Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis

Abstract

AbstractYeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations inS. cerevisiaethat conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. In addition to three aneuploid strains, we detected missense alleles of theHXK2, REG1, GLC7andSNF1genes that were shown to confer significant resistance to 2-deoxyglucose. All three missense alleles inHXK2resulted in significantly reduced catalytic activity. Missense alleles affecting the Snf1 kinase pathway (REG1,GLC7andSNF1)exhibited different capacities to affect the regulation of invertase expression. Of the seven missense alleles identified in this study, all but one affected hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both theDOG1andDOG2mRNA was elevated after treatment with 2-deoxyglucose. Deletion of theHXK2andREG1genes confers resistance to 2-deoxyglucose and causes increased expression of theDOG2mRNA. We conclude that Snf1 kinase-mediated regulation of the endocytosis of the hexose transporters and regulation ofDOG2expression are important mechanisms for resistance to 2-deoxyglucose. However, the dominantSNF1-G53Rallele can confer additional 2-deoxyglucose resistance in cells that are genetically compromised in both the endocytosis andDOGpathways. Thus at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.Author SummaryYeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Another similarity between yeast cells and human tumor cells is that both cells can acquire resistance to 2-deoxyglucose, an outcome that can limit the usefulness of some cancer therapeutics. In this study, we used bakers’ yeast as a model organism to better understand the mechanism of toxicity and acquisition of resistance to 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were isolated and identified by whole genome sequencing, a technology that was not available until recently. Our studies indicate that 2-deoxyglucose becomes toxic after it is phosphorylated by an enzyme called hexokinase. One important route to resistance is to reduce hexokinase activity. Other parallel pathways to resistance include increased expression of a hydrolase that degrades the toxic metabolite, altered localization of glucose transporters and altered glucose signal transduction pathways.