M. Nagahashi1, M. Nakajima1, T. Saito2, M. Komatsu2, T. Soga3, R. Zhao4, H. Zhou4, S. Okuda5, K. Takabe6, T. Wakai1 1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, NIIGATA, Japan 2Niigata University Graduate School Of Medical And Dental Sciences,Department Of Biochemistry,Niigata, NIIGATA, Japan 3Keio University,Institute For Advanced Biosciences,Tsuruoka, YAMAGATA, Japan 4Virginia Commonwealth University School Of Medicine,Department Of Microbiology And Immunology, Medical College Of Virginia Campus,Richmond, VA, USA 5Niigata University Graduate School Of Medical And Dental Sciences,Bioinformatics,Niigata, NIIGATA, Japan 6Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA
Introduction: Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that regulates many physiological and pathological processes. S1P exerts its function either intracellularly or extracellularly after produced by sphingosine kinases (SphK1 and SphK2) inside the cells. Previous studies suggested that S1P and SphKs play important roles in cancer cell survival. Indeed, expression of SphK1 has been reported to be associated with worse prognosis of breast cancer patients. We hypothesized that SphK1 affects cancer cell-specific metabolism, such as “Warburg effect” in which cancer cells predominantly produce energy by a high rate of glycolysis, and plays a role in cancer cell survival. The aim of this study is to test the hypothesis that SphK1 regulates breast cancer cell metabolism by metabolome analysis.
Methods: SphK1 was downregulated by SphK1-shRNA in 4T1 murine mammary adenocarcinoma cell line. Proliferation and migration assays were performed to examine the cell biology. Metabolic changes in 4T1 cells were analyzed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) between sh-control (sh-Ct) or sh-SphK1 treated cells.
Results: Proliferation assays revealed significantly less cell proliferation of 4T1 sh-SphK1 cells compared to sh-Ct cells under serum free condition. Cell migration was also suppressed with down-regulation of SphK1. CE-TOFMS analysis revealed the metabolomics profiles of 4T1 sh-SphK1 cells, which were dramatically changed in the glycolysis pathway, amino acid synthesis, and tricarboxylic acid (TCA) cycle compared to the sh-Ct cells. The levels of most nucleotides such as ATP, ADP, GTP, and GDP in 4T1 sh-SphK1 cells were significantly lower than those in sh-Ct cells. Moreover, 4T1 sh-SphK1 cells contained lower amount of glutathione (GSH) than sh-Ct cells, which indicate an important role of SphK1 in not only cell survival, but also oxidative stress and drug resistance.
Conclusion: Our results indicate that SphK1 plays pivotal roles in cancer specific metabolism, which strengthen resistance to oxidative stress and cancer cell survival. SphK1 will be a promising target for patients with breast cancer.