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. 2016 Dec 1;76(23):7012-7023.
doi: 10.1158/0008-5472.CAN-16-1371. Epub 2016 Oct 10.

Genetic Polymorphisms in the Long Noncoding RNA MIR2052HG Offer a Pharmacogenomic Basis for the Response of Breast Cancer Patients to Aromatase Inhibitor Therapy

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Free PMC article

Genetic Polymorphisms in the Long Noncoding RNA MIR2052HG Offer a Pharmacogenomic Basis for the Response of Breast Cancer Patients to Aromatase Inhibitor Therapy

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

Abstract

Genetic risks in breast cancer remain only partly understood. Here, we report the results of a genome-wide association study of germline DNA from 4,658 women, including 252 women experiencing a breast cancer recurrence, who were entered on the MA.27 adjuvant trial comparing the aromatase inhibitors (AI) anastrozole and exemestane. Single-nucleotide polymorphisms (SNP) of top significance were identified in the gene encoding MIR2052HG, a long noncoding RNA of unknown function. Heterozygous or homozygous individuals for variant alleles exhibited a ∼40% or ∼63% decrease, respectively, in the hazard of breast cancer recurrence relative to homozygous wild-type individuals. Functional genomic studies in lymphoblastoid cell lines and ERα-positive breast cancer cell lines showed that expression from MIR2052HG and the ESR1 gene encoding estrogen receptor-α (ERα) was induced by estrogen and AI in a SNP-dependent manner. Variant SNP genotypes exhibited increased ERα binding to estrogen response elements, relative to wild-type genotypes, a pattern that was reversed by AI treatment. Further, variant SNPs were associated with lower expression of MIR2052HG and ERα. RNAi-mediated silencing of MIR2052HG in breast cancer cell lines decreased ERα expression, cell proliferation, and anchorage-independent colony formation. Mechanistic investigations revealed that MIR2052HG sustained ERα levels both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated, proteasome-dependent degradation of ERα. Taken together, our results define MIR2052HS as a functionally polymorphic gene that affects risks of breast cancer recurrence in women treated with AI. More broadly, our results offer a pharmacogenomic basis to understand differences in the response of breast cancer patients to AI therapy. Cancer Res; 76(23); 7012-23. ©2016 AACR.

Conflict of interest statement

No potential conflicts of interest were disclosed by the authors.

Figures

Figure 1
Figure 1
A, GWAS Manhattan Plot. B, Locus zoom of the chromosome 8 region surrounding the MIR2052HG gene.
Figure 2
Figure 2
A. Schematic of estrogen response elements (EREs) around rs4476990 and rs3802201. The EREs are indicated as boxes and the SNPs are indicated as red circles. B. MIR2052HG mRNA expression in lymphoblastoid cell lines (LCLs) with wild type SNP (W) and variant SNP (V) genotypes for both rs4476990 and rs3802201 after exposure to increasing concentrations of E2. Error bars represent SEM. *P<0.05. C. and D., ChIP assay using six LCLs with known genotypes for rs4476990 and rs3802201 SNPs. Error bars represent SEM of three independent experiments. Representative PCR products are visualized on agarose DNA gels.
Figure 3
Figure 3
A and B. MIR2052HG mRNA expression in lymphoblastoid cell lines (LCLs) withWT and V genotypes for both rs4476990 and rs3802201 after exposure to androstenedione alone and with increasing concentrations of exemestane or anastrozole. C and D. mRNA expression for ESR1 in LCLs with the same conditions as A and B. *P<0.05, **P<0.01.
Figure 4
Figure 4
A, Knock down of MIR2052HG by antisense oligonucleotides (ASO1 and ASO2) down-regulated ERα protein. The histogram shows knock down efficiency in CAMA1 and MCF7/AC1 cells. B, Knock down of MIR2052HG decreased ESR1 mRNA expression levels in CAMA1 and MCF7/AC1 cell lines. C and D. Knock down of MIR2052HG decreased proliferation and colony formation in CAMA1 and MCF7/AC1 cells. The representative colony formation pictures from triplicate experiments are shown. The colony formation rates are quantified as percentages. Error bars represent SEM; ** P< 0.01 compared to baseline (negative control). E, Overexpression of MIR2052HG increased ERα protein levels. Overexpression efficiency was determined by qRT-PCR. Overexpression of MIR2052HG increased the proliferation and colony formation and ERα protein levels in MCF7/AC1 cells. F. Overexpression of MIR2052HG conferred resistance to AIs (exemestane and anastrozole) and 4-hydroxy-tamoxifen treatments in MCF7/AC1 compared to negative controls. The assay was performed as described in C and D. Error bars represent SEM. The concentrations for androstenedione (A), exemestane (EXE, anastrozole (ANA), estradiol (E2) and 4-hydroxy-tamoxifen (TAM) are indicated.
Figure 5
Figure 5
A, Knock down of MIR2052HG shortened ERα protein half-life. CAMA1 cells were transfected with MIR2052HG specific antisense oligonucleotides (ASOs) or a negative control ASO and then treated with cycloheximide (CHX). The representative western blotting results from three independent experiments are shown. The knock down efficiency was determined by qRT-PCR. B. Quantitative intensities of ERα are mean values with SEM (error bars) from three independent experiments. C. MIR2052HG regulated ERα stability in a proteasome-dependent manner. MIR2052HG was knocked down with ASOs in CAMA1 cells that were treated with MG132 or bortezomib. D. Knock down of MIR2052HG promoted the ubiquitination of ERα. 293T cells were transfected with HA-Ub plasmid and FLAG-ERα plasmid, and then transfected with either the MIR2052HG specific ASOs or the negative control ASO followed by MG132. ERα proteins were immunoprecipitated and analyzed by western blotting. Knock down efficiency in 293T cells was determined by qRT-PCR. E. MIR2052HG regulated ERα transcription through the AKT-FOXO3 pathway. Knock down of MIR2052HG increased AKT phosphorylation and decreased FOXO3 phosphorylation and FOXO3 total level in MCF7/AC1 and CAMA1 cells. F. Overexpression of FOXO3 in MIR2052HG knocked-down MCF7/AC1 cells reversed ERα protein and mRNA levels. Overexpression of FOXO3 was determined by western blotting. HA: expression tag.

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