Antisense glutaminase inhibition modifies the O‐GlcNAc pattern and flux through the hexosamine pathway in breast cancer cells

Abstract

Glutamine behaves as a key nutrient for tumors and rapidly dividing cells. Glutaminase is the main glutamine-utilizing enzyme in these cells, and its activity correlates with glutamine consumption and growth rate. We have carried out the antisense L-type glutaminase inhibition in human MCF7 breast cancer cells, in order to study its effect on the hexosamine pathway and the pattern of protein O-glycosylation. The antisense mRNA glutaminase expressing cells, named ORF19, presented a 50% lower proliferation rate than parental cells, showing a more differentiated phenotype. ORF19 cells had an 80% reduction in glutamine:fructose-6-P amidotransferase activity, which is the rate-limiting step of the hexosamine pathway. Although the overall cellular protein O-glycosylation did not change, the O-glycosylation status of several key proteins was altered. O-glycosylation of O-GlcNAc transferase (OGT), the enzyme that links N-acetylglucosamine to proteins, was fivefold lower in ORF19 than in wild type cells. Inhibition of glutaminase also provoked a 10-fold increase in Sp1 expression, and a significant decrease in the ratio of O-glycosylated to total protein for both Sp1 and the Rpt2 proteasome component. These changes were accompanied by a higher Sp1 transcriptional activity. Proteome analysis of O-glycosylated proteins permitted the detection of two new OGT target proteins: the chaperonin TCP-1 theta and the oncogene Ets-related protein isoform 7. Taken together, our results support the hexosamine pathway and the O-glycosylation of proteins being a sensor mechanism of the nutritional and energetic states of the cell.

Glucose Deprivation-induced Increase in Protein O-GlcNAcylation in Cardiomyocytes Is Calcium-dependent

“…The majority of studies focusing on the role of O-GlcNAc in regulating cellular function have been in the context of chronic diseases including cancer, age-related diseases, diabetes, and diabetic complications (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). However, in 2004 Zachara et al (15) demonstrated for the first time that there was an acute increase in cellular O-GlcNAc levels in response to a range of stress stimuli and importantly that inhibition of this response increased cell death and conversely that augmenting this response increased tolerance of cells to stress.…”

Early Detection of Breast Cancer Using Posttranslationally Modified Biomarkers

“…Although studies have revealed that CA-125 is not tumor specific, 20,21 the strategy of combining multiple tumor markers found in serum has been shown to facilitate the detection of early stage tumors and accurately identify tumor recurrence. 22,23 Unfortunately, serum markers cannot be used to determine tumor volume or location, which is critical information in the clinical management of cancer patients.…”

Reprogrammed Cellular Metabolism and O -GlcNAc Modification in Cancer

“…Furthermore, oncogenes such as MYC, NF-kB, AKT, tyrosine kinases and tumour suppressor genes including TP53 and PTEN that have been linked to increased glycolysis, have also been implicated in the upregulation of glutamine [28]. Glutamine function as a source of both nitrogen and carbon and contains amino and amido nitrogens, which are transferred to metabolic intermediates in the synthesis of nucleic acids, proteins, and hexosamines making it a crucial nutrient during cell proliferation [29,30]. It is not surprising that novel imaging strategies focusing on glutamine that could provide a valuable complement to 18 F-FDG PET, because glutamine complements glucose in the metabolic platforms that support tumour growth at the cellular level [29].…”

“…It would be very challenging to distinguish the target specific effect from nontarget effect of such global approaches. Although it has been proposed that increased glucose uptake/glycolysis in cancer cells may lead to increased glucose flux through HBP and subsequently increased O-GlcNAc modification in a number of proteins [7,30]. However, to the best of my knowledge no attempt has been made to study the effect of inhibitors of glycolysis on HBP, O-GlcNAc level and O-GlcNAc protein modification in cancer cells.…”

“…However, to the best of my knowledge no attempt has been made to study the effect of inhibitors of glycolysis on HBP, O-GlcNAc level and O-GlcNAc protein modification in cancer cells. Furthermore, such hypothesis is not tenable to explain the simultaneous downregulation of O-GlcNAc modification in a number of proteins in cancer cells [30]. Although not well understood, such findings are not surprising, keeping in mind that O-GlcNAc modification in protein is a highly regulated process and involved not only O-GlcNAc level, O-GlcNAc cycling enzymes OGT and OGA but also their interacting proteins and various modification status of the substrate itself [9,15].…”