, this association in the primary prevention of breast cancer is unknown. Methods: We conducted a nested case–control study in the context of the NSABP P1 and P2 prevention trials to determine the impact of CYP2D6 genotype, CYP2D6 inhibitor use, and metabolizer status (CYP2D6 genotype combined with CYP2D6 inhibitor use), on breast cancer events. Women who developed breast cancer (both noninvasive and invasive) while on 5 years of selective estrogen receptor modulators therapy (cases) were matched to controls free of breast cancer. Comprehensive CYP2D6 genotyping was conducted for alleles associated with absent (*3, *4, *5, and *6), reduced (*10, *17, and *41), and increased (*1XN and *2XN) enzyme activity. Information regarding the use of CYP2D6 inhibitors was recorded. Results: A total of 591 cases were matched to 1,126 controls and DNA was genotyped in more than 97%. In patients treated with tamoxifen, there was no association of CYP2D6 genotype [OR (extensive/poor metabolizer): 0.90; 95% CI: 0.46–1.74, P = 0.74), use of a potent CYP2D6 inhibitor (OR 0.92; 95% CI: 0.575–1.486), or CYP2D6 metabolizer status (OR 1.03; 95% CI: 0.669–1.607) with breast cancer occurrence. Likewise, there was no association between any CYP2D6 metabolism parameter with breast cancer events in raloxifene-treated patients. Conclusion: In the NSABP P1 and P2 clinical trials, alterations in CYP2D6 metabolism are not associated with either tamoxifen or raloxifene efficacy. Clin Cancer Res; 17(21); 6944–51. ©2011 AACR.