Abstract

O-GlcNAcylation (addition of N-acetyl-glucosamine on serine or threonine residues) is a post-translational modification that regulates stability, activity or localization of cytosolic and nuclear proteins. O-linked N-acetylgluocosmaine transferase (OGT) uses UDP-GlcNAc, produced in the hexosamine biosynthetic pathway to O-GlcNacylate proteins. Removal of O-GlcNAc from proteins is catalyzed by the β-N-Acetylglucosaminidase (OGA). Recent evidences suggest that O-GlcNAcylation may affect the growth of cancer cells. However, the consequences of O-GlcNAcylation on anti-cancer therapy have not been evaluated. In this work, we studied the effects of O-GlcNAcylation on tamoxifen-induced cell death in the breast cancer-derived MCF-7 cells. Treatments that increase O-GlcNAcylation (PUGNAc and/or glucosoamine) protected MCF-7 cells from death induced by tamoxifen. In contrast, inhibition of OGT expression by siRNA potentiated the effect of tamoxifen on cell death. Since the PI-3 kinase/Akt pathway is a major regulator of cell survival, we used BRET to evaluate the effect of PUGNAc+glucosamine on PIP3 production. We observed that these treatments stimulated PIP3 production in MCF-7 cells. This effect was associated with an increase in Akt phosphorylation. However, the PI-3 kinase inhibitor LY294002, which abolished the effect of PUGNAc+glucosamine on Akt phosphorylation, did not impair the protective effects of PUGNAc+glucosamine against tamoxifen-induced cell death. These results suggest that the protective effects of O-GlcNAcylation are independent of the PI-3 kinase/Akt pathway. As tamoxifen sensitivity depends on the estrogen receptor (ERα) expression level, we evaluated the effect of PUGNAc+glucosamine on the expression of this receptor. We observed that O-GlcNAcylation-inducing treatment significantly reduced the expression of ERα mRNA and protein, suggesting a potential mechanism for the decreased tamoxifen sensitivity induced by these treatments. Therefore, our results suggest that inhibition of O-GlcNAcylation may constitute an interesting approach to improve the sensitivity of breast cancer to anti-estrogen therapy.

Highlights

  • Growth and proliferation of cancer cells tightly depend on their nutritional environment, on glucose availability, which is necessary for increased biosynthesis of cellular components associated with proliferation [1]

  • Similar results were observed after 48 h of culture with the different agents (Figure 2B). These results suggest that treatment with PUGNAc and GlcN protects MCF-7 cells form 4OH-Tamoxifen-induced cell death

  • To determine whether PUGNAc+GlcN protective effects against 4-OH-Tamoxifen-induced cell death were mediated by their stimulatory effect on PI-3 kinase/Akt pathway, we evaluated the effects of these treatments in presence of LY294002, a PI-3 kinase inhibitor

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Summary

Introduction

Growth and proliferation of cancer cells tightly depend on their nutritional environment, on glucose availability, which is necessary for increased biosynthesis of cellular components associated with proliferation (e.g. membranes, proteins and nucleic acids) [1]. Nutritional and metabolic conditions are known to influence tumour development. Food restriction has inhibitory effects on the growth of certain tumours [3], whereas in diet-induced obesity models, overfeeding is associated with accelerated development of tumours [4]. Nutritional conditions can modulate tumour development by modifying insulin and IGF-1 concentrations, which affect signalling pathways involved in cell growth, proliferation and apoptosis. At the cellular level, glucose can directly regulate signalling pathways and multiple biological processes through O-GlcNAc glycosylation (O-GlcNAcylation) of cytosolic and nuclear proteins [5].

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