The Mechanism Of SLC6A8-mediated Creatine Uptake On Hypoxic TNBC Cell Survival Via Ameliarating Oxidative Stress

Background:As the most aggressive subtype of breast cancer,triple-negative breast cancer（TNBC）is a major threat to female health.Hypoxia is a key feature of TNBC.Metabolic adaptation promotes progression of TNBC cells that are located within the hypoxic tumor regions.However,it is not well understood regarding the precise molecular mechanisms underlying the regulation of metabolic adaptions by hypoxia.Methods:To analyze the gene expression profiles in TNBC cells MDA-MB-231（20%O₂ vs 1%O₂）.Quantitative real-time PCR was conducted to detect the expression of slc6a8,the creatine transporter protein encoding gene,in breast cancer cells and tissues.Immunohistochemistry was performed to detect SLC6A8 protein abundances in tumor tissues.Clinicopathologic correlation and overall survival were evaluated by chi-square test and Kaplan-Meier analysis,respectively.Cell viability assay and flow cytometry analysis with Annexin V/PI double staining were performed to investigate the impact of SLC6A8-mediated uptake of creatine on viability of hypoxic TNBC cells.TNBC orthotopic xenograft model was used to evaluate the effect of creatine in vivo.Results:SLC6A8 was aberrantly upregulated in TNBC cells in hypoxia.SLC6A8 was dramatically enhanced in TNBC tissues and its level was tightly associated with higher histological grading,advanced TNM stage and poorer overall survival of TNBC patients.We found that SLC6A8 was transcriptionally upregulated by p65/NF-κB and mediated accumulation of intracellular creatine in hypoxia.SLC6A8-mediated accumulation of creatine promoted survival and inhibited apoptosis via maintaining redox homeostasis in hypoxic TNBC cells.Furthermore,creatine was required to facilitate tumor growth in xenograft mouse models.Mechanistically,intracellular creatine bolstered cell antioxidant defense by reducing mitochondrial activity and oxygen consumption rates to reduce accumulation of intracellular reactive oxygen species,ultimately leading to activation of AKT-ERK signaling.The AKT-ERK signaling-mediated upregulation of Ki-67 and Bcl-2,and downregulation of Bax and cleaved Caspase-3 contributed to increased cell viability of hypoxic TNBC cells.Conclusions:Our study indicates that SLC6A8-mediated creatine accumulation plays an important role in promoting TNBC progression,and may provide a potential therapeutic option for treatment of SLC6A8 high expressed TNBC.