Abstract 2872: Estrogen withdrawal and breast cancer development in the presence of the KRAS-variant

Background: The KRAS-variant is a germ-line, microRNA binding site mutation in the KRAS oncogene, which predicts increased breast cancer risk and aggressive breast tumor biology. We have recently shown clinically that estrogen withdrawal enhances the risk of breast cancer (BC) development and predicts aggressive tumor biology for women with the KRAS-variant. To better understand our clinical results, we have studied the impact of the KRAS-variant and estrogen withdrawal in a newly created isogenically matched normal breast epithelial line, with (MCF10aKRAS+/-; MUT1 and MUT2) versus without (MCF10aKRAS-/-; PAR) the KRAS-variant. Methods: We evaluated phenotypic alterations in appearance, gene and microRNA expression and growth in the lines with the KRAS-variant versus the parental line. We further characterized the impact of estrogen withdrawal on cellular transformation. We used MTT (proliferation), 3-D culture on Matrigel™ (acini formation), and soft agar colony formation (anchorage independent growth) to evaluate growth and transformation differences. We used charcoal stripped media, as well as other clinically used anti-estrogen medications to simulate estrogen withdrawal. We also investigated the underlying molecular changes that lead to transformation in our isogenic MUT cell lines by performing targeted qRT-PCR, western blotting, and Immunocytochemistry. Results: When grown under standard culture conditions, both MUT lines exhibited a fibroblast-like, mesenchymal appearance. Consistent with an epithelial to mesenchymal transition (EMT), the MUT lines showed downregulation of epithelial markers (E-cadherin and Occludin) and upregulation of mesenchymal markers (vimentin, and fibronectin). Furthermore, in 3D culture MUT cells formed small irregularly shaped spheroids, whereas MCF10a PAR cells formed well-organized, polarized spheroids, supporting a mesenchymal phenotype for MUT lines. In anchorage independent growth analysis, MUT1 and MUT 2 cell lines did not form colonies in soft Agar when grown in regular growth culture conditions. However, both MUT cell lines exhibited enhanced transformation with estrogen withdrawal, with a 2-fold increase in colony formation rate in the presence of tamoxifen, a 6.2 fold increase in colony formation rate in charcoal stripped media, and a 7.9 fold increase in colony formation rate with the combination. Conclusions: These findings confirm that normal breast epithelial cells with the KRAS-variant are biologically different than non-variant cells, and that estrogen withdrawal increases their transformation. Studies are ongoing to determine the impact of additional anti-estrogen medications used clinically on the rate of transformation in these lines, as well as to better characterize baseline and oncogenic changes in the presence of the KRAS-variant. Citation Format: Song-Yi Jung, David Salzman, Joanne B. Weidhaas. Estrogen withdrawal and breast cancer development in the presence of the KRAS-variant. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2872. doi:10.1158/1538-7445.AM2015-2872