Abstract 1930: Impacts of the KRAS-variant on breast cell biology
Authors: Jung Song-Yi , Weidhaas Joanne 
Published: July 2016

Abstract
Background: The KRAS-variant is a germ-line, microRNA binding site mutation in the KRAS oncogene, which is associated with an increased risk of cancer and altered response to cancer treatment. We investigated the hypothesis that normal cells with the KRAS-variant would be biologically unique.
Methods: We created perfectly matched, isogenic normal breast epithelial lines with (MCF10AKRAS+/−, MT) and without (MCF10AKRAS-/−, WT) the KRAS-variant. We also created isogenically matched MCF10A cells with p53 gene-specific stable knockdown with (MLPKRAS+/−) and without (MLPKRAS-/−) the KRAS-variant. We evaluated phenotypic alterations in appearance, gene and microRNA expression and growth in the KRAS-variant vs the parental lines. We used 3-D culture on Matrigel™ (acini formation), and soft agar colony formation (anchorage independent growth) to evaluate growth and transformation. We also investigated EMT and stem cell analysis by performing targeted qRT-PCR, western blotting and flow cytometry.
Results: We found that KRAS-variant normal breast epithelial lines (MT) exhibited a mesenchymal phenotype, exhibiting a spindle-like morphology vs the parental (WT) cells, consistent with a baseline epithelial to mesenchymal transition (EMT). In agreement with their appearance, MT cells had significantly lower E-Cadherin and Occludin, and significantly higher Fibronectin and Vimentin than the WT line. We also found that mir-200c was dramatically down-regulated in the MT line, being 828-fold suppressed. miR-200c has been shown to regulate EMT induction and self-renewal and proliferation of stem cells. We thus next investigated whether the MT line had an increased stem cell component. By sorting cells for CD44+/CD24low/− fraction and ALDH1 positivity, we found that MT cells had a significantly higher proportion of CD44+/CD24low/− cells, and higher levels of ALDH1positive cells. In 3-D culture, MT cells formed small irregularly shaped spheroids, whereas WT cells formed well-organized, polarized spheroids, supporting a mesenchymal phenotype. In the MLPKRAS+/− cells we also found a higher stem cell component, however we did not see an EMT phenotype. However, MLPKRAS+/− cells formed disorganized acinar structures, similar to transformed cells, while the MLPKRAS-/− cells formed single, normal round acini. We next plated these cell line in soft agar to test for baseline transformation. While the KRAS-variant normal breast epithelial cells were not transformed at baseline, MLPKRAS+/− cells exhibit colony formation in soft agar, consistent with transformation.
Conclusions: These findings indicate that KRAS-variant normal cells are biologically different than cells without the KRAS-variant, but are not transformed. In contrast, p53 suppression in addition to the KRAS-variant is associated with cellular transformation, similar to findings with mutated KRAS. Studies are ongoing to better characterize baseline and oncogenic changes in the presence of the KRAS-variant.
Citation Format: Song-Yi Jung, Joanne Weidhaas. Impacts of the KRAS-variant on breast cell biology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1930.