Abstract 4281: Adaptive post-translational regulation of the thiamine transporter THTR1 in breast cancer cells during hypoxic stress

Abstract

Abstract Purpose: The cellular metabolic re-programming within hypoxic tumor microenvironments involves adaptations that alter the cellular dependence for glycolysis and the pentose phosphate pathway. However, enzymatic activity within these pathways requires an adequate supply and cellular pool of enzyme cofactors. In particular, thiamine is an essential cofactor within key metabolic pathways that are responsible for energy production and anabolic precursor synthesis. Previously, we have reported on the hypoxia-mediated transcriptional regulation of the thiamine transporters within breast cancer cells. Therefore, the purpose of this study was to further our understanding of hypoxia-mediated regulation of thiamine transporters, by examining the intracellular localization and plasma membrane expression of THTR1 during hypoxic stress. Methods: Immunofluorescence was employed to visualize the intracellular localization of THTR1 (SLC19A2) within the non-transformed breast epithelial cell line, MCF10A and breast cancer cells, BT20 and BT474 after 1, 3 and 7 days of 1% O2 exposure. Cell surface biotinlyation assays were utilized to assess changes in the plasma membrane expression of THTR1 after hypoxia exposure. In addition, total THTR1 expression was determined using Western blotting of whole cell lysates and qRT-PCR. Results: The cellular distribution of THTR1 was predominantly localized in the plasma membrane for the non-transformed MCF10A cells, but was predominantly intracellular in the breast cancer cells, BT20 and BT474. No change in total mRNA or cellular THTR1 protein expression was found after hypoxic exposure. However, THTR1 exhibited a time dependent increase in plasma membrane localization after 1% O2 exposure. This observation was confirmed with an increase in THTR1 plasma membrane expression using cell surface biotinylation pull down assays. Conclusions: We conclude that hypoxic stress induces the intracellular trafficking of the thiamine transporter THTR1 to the plasma membrane. These results suggest that post-translational modification of THTR1 during hypoxic exposure may be an essential adaptive cellular response to increase intracellular supply of thiamine to support glycolytic metabolism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4281. doi:1538-7445.AM2012-4281