Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells

Rafaela Muniz De Queiroz,Rashna Madan,Jeremy Chien,Wagner Barbosa Dias,Chad Slawson

doi:10.1074/jbc.m116.734533

Abstract

O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked β-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function. Because ovarian cancer has a high frequency of p53 mutation rates, we decided to investigate the relationship between O-GlcNAcylation and p53 function in ovarian cancer. We measured a significant decrease in O-GlcNAcylation of tumor tissue in an ovarian tumor microarray. Furthermore, O-GlcNAcylation was increased, and OGA protein and mRNA levels were decreased in ovarian tumor cell lines not expressing the protein p53. Treatment with the OGA inhibitor Thiamet-G (TMG), silencing of OGA, or overexpression of OGA and OGT led to p53 stabilization, increased nuclear localization, and increased protein and mRNA levels of p53 target genes. These data suggest that changes in O-GlcNAc homeostasis activate the p53 pathway. Combination treatment of the chemotherapeutic cisplatin with TMG decreased tumor cell growth and enhanced cell cycle arrest without impairing cytotoxicity. The effects of TMG on tumor cell growth were partially dependent on wild type p53 activation. In conclusion, changes in O-GlcNAc homeostasis activate the wild type p53 pathway in ovarian cancer cells, and OGA inhibition has the potential as an adjuvant treatment for ovarian carcinoma.

Highlights

Like most post-translational modifications, O-GlcNAcylation can alter the function, stability, or localization of a protein
Using a tumor microarray containing 48 ovarian tumors samples and nine normal tissue samples, we evaluated the changes in O-GlcNAcylation, O-linked ␤-N-acetylglucosamine transferase (OGT), and OGA between normal and cancer tissue
We found that in all groups O-GlcNAcylation in the cytoplasm was very low with higher amounts of O-GlcNAcylation occurring in the nucleus

Summary

Introduction

Like most post-translational modifications, O-GlcNAcylation can alter the function, stability, or localization of a protein. Cancer cells exhibit important metabolic differences from normal cells known as the Warburg effect This phenomenon is characterized by a high rate of glycolytic flux and increased glucose uptake by tumor cells [12]. O-GlcNAcylation was proposed as a prognostic factor for prostate cancer and leukemia with high O-GlcNAc levels in leukemia patients leading to a better prognosis [18]; in prostate cancer higher O-GlcNAc correlates with a poor prognosis [19]. All these conflicting data show that O-GlcNAc can have different roles in different tissues, contributing to or impairing tumor development and treatment. Mutations in p53 correspond to poor prognosis and low response to chemotherapy [25]

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