The Effects Of C-Myc/SLC1A5 Signaling Pathway-mediated Reprogramming Of Glutamine Metabolism On The Growth And Radiosensitivity In Nasopharyngeal Carcinoma

Reprogrammed cellular metabolism is one of the most important biological characters of human tumor cells. As far back as 1920s, Otto Warburg had been put forward the well-known theory named "Warburg effect", in which tumor cells were considered to exhibit an increased dependence on anaerobic glycolysis for ATP generation, even in aerobic conditions. With more and more research available, many studies reported that upregulated glutaminolysis has also been recognized as a key character of tumor metabolism, along with increased anaerobic glycolysis. Under a series of interior and exterior factors, glutamine metabolism is reprogrammed in many cancer cells, the exogenous gluatmine is transported into cytoplasm and then take part in a series of metabolic pathway. As carbon/nitrogen source provider in ATP production and macromolecular synthesis, gluatmine not only maintaines redox homeostasis, but also modulates signal transduction pathways, finally promoting cancer cell growth and proliferation. Furthermore, there is a correlation between the alteration of gluatmine metabolism and the level of malignancy in breast cancer. Unfortunately, the relationship between gluatmine metabolism and the development of nasopharyngeal carcinoma (NPC) has not been reported. This thesis is divided into three parts. Firstly, we observed the effects of exogenous gluatmine on growth, proliferation, apoptosis, and radiosensitivity. Secondly, the biologic function of SLC1A5, a potential molecular target for increasing radiosensitization, was aslo analyzed in NPC. Thirdly, we explored the underlying molecular mechanisms of glutamine addiction of nasopharyngeal carcinoma cells.Previous studies have reported that MYC-amplified cancer cells became dependent on glutamine anapleurosis for maintaining the mitochondrial integrity and TCA cycle function. In our preliminary studies, the western blot results also revealed that there were significant differences in protein expression of c-Myc among different types of NPC cell lines, indicating some types of NPC cell lines might depend strongly on exogenous gluatmine. In order to validate the hypothesis, NPC cell lines were treated with different doses of exogenous gluatmine, and the changes in cell biology and radiosensitivity were determined by using a series of biochemical method, including methods of cell counting kit-8, trypanblau dying with cell counting, clonogenic assay, Annexin V-FITC/PI apotosis kit, glucose consumption testing, ROS detection kit, western blot and so on. Our results showed that glutamine deprivation reduced the ability of proliferation, repressed the growth, induced G1 phrase arrest and apoptosis, slowed down the rate of glucose metabolism, and induced the imbalance of redox and enhanced the sensitivity to radiotherapy in c-Myc-high NPC cell lines. However, there were no apparent changes of the phenomena mentioned above in c-Myc-low NPC cell lines, and along with the up-regulated expression of gluatmine synthetase.Previous studies have supplied evidence that overexpression of c-Myc protein relates to poor local control and recurrence in nasopharyngeal carcinoma. We also observed that the c-Myc-amplified NPC cell lines presented several featues such as rapid growth and proliferation, resistance to radiotherapy. Based on the preliminary results of glutamine addition in NPC cells, we proposed that targeting glutamine metabolism-related genes combine with radiotherapy might improve the NPC treatment effectiveness. After the construction of shRNA lentiviral vector targeting SLC1A5 was completed, the effects of SLC1A5 silencing on growth and radiosensitivity of glutamine-addicted NPC cell line were determined. Our results showed that downregulation of SLC1A5 by synthetic shRNA could significantly inhibit growth and increase radiosensitivity in vivo and in vitro.As a transcription factor, c-Myc directly binds the promoter of glutamine transport SLC1A5 and speed up glutamine metabolism in human glioma line SF188. Furthermore, Epstein-barr virus (EB) infection was considered as one of major carcinogenic factors in NPC, and EB-associated proteins had reported to take part in regulation of glucose metabolism in NPC. In order to explore the modulation of c-Myc or EB-associated proteins on glutamine metabolism, we constructed lentiviral vector targeting c-Myc and overexpressing LMP1, LMP2A and EBNA1, our results demonstrated that downregulation of c-Myc gene expression could decrease the protein expression of SLC1A5 and inhibit the uptake of glutamine and the growth of NPC cell line CNE2. Although the expression of exogenous LMP1, LMP2A and EBNA1 was high, no changes in key proteins of glutaminolysis and the consumption of glutamine were observed.