Dudenkov, Tanda M.

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.

Anastrozole is a widely prescribed aromatase inhibitor for the therapy of estrogen receptor positive (ER+) breast cancer. We performed a genome‐wide association study (GWAS) for plasma anastrozole concentrations in 687 postmenopausal women with ER+ breast cancer. The top single‐nucleotide polymorphism (SNP) signal mapped across SLC38A7 (rs11648166, P = 2.3E‐08), which we showed to encode an anastrozole influx transporter. The second most significant signal (rs28845026, P = 5.4E‐08) mapped near ALPPL2 and displayed epistasis with the SLC38A7 signal. Both of these SNPs were cis expression quantitative trait loci (eQTL)s for these genes, and patients homozygous for variant genotypes for both SNPs had the highest drug concentrations, the highest SLC38A7 expression, and the lowest ALPPL2 expression. In summary, our GWAS identified a novel gene encoding an anastrozole transporter, SLC38A7, as well as epistatic interaction between SNPs in that gene and SNPs near ALPPL2 that influenced both the expression of the transporter and anastrozole plasma concentrations.

Abstract Objective: Response to menopausal hormone therapy (MHT) shows individual variation. SLCO1B1 encodes the OATP1B1 transporter expressed in the liver that transports many endogenous substances, including estrone sulfate, from the blood into hepatocytes. This study evaluated the relationship between genetic variation in SLCO1B1 and response to MHT in women enrolled in the Kronos Early Estrogen Prevention Study (KEEPS) at Mayo Clinic, Rochester, MN. Methods: KEEPS participants were randomized to oral conjugated equine estrogen (n = 33, oCEE), transdermal 17β-estradiol (n = 33, tE2), or placebo (n = 34) for 48 months. Menopausal symptoms (hot flashes, night sweats, insomnia, palpitations) were self-reported before treatment and at 48 months. Estrone (E1), E2, and sulfated conjugates (E1S, E2S) were measured using high-performance liquid chromatography-tandem mass spectrometry. SLCO1B1 rs4149056 (c.521T>C, p.Val174Ala) was genotyped using a TaqMan assay. Results: After adjusting for treatment, there was a significant association between the SLCO1B1 rs4149056 TT genotype (encoding normal function transporter) and lower E1S, E1S/E1, and E2S (P = 0.032, 0.010, and 0.008, respectively) compared with women who were heterozygous (TC) or homozygous (CC) for the reduced function allele. The interactions between genotype, treatment, and E2S concentration were stronger in women assigned to tE2 (P = 0.013) than the women taking oCEE (P = 0.056). Among women assigned to active treatment, women with the CT genotype showed a significantly greater decrease in night sweats (P = 0.041) than those with the TT genotype. Conclusions: Individual variation in sulfated estrogens is explained, in part, by genetic variation in SLCO1B1. Bioavailability of sulfated estrogens may contribute to relief of night sweats.

Abstract BACKGROUND: Based on prospective clinical trials, there is no evidence for differences in efficacy between the 3 aromatase inhibitors (AIs) anastrozole, exemestane, and letrozole. The purpose of this study was to identify germline genetic variants associated with response to AIs and to help identify novel mechanisms associated with drug disease efficacy. METHODS: A genome-wide association study (GWAS) was performed for 624 patients (Steroids 2015;99:32-38) to identify SNPs associated with estrogen level change in women with estrogen receptor (ER) positive breast cancer treated with anastrozole. Replication of associated SNPs was performed in a GWAS from the MA.27 trial that compared adjuvant anastrozole and exemestane treatment of post-menopausal women with ER+ breast cancer. Functional studies were subsequently performed to determine SNP effects and underlying mechanisms. RESULTS: Our initial GWAS identified SNPs within CSMD1 that were associated with changes in estrogen levels during anastrozole therapy. An additional SNP in CSMD1 was also associated with breast cancer events in CCTG MA.27. Functionally, we showed that CSMD1 regulates CYP19 expression in a SNP-, and in an anastrozole- dependent fashion. These phenomena were not observed for either letrozole or exemestane. In MA.27, an anastrozole- specific effect was also seen with the minor allele having a protective effect on time to distant metastasis (HR=0.49, p=0.00259), but this was not the case for exemestane (HR=0.71, p=0.111). Our in vitro functional studies indicated that overexpression of CSMD1 sensitized anastrozole or letrozole resistant cells to anastrozole but not to the other two AIs. The SNP in CSMD1 that was associated with increased CSMD1 and CYP19 expression levels increased anastrozole sensitivity, but not letrozole or exemestane in lymphoblastoid cell lines (LCLs) homozygous for either WT or variant CSMD1 SNP genotypes. Based on these observations, we explored whether anastrozole has additional mechanisms beyond its function as a CYP19 inhibitor. Utilizing an estrogen response element (ERE) luciferase reporter assay in a CYP19 CRISPR knockout breast cancer T47D cell line and a surface plasmon resonance (SPR) assay, we found that anastrozole can also function as an ERα agonist, and can bind to, and result in, proteasome dependent ERα degradation, especially in the presence of E2. Treatment of these CYP19 CRISPR knockout cells with anastrozole in the presence of increasing concentrations of E2 results in greater sensitivity compared with anastrozole alone, while the addition of E2, as expected, does not improve letrozole or exemestane sensitivity. These same observations were also seen in letrozole and anastrazole resistant cells. CONCLUSIONS: Our findings suggest that anastrozole might be more effective than letrozole or exemestane in patients with the CSMD1 SNP. Furthermore, anastrozole can function as an ERα agonist, binding to ERα and resulting in its degradation, especially in the presence of E2. These findings should help to make it possible to develop precision endocrine therapies for women who are candidates for AIs. Citation Format: Cairns J, Ingle J, Dudenkov T, Kalari K, Buzdar A, Kubo M, Robson M, Ellis M, Goss P, Shepherd L, Goetz M, Weinshilboum R, Wang L. CSMD1 SNPs selectively affect anastrozole response in postmenopausal breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD1-04.