Pharmacogenomics of aromatase inhibitors in postmenopausal breast cancer and additional mechanisms of anastrozole action

Aromatase inhibitors (AIs) reduce breast cancer recurrence and prolong survival, but up to 30% of patients exhibit recurrence. Using a genome-wide association study of patients entered on MA.27, a phase III randomized trial of anastrozole versus exemestane, we identified a single nucleotide polymorphism (SNP) in CUB And Sushi multiple domains 1 (CSMD1) associated with breast cancer–free interval, with the variant allele associated with fewer distant recurrences. Mechanistically, CSMD1 regulates CYP19 expression in an SNP- and drug-dependent fashion, and this regulation is different among 3 AIs: anastrozole, exemestane, and letrozole. Overexpression of CSMD1 sensitized AI-resistant cells to anastrozole but not to the other 2 AIs. The SNP in CSMD1 that was associated with increased CSMD1 and CYP19 expression levels increased anastrozole sensitivity, but not letrozole or exemestane sensitivity. Anastrozole degrades estrogen receptor α (ERα), especially in the presence of estradiol (E2). ER+ breast cancer organoids and AI- or fulvestrant-resistant breast cancer cells were more sensitive to anastrozole plus E2 than to AI alone. Our findings suggest that the CSMD1 SNP might help to predict AI response, and anastrozole plus E2 serves as a potential new therapeutic strategy for patients with AI- or fulvestrant-resistant breast cancers.

This research was performed after approval by local institutional review boards in accordance with assurances filed with, and approved by, the Department of Health and Human Services. The primary outcome in our pharmacogenomics studies with MA.27 patients was the STEEP endpoint of Breast Cancer-Free Interval BCFI (5).

Cell lines
Human ER+ breast cancer cell lines ZR-75-1 and T47D were obtained from American Type

Gene set enrichment analysis
The biological information and attribution embedded in the treatment-associated gene set were analyzed using a web-based integrated data mining system, WebGestalt. Biochemical pathways and functions linked to the resveratrol gene set were specifically queried and navigated by the KEGG pathway enrichment analysis tool in WebGestalt (12). The gene set enrichment analysis searches for combinations of features that show a significant difference in means between two classes that is not an artifact or data noise. Significance in the identified gene sets is confirmed by replacing chosen significant features (i.e., gene sets) with other genes chosen at random, followed by testing and re-analyzing to see whether the significant difference still persists. The validated significant gene sets were then organized based on the KEGG biochemical pathways in a KEGG Table. The top 10 pathways with an adjusted P-value less than 0.01 were selected.
RNA was extracted using the QIAGEN RNeasy kit (Germantown, MD). RNA was measured by NanoDrops300 (Thermo fisher, Rockford, IL). QRT-PCR reactions were prepared as per the manufacturer's protocol. Samples were run using StepOnePlus real-time PCR system (ABI).

Chromatin Immunoprecipitation (ChIP) assays
ChIP assays were performed using EpiTect ChIP OneDay kit (Qiagen). Approximately 2×10 7 LCLs per every sample (different SNP genotypes with E2 or E2 plus 4-OH TAM treatment groups; androstenedione or androstenedione plus AI) were collected for the ChIP assay. The details for the assay have been described (8).

Cell proliferation assays
For cell proliferation after E2 or AI treatment, cells were starved in culture medium with 5% charcoal stripped FBS for 24 hours followed by various treatments. Specifically, 3000 cells were

Co-Immunoprecipitation Assay
Cells were lysed with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing 50 mM β-glycerophosphate, 10 mM NaF, and 1 mg/ml each of pepstatin A and aprotinin on ice for 25 min. After centrifugation, cell lysates were incubated with 2 μg antibody and protein A sepharose beads (Amersham Biosciences) for 3 h at 4°C. After centrifugation, the beads were then washed four times with NETN buffer and immunoprecipitates were separated by SDS-PAGE. Immunoblotting was performed following standard procedures.

Generation of CYP19 Knockout cells by CRISPR/Cas9 technology
CYP19 CRISPR/Cas9 KO plasmid and HDR plasmid were obtained from Santa Cruz Biotechnology. 2 x 10 5 T47D cells were seeded in 6-well tissue culture plate, and were transfected with 1 μg of CYP19 CRISPR/Cas9 KO plasmid and 1 μg of HDR plasmid using Lipofectamine 2000. 72 hrs later, puromycin selection was performed for the following 4 weeks to isolate CYP19 CRISPR/Cas9 KO single clones. CYP19 mRNA and protein were analyzed to confirm the knockout efficiency.

Luciferase activity assay
Transcription activity of ERα was measured using the dual luciferase assay with the Cignal ERE Reporter Assay Kit (Qiagen). CYP19 CRISPR knockout and control T47D cells were cultured in charcoal stripped serum medium for 48 hours and then switched to serum free medium in 96well plates for 2 days. Cells were then transfected with either ERE reporter (inducible ERαresponsive GFP reporter), negative control (GFP reporter construct with GFP expression controlled by a minimal promoter), or Positive control (constitutively expressing GFP construct) constructs using the Lipofectamine 2000 transfection reagent. After 16-24 hours of transfection, cells were treated with E2, AI, or E2 plus AI for 48 hrs. Luciferase assay was performed using the Dual-Luciferase Reporter Assay System from Promega following the manufacturer's protocol.

RNA-Seq analysis and normalization
RNA was prepared from cells using the TRIzol (Life Technologies) extraction kit. Genomic DNA was removed using the Ambion DNA-free kit. NuGEN Encore reagents were used for library preparation of total RNA samples. One microgram of total RNA input was used for each sample. The libraries were sequenced on an Illumina HiSeq 2000 sequencing system using 100bp single-ended reads. After removing the poor-quality bases from FASTQ files for the whole transcriptome sequencing, paired-end reads are aligned by reads that were aligned to the human reference genome UCSC hg19 with Tophat 2.0.14 and the bowtie 2.2.6 aligner option.
Transcript abundance was estimated using a count-based method with htseq-count. Gene counts were used as input for TMM (Trimmed Mean of M values) normalization of the R package edgeR 3.18.1, and the normalized counts were transformed to log2-counts per million (logCPM) by applying voom from the R package limma to account for higher variability at low expression levels. Genes with zero read counts across all samples were removed.
After incubation for an additional 72 hours, 100 µL of CellTiter-Glo reagent was added to each well, followed by mixing contents for 2 minutes on an orbital shaker to induce cell lysis. Plates were incubated at 37°C for additional one hour, and the luminescent signal was measured in a Safire2 plate reader.