Abstract 2207: High-dose omega-3 fatty acid supplementation is associated with a decrease in FOXA1 in benign breast tissue
Authors: Fabian Carol J. , Metheny Trina , Phillips Teresa A. , Danley Marsha , Kimler Bruce F. 
Published: June 2022

Abstract
Background: FOXA1 is a pioneer transcription factor necessary for full expression of estrogen receptor activity. In collaboration with Grainyhead-like 2 (GRHL2), increased FOXA1 activity can result in endocrine resistance through increase of the endoplasmic reticulum protein AGR2 and its receptor LYPD3. Hyperglycemia and hyperinsulinemia increase FOXA1. Since eicosapentaenoic and docosahexaenoic omega-3 fatty acids (EPA and DHA) improve insulin sensitivity and reduce IGFR-related signaling, we explored the effect of EPA + DHA supplementation on FOXA1 and AGR2 expression in benign breast epithelium.
Methods: Reserved excess cells from fine needle aspirations performed pre and post 6 months of supplementation given to postmenopausal women at increased risk for breast cancer were utilized. Cells had been stored at -20 C in a methanol fixative for ~10 y (Fabian et al, Ca Prev Res 2015). Of the 35 women entered, 28 had reserved excess cells available for blocks and after obtaining 4-5 sections per block, 12 had sufficient epithelial cells on both pre and post intervention slides as determined by H and E staining to proceed with immunohistochemistry assessment. Of the 12, median age was 53 and 7/12 were on low dose hormone replacement during the study. Slides were stained with FOXA1 (2F83 Mouse Monoclonal Antibody) at a 1:50 dilution and AGR2 rabbit polyclonal (Proteintech,12275-1-AP) at a 1:800 dilution. F0XA1 exhibits a nuclear staining pattern whereas AGR2 is predominately cytoplasmic. IHC staining was assessed individually by two readers (TM and TP). The most abnormal cell clusters were preferentially assessed. Up to 500 cells are evaluated for percent positive stain and staining intensity. An Allred score was computed as the sum of a categorical score for percent positive cells and a categorical score for the predominant staining intensity.
Results: For FOXA1 there was a statistically significant decrease in percent positive cells (11/12, p=0.019) and Allred score (9/12, p=0.022) by Wilcoxon signed rank test. Of the 11 which showed a decrease in FOXA1, seven were on HRT throughout the study (usually estrogen alone). There was no statistically significant change in AGR2 IHC. However, there was a robust linear relationship between stain positivity for FOXA1 and AGR2 (p&lt;0.001), as well as for the change in percent positive cells over the course of the intervention (p&lt;0.003). There was no statistically significant correlation between change in FOXA1 and change in Ki-67 immunocytochemical staining (performed as part of the original study), nor for a variety of serum measures of insulin sensitivity or resistance including HOMA-IR and HOMA %B.
Conclusions: If this finding is confirmed in other studies, FOXA1 protein expression may be a useful tissue response biomarker in breast cancer prevention trials of omega-3 fatty acids.
Citation Format: Carol J. Fabian, Trina Metheny, Teresa A. Phillips, Marsha Danley, Bruce F. Kimler. High-dose omega-3 fatty acid supplementation is associated with a decrease in FOXA1 in benign breast tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2207.