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. 2017 Aug 21;3:30.
doi: 10.1038/s41523-017-0036-4. eCollection 2017.

Breast Cancer Chemoprevention Pharmacogenomics: Deep Sequencing and Functional Genomics of the ZNF423 and CTSO Genes

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Free PMC article

Breast Cancer Chemoprevention Pharmacogenomics: Deep Sequencing and Functional Genomics of the ZNF423 and CTSO Genes

Duan Liu et al. NPJ Breast Cancer. .
Free PMC article

Abstract

Our previous GWAS using samples from the NSABP P-1 and P-2 selective estrogen receptor modulator (SERM) breast cancer prevention trials identified SNPs in ZNF423 and near CTSO that were associated with breast cancer risk during SERM chemoprevention. We have now performed Next Generation DNA sequencing to identify additional SNPs that might contribute to breast cancer risk and to extend our observation that SNPs located hundreds of bp from estrogen response elements (EREs) can alter estrogen receptor alpha (ERα) binding in a SERM-dependent fashion. Our study utilized a nested case-control cohort selected from patients enrolled in the original GWAS, with 199 cases who developed breast cancer during SERM therapy and 201 matched controls who did not. We resequenced approximately 500 kb across both ZNF423 and CTSO, followed by functional genomic studies. We identified 4079 SNPs across ZNF423 and 3876 across CTSO, with 9 SNPs in ZNF423 and 12 in CTSO with p < 1E-02 that were within 500 bp of an ERE motif. The rs746157 (p = 8.44E-04) and rs12918288 SNPs (p = 3.43E-03) in intron 5 of ZNF423, were in linkage equilibrium and were associated with alterations in ER-binding to an ERE motif distant from these SNPs. We also studied all nonsynonymous SNPs in both genes and observed that one nsSNP in ZNF423 displayed decreased protein expression. In conclusion, we identified additional functional SNPs in ZNF423 that were associated with SNP and SERM-dependent alternations in ER binding and transcriptional regulation for an ERE at a distance from the SNPs, thus providing novel insight into mechanisms of SERM effect.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
The SERM-dependent, SNP-dependent “reversal” of estradiol induction of ZNF423 and BRCA1 expression in LCLs. a Estradiol (E2) and 4-hydroxytamoxifen (4OH-TAM) dose response curves for expression of ZNF423 (a) and BRCA1 (b) in LCLs with known genotypes for the ZNF423 intron 2 rs9940645 SNP. Values are mean ± SEM for 8 determinations. (modified from Fig. 3, Ingle et al. Cancer Discovery 2013)
Fig. 2
Fig. 2
ChIP assays showing fold changes in ERα binding to DNA sequences containing ZNF423 SNPs (panels a and c) or CTSO SNPs (panel b and d). ChIP assays were performed in LCLs homozygous for WT (W/W) or variant (V/V) genotypes for these SNPs after exposure to E2 (0.01 nM) or E2 (0.01 nM) plus 4OH-TAM (0.01 μM). Percentage of ChIP DNA/input was determined by qPCR. The level of enrichment was expressed as relative enrichment above vehicle treatment. The values shown represent mean ± SEM for six determinations.*P < 0.05, **P < 0.005 comparing WT and V SNP genotype cell lines at the same concentrations of E2 and 4OH-TAM
Fig. 3
Fig. 3
ChIP assay results for the rs746157 and rs12918288 SNPs in ZNF423 intron 5. a The figure shows a schematic representation of the ERE motif located near the rs12918288 and rs746157 SNPs in intron 5 of ZNF423. b Bar graphs showing quantitative ERα ChIP results for the area of ZNF423 containing rs746157 and rs12918288. The values shown represent mean ± SEM for six determinations. c Agarose gel assay showing differential binding for the region including the rs746157 and rs12918288 SNPs for LCLs homozygous for WT (W/W) or variant (V/V) genotypes for these two SNPs after exposure to E2 (0.01 nM) or E2 (0.01 nM) plus 4OH-TAM (0.01 μM)
Fig. 4
Fig. 4
The induction of ZNF423 and BRCA1 mRNA expression in a SNP-dependent, SERM-dependent fashion for LCLs homozygous for WT and variant ZNF423 rs746157 and rs12918288 genotypes. a The figure shows the effect of E2 and E2 plus 4OH-TAM dose response curves on the expression for ZNF423 and BRCA1 in LCLs homozygous for WT and variant genotypes for the rs746157 and rs12918280 SNPs in ZNF423 intron 5. Values are mean ± SEM for 3 determinations. All of these LCLs were homozygous WT for the intron 2 rs9940645 SNP. b Bar graphs showing mRNA expression for ZNF423 and BRCA1 at the optimal concentrations for E2 (0.01 nM) and E2 (0.01 nM) plus 4OH-TAM (0.01 μM) shown in (a). Compare with Fig. 1b. Data represented as mean ± SEM
Fig. 5
Fig. 5
a Levels of protein expression in COS-1 cells for ZNF423 WT and variant allozymes. b Levels of mRNA expression in COS-1 cells for the Q617 ZNF423 variant allozyme compared with that for the WT allozyme. Data represented as mean ± SEM for 6 determinations. EV = empty vector

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