p53 regulation on GLT1D1-induced autophagy

Abstract

Autophagy is a type of programmed cell death, which is an evolutionary conserved selfdigestive mechanism important in maintaining cellular homeostasis. It involves cellular degradation of cytoplasmic components through the formation of autophagosome vesicles and subsequent fusion with lysosomes in response to nutrient deprivation or metabolic stresses. Recently, autophagy has been shown to play key roles in suppression of various human cancers, including breast cancer. Breast cancer is the most common cancer and one of the leading causes of cancer-related mortality in females, with nearly one million new cases reported worldwide each year. In particular, New Zealand has one of the highest rates of breast cancer, accounting for almost 30% of all newly diagnosed cancers each year. Mutations in p53, a critical tumour suppressor gene involved in the regulation of various cellular functions, are associated with mammary carcinogenesis, and it has been demonstrated to be associated with activation of autophagy in breast cancer. Our preliminary studies have shown that overexpression of a novel human protein, glycosyltransferase 1 domain containing 1 (GLT1D1) results in significant autophagic cell death in breast cancer cells, and that the transcription of p53 is increased by GLT1D1. Therefore, my master’s project aimed to identify the mechanisms of p53 regulation on GLT1D1-induced autophagy in mammary carcinoma. Two breast cancer cell lines, MCF-7 (endogenous p53) and BT-549 (mutant p53) were used to generate stable cell lines with endogenous p53 knock-down by RNA interference approaches. We demonstrated that endogenous p53 localised predominantly to the nucleus was associated with autophagy induction. Mutant p53 was localised in both the nucleus and the cytoplasm, and the cytoplasmic mutant p53 was particularly associated with autophagy inhibition. In addition, the roles of p53 in GLT1D1-induced autophagy were studied by transient forced expression of GLT1D1 in each stable cell line. We demonstrated that endogenous p53 increased LC3-II autophagosome protein expression and caused accumulation of DsRed2-LC3B tagged autophagosome in cells expressing GLT1D1, which are characteristics of autophagy induction. Cytoplasmic mutant p53 also increased LC3-II levels while decreasing the number of autophagosomes in GLT1D1-transfected cells. Thus, this increase was likely due to autophagy inhibition. More studies are required for better understanding of p53 and potential roles of GLT1D1 in the regulation of autophagy. Full elucidation of molecular pathways that control p53- mediated GLT1D1-induced autophagy may provide opportunities for the development of novel therapeutic approaches for breast and other cancers.