Abstract P5-04-02: The <i>KRAS</i>-variant induces an EMT in normal breast epithelial cells
Authors: S-Y Jung, D Salzman, K Nguyen, E Pak, J King, S Nallur, J Weidhaas
Journal: Cancer Research (2018)

Abstract Background: The KRAS-variant is a functional, microRNA binding site variant in the 3'untranslated regions (3'UTR) of the KRAS oncogene, which predicts increased cancer risk for certain populations of patients, including an increased risk of triple negative breast cancer [1], and multiple primary breast cancers [2]. Tumors in KRAS-variant patients exhibit altered biology with a KRAS-addicted signature, an estrogen negative basal-like gene expression pattern, and a unique response to cancer therapy. Our goal was to better understand the underlying normal cellular biology associated with the KRAS-variant using an isogenic normal epithelial cell line model. Methods: Perfectly matched, isogenic normal breast epithelial cell lines with (MCF10AKRAS+/-, MT1 and MT2) versus without the KRAS-variant (MCF10AKRAS-/-, WT) were created and used in these studies [2]. The underlying molecular changes leading to EMT, stem cell phenotypes and altered microRNA expression were evaluated by targeted qRT-PCR, immunofluorescence, immunoblotting, immunoprecipitation, flow cytometry, global microRNA array and human phospho-kinase array analysis. Results: We found that KRAS-variant MT cells display a mesenchymal phenotype. We confirmed the downregulation of epithelial markers (E-cadherin and Occludin), upregulation of mesenchymal markers (Vimentin, Fibronectin and N-cadherin) and transcription factors (SNAIL and ZEB1) involved in EMT, as well as decreased level of miR-200c (1000-fold), consistent with EMT induction in the presence of the KRAS-variant. We found that KRAS-variant MT cells also exhibited elevated TGFß, consistent with our findings in KRAS-variant patients [3]. We further found that TGFβ signaling in KRAS-variant MT cells appears to lead to EMT through non-canonical signaling through MAPK (ERK) and PI3K (AKT) and AKT/GSK3β/Snail pathways. Conclusions: KRAS-variant MT normal epithelial cells exhibit an EMT phenotype and genotype, apparently through a non-canonical TGFß/AKT-mediated pathway. This is the first evidence to our knowledge of the powerful impact that a 3'UTR germ-line mutation can have on epithelial normal cellular biology, opening up numerous avenues for further elucidation of germ-line human variants in biology and their clinical applications to breast cancer risk. References: 1. Paranjape et al. A 3'-untranslated region KRAS variant and triple-negative breast cancer: a case-control and genetic analysis. Lancet Oncology, 2011. 12(4): p. 377-86 2. McVeigh et al. Estrogen withdrawal, increased breast cancer risk and the KRAS-variant. Cell Cycle, 2015. 14(13): p. 20919.PMC461452 3. Weidhaas, J.B. et al. The KRAS-variant and Cetuximab Response in Head and Neck Squamous Cell Cancer, A Secondary Analysis of a Randomized Cinical Trial. JAMA Oncology, 2017. 3(4): p. 483-491 Citation Format: Jung S-Y, Salzman D, Nguyen K, Pak E, King J, Nallur S, Weidhaas J. The KRAS-variant induces an EMT in normal breast epithelial cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-04-02.