Abstract 790: Vitamin K pathway functions in triple-negative breast cancer

Abstract

Abstract The vitamin K pathway comprises three major enzymes: gamma-glutamyl carboxylase (GGCX) and the vitamin K oxidoreductases VKORC1 and VKORC1L1. GGCX requires the reduced form of vitamin K as a co-factor to post-translationally generate γ-carboxylated (GLA) residues in proteins. Either VKORC1 or VKORC1L1 can reduce the oxidized form of vitamin K to maintain pathway activity. Interrogation of TCGA data indicates a subset of invasive breast cancer tumors harbor mutations in GGCX (8%), VKORC1 (9%), and/or VKORC1L1 (9%), with nearly all alterations being amplifications. As these data suggest upregulation of these genes is associated with aggressive breast cancer, we measured their expression in a variety of established breast cancer cell lines. qPCR results correlated with the TCGA data indicating the highest expression of GGCX, VKORC1 and VKORC1L1 expression in the two triple-negative breast cancer (TNBC) cell lines, Hs578T and SUM159PT. Since the function of these genes is to generate GLA residues in proteins, we used Western blotting to detect GLA-modified proteins in TNBC cells cultured in the presence or absence of vitamin K. In lysates from cells maintained in vitamin K, multiple bands representing GLA-modified proteins were observed between 40-60 kDa and 80 kDa. These bands were not present in lysates of cells grown in standard media that are typically devoid of vitamin K. Treatment with the VKOR antagonist, warfarin, decreased expression of these GLA-modified proteins in cells cultured in vitamin K. Thus, our data suggest that vitamin K acts in TNBC cells to produce GLA-modified proteins, and that these effects are abrogated by warfarin. To confirm the presence of a functional vitamin K pathway, we used a vitamin K-dependent reporter protein (FIXgla-PC) to measure γ-carboxylation activity. TNBC cells stably transfected with the FIXgla-PC reporter were cultured in the presence and absence of vitamin K, with or without warfarin, and secretion of γ-carboxylated FIX-gla was measured by ELISA. FIX-gla γ-carboxylation was enhanced in the presence of vitamin K and blocked by warfarin in both Hs578T and SUM159PT cell lines. These data confirm that the classical vitamin K pathway is activated in these representative TNBC cell lines when cultured in vitamin K-supplemented media. To assess the biologic relevance of this pathway in TNBC, mammosphere assays were performed with Hs578T and SUM159PT cells. Both cell lines formed significantly more mammospheres when cultured in the presence of vitamin K, suggesting that γ-carboxylation may enhance stemness. Further studies to identify the GLA-modified proteins generated in TNBC cells treated with vitamin K and their mechanisms of action may lead to new therapeutic targets for aggressive TNBC. Citation Format: Sarah Beaudin, Leila Kokabee, JoEllen Welsh. Vitamin K pathway functions in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 790.