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. 2013 Jul;3(7):812-25.
doi: 10.1158/2159-8290.CD-13-0038. Epub 2013 Jun 13.

Selective Estrogen Receptor Modulators and Pharmacogenomic Variation in ZNF423 Regulation of BRCA1 Expression: Individualized Breast Cancer Prevention

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Free PMC article

Selective Estrogen Receptor Modulators and Pharmacogenomic Variation in ZNF423 Regulation of BRCA1 Expression: Individualized Breast Cancer Prevention

James N Ingle et al. Cancer Discov. .
Free PMC article

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.

Conflict of interest statement

Potential conflicts of interest: The authors declare no potential conflicts of interest

Figures

Figure 1
Figure 1
Panel A. Manhattan plot of p-values for conditional logistic regression adjusted for nine eigenvectors. (Black: p-value ≥1E-4, Blue: p-value <1E-4 to 1E-5, Red: p-value < 1E-5). Panel B. Chromosome 16 region of interest: Manhattan plot of –log10 p-values from conditional logistic regression adjusted for nine eigenvectors for Platform SNPs (genotyped using Illumina Human610-Quad BeadChip) (blue circles), Fine mapped SNPs (imputed using HapMap2 and then genotyped) (red circles), HapMap2 imputed SNPs (green circles) and 1K Imputed SNPs (imputed using 1000 Genomes database) (black circles).Panel C. Chromosome 4 region of interest: Manhattan plot of –log10 p-values from conditional logistic regression adjusted for nine eigenvectors for Platform SNPs (genotyped using Illumina Human610-Quad BeadChip) (blue circles), Fine mapped SNPs (imputed using HapMap2 and then genotyped) (red circles), HapMap2 imputed SNPs (green circles) and 1K Imputed SNPs (imputed using 1000 Genomes database) (black circles).
Figure 2
Figure 2
Panels A and B. mRNA expression relative to ERα (in %) for ZNF423 (panel A) and BRCA1 (panel B) for lymphoblastoid cell lines (LCLs) of known genotypes for variant (V) and wild type (WT) chromosome 16 ZNF423 SNPs (3 cell lines each) incubated for 24 h with estradiol (E2) concentrations that varied from 0.00001 to 0.01 nM. Error bars represent standard error of the mean (SEM). Panel C. ZNF423 mRNA expression relative to baseline in Hs578T cells after ZNF423 overexpression (OE) (upper left graph) or ZNF423 knockdown (KD) (upper right graph) together with corresponding BRCA1 transcriptional activity measured by luciferase activity at 48 h (lower graphs). Error bars represent SEM. Panel D. Chromatin immunoprecipitation assay using human Hs578T cells stably transfected with ERα showing binding of ZNF423 to a region containing a ZNF423 consensus binding sequence located 1735 to 1518 bp from the ATG in BRCA1.
Figure 3
Figure 3
Panels A and B. mRNA expression for ZNF423 (panel A) and BRCA1 (panel B) for lymphoblastoid cell lines with WT/WT (8 cell lines), WT/V (7 cell lines), and V/V (8 cell lines) genotypes for the chromosome 16 ZNF423 SNPs after exposure to estradiol (E2) alone or E2 with increasing concentrations of 4-hydroxytamoxifen (4-OH-TAM). Error bars represent standard error of the mean (SEM). Panel C. Schematic depiction of the ZNF423 intron 2 area near the rs9940645 SNP. The locations of estrogen response elements (EREs) are shown as boxes, and arrows show the locations of primers used to perform the ChIP amplifications. Panel D. Chromatin immunoprecipitation assay for the area of ZNF423 that contains the rs9940645 SNP. WT, wild type; V, variant; E2, estradiol; 4-OH TAM, 4-hydroxytamoxifen. Panel E. Dual luciferase reporter-gene assays showing the effect of 4-hydroxytamoxifen (4-OH-TAM) (left graph) and raloxifene (right graph). A 500 bp DNA sequence that included either WT or variant SNP sequence for rs9940645 and all of the EREs shown in Figure 4C was cloned into the pGL3 basic reporter plasmid. A 1500 bp region of the ZNF423 promoter was then cloned downstream of the 500 bp segment, and these constructs were used to transfect IGROV-1 cells that were treated with 0.1 nM E2, with or without 10−7 μM 4-hydroxytamoxifen (left graph) or 10−7 M raloxifene (right graph). Error bars represent SEM.
Figure 4
Figure 4
Panel A. Comet assays for knockdown (KD) of ZNF423 with or without BRCA1 overexpression (OE) and for BRCA1 KD. Panel B. CTSO mRNA expression relative to ERα (in %) in lymphoblastoid cell lines (LCLs) of known genotypes for variant (V) and wild type (WT) chromosome 4 SNPs (3 cell lines each) incubated for 24 hours at varying estradiol (E2) concentrations from 0.0001 to 0.01 nM. Error bars represent standard error of the mean (SEM). Panel C. BRCA1 mRNA expression relative to ERα (in %) in the same LCLs shown in panel B. Error bars represent SEM. Panel D. Comet assays for KD of CTSO with or without BRCA1 OE and BRCA1 KD.

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