Antisense glutaminase inhibition modifies the O-GlcNAc pattern and flux through the hexosamine pathway in breast cancer cells.
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.
Donadio
Ana C
AC
Departamento de Bioquímica Clínica, Centro de Investigaciones en Bioquímica Clínica e Inmunología, CIBICI-CONICET, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de Torre y Medina Allende, Ciudad Universitaria, Córdoba, Argentina.
Lobo
Carolina
C
Tosina
Marta
M
de la Rosa
Vanessa
V
Martín-Rufián
Mercedes
M
Campos-Sandoval
José A
JA
Matés
José M
JM
Márquez
Javier
J
Alonso
Francisco J
FJ
Segura
Juan A
JA
D000818:Animals; D001943:Breast Neoplasms / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D015870:Gene Expression / Q000235:genetics; D005972:Glutaminase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D006595:Hexosamines / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics; D040901:Proteomics; D034622:RNA Interference; D016329:Sp1 Transcription Factor / Q000096:biosynthesis* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D006595:Hexosamines; D016329:Sp1 Transcription Factor; D005973:Glutamine; D014537:Uridine Diphosphate N-Acetylglucosamine; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D005972:Glutaminase