Abstract P1-10-04: AGR2, an estrogen response gene associated with tamoxifen resistance, is modulated by acolbifene in premenopausal women at high risk for development of breast cancer
Authors: Fabian Carol J. , Phillips Teresa A. , Kimler Bruce F. 
Published: March 2022

Anterior Gradient 2 is an estrogen early response gene (AGR2) and protein (AGR-2). High expression is associated with endoplasmic reticulum stress and endocrine therapy resistance. Developmentally, AGR2 is associated with estrogen-mediated epithelial proliferation and lobuloalveolar development. In the adult, AGR2 is upregulated by inflammatory and metabolic stress signaling from NFKB, HSP90, IGFR-1, and FOXA1, in addition to estrogen bound to its receptor. The Selective Estrogen Receptor Modulator (SERM) Full dose tamoxifen has been reported to upregulate AGR2 in breast cancer but Selective Estrogen Receptor Down Regulators (SERDs) and aromatase inhibitors do not. AGR-2 is also a secreted protein, with increased tumor levels associated with metastases and decreased disease-free survival in tamoxifen-treated patients. We assessed AGR2 expression in reserved benign breast tissue from baseline and off-study specimens acquired by random periareolar fine needle aspiration (RPFNA) of premenopausal women at high risk for development of breast cancer who had participated in an early phase prevention trial of the SERM acolbifene (20 mg daily for 6 months). This pilot had demonstrated reduced Ki-67 and down-regulation of several estrogen response genes (Fabian et al. Ca Prev Res 2015). Methods Total RNA was extracted from aliquots of frozen benign breast tissue using TRIzol LS (Life Technologies) and purified using RNeasy MinElute Cleanup Kit (Qiagen). RNA was amplified using MessageAmpII aRNA amplification kit (Life Technologies) and reverse transcribed to cDNA using SMARTScribe Reverse Transcriptase (Takara Bio USA, Inc.) and random nonamer primers. Real-time quantitative PCR (qPCR) was performed using hydrolysis probes with baseline and postintervention specimens assessed together. PCR reactions were run on an Applied Biosystems Prism 7000 Sequence Detection System. The cycle threshold mean value for each transcript (from duplicate assays per specimen) was normalized using three reference transcripts, plus two epithelial cell transcripts. Relative levels of each transcript were calculated using the ΔΔCt method. The ratio (postintervention: baseline) of final values indicated upregulation (value &gt; 1) or downregulation (value &lt; 1). Results There were 17 available paired specimens for analysis for AGR2 gene expression which was downregulated in 13 with nine by more than a 50% reduction; for the four cases of up-regulation, three were increased by more than two-fold (p=0.076, Wilcoxon signed ranks test). Several estrogen response genes (TFF1, PGR, and the ratio of ESR1:ESR2) exhibited significant down-regulation (p≤0.007). Further, there was no evidence of linear correlation (p&gt;0.28; Spearman’s rho) between change in AGR2 and any of these other estrogen response genes. Lastly, when an Estrogen Response Gene Index (ERGI) was constructed using the average log2(fold change) for TFF1, PGR, and the ratio of ESR1:ESR2, down regulation was observed in 16 of the 17 paired specimens with only one increase. ERGI and AGR2 change indicate favorable modulation by acolbifene and appear to provide independent information. Conclusions These results suggest that change in expression of benign breast AGR2 might be a useful addition to change in expression of other classic estrogen response genes in evaluating the potential of novel SERMs such as acolbifene compared to standard SERMs like tamoxifen in early phase prevention trials.

Citation Format: Carol J. Fabian, Teresa A. Phillips, Bruce F. Kimler. AGR2, an estrogen response gene associated with tamoxifen resistance, is modulated by acolbifene in premenopausal women at high risk for development of breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-10-04.