Abstract C60: Vitamin C inhibits the survival and growth of colorectal cancer cells with KRAS or BRAF mutations

Abstract

Abstract Although more than half of colorectal cancer patients have either KRAS or BRAF mutations, targeted therapies for these subgroups of cancer patients are lacking. We previously showed that isogenic human colorectal cancer cell (CRC) lines harboring a KRAS or BRAF mutation up-regulate the expression of glucose transporter 1 (GLUT1), and increase glucose uptake and glycolysis. In this study, we go on to show that KRAS or BRAF mutant cells exhibit a significant increase in the uptake of vitamin C, mainly in its oxidized form, dehydroascorbic acid (DHA), relative to their wild-type counterparts through GLUT1. Notably, CRCs did not uptake vitamin C in its reduced form, possibly due to the fact that its known transporters, sodium vitamin C cotransporters (SVCTs) were generally not present in CRCs. Deletion of GLUT1 in mutant cells, overexpression of GLUT1 in wild-type cells and competition assays between glucose and DHA confirmed that GLUT1 is both necessary and sufficient for the uptake of vitamin C in CRCs. Both mutant and wild-type cells were able to grow in low glucose (1-2 mM) at a similar rate. In the same condition, we found vitamin C to be selectively toxic to KRAS and BRAF mutant cells, causing them to undergo apoptosis. In contrast, in high glucose levels (>10 mM), a similar concentration of vitamin C had no effect on the survival and growth of mutant cells. In addition, vitamin C significantly inhibited the growth of cancer cells with KRAS or BRAF mutations in vivo xenografts models. To find the mechanism by which vitamin C induces cell death in KRAS or BRAF mutant cells, we performed targeted metabolomics using LC/MS. Both unlabeled total metabolite analysis and flux analysis with C13-Glucose isotope strongly suggested that vitamin C re-routes glucose usage from aerobic glycolysis and non-oxidative pentose phosphate pathway (PPP) to the oxidative PPP to compensate for the lower levels of NADPH which was rapidly consumed to reduce GSSH to GSH. Having taken up exclusively DHA, the oxidized state of vitamin C, the mutant cells immediately use GSH to reduce Vitamin C, resulting in the accumulation of GSSH. Shunting glucose to the oxidative PPP reduces aerobic glycolysis, lowering ATP levels, and decreases glucose anabolism, disfavoring the synthesis of nucleic acids, lipid and amino acids, all needed for the survival and growth of mutant cells. Surprisingly the level of reactive oxidative stress (ROS) was largely unchanged following vitamin C treatment possibly because increased levels of vitamin C served as an anti-oxidant in mutant cells. There are currently more than three clinical trials phase I/II examining the effect of high dose vitamin C via intravenous administration in patients against lymphoma or solid cancers including CRCs. Our findings will provide proof of concept that KRAS or BRAF mutations in CRC can be predictive biomarkers for vitamin C therapy. Citation Format: Jihye Yun, Adam Kavalier, Jatin Roper, Steve Gross, Carlo Rago, Nickolas Papadopoulos, Bert Vogelstein, Lewis Cantley. Vitamin C inhibits the survival and growth of colorectal cancer cells with KRAS or BRAF mutations. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C60.