M. Nagahashi1, M. Nakajima1, M. Abe2, T. Saito3, M. Komatsu3, T. Soga4, J. Tsuchida1, K. Yuza1, K. Takabe5,6, K. Sakimura2, T. Wakai1 1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata City, NIIGATA, Japan 2Brain Research Institute, Niigata University,Department Of Cellular Neurobiology,Niigata City, NIIGATA, Japan 3Niigata University Graduate School Of Medical And Dental Sciences,Department Of Biochemistry,Niigata City, NIIGATA, Japan 4Keio University,Institute For Advanced Biosciences,Tsuruoka City, YAMAGATA, Japan 5Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 6University At Buffalo Jacobs School Of Medicine And Biomedical Sciences, The State University Of New York,Department Of Surgery,Buffalo, NY, USA
Introduction:
Cancer cells reprogram their metabolism to promote proliferation, survival, and long-term maintenance. The common feature of this altered metabolism is the increased glucose uptake and fermentation of glucose to lactate, which has been known as “Warburg effect”. However, the mechanism how cancer cells regulate their metabolism has not yet revealed. 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. Previously our group and others have demonstrated that S1P and SphKs play important roles in cancer cell survival. We have recently published that expression of SphK1 associates with worse prognosis of breast cancer patients. Thus, we hypothesized that SphKs regulates cancer cell-specific metabolism, including “Warburg effect”.
Methods:
SphK1 or SphK2 knock-out (KO) E0771 murine breast cancer cell lines were generated with a CRISPR/Cas9 mediated targeted deletion of the SphK1 or SphK2 gene. Proliferation was assessed by WST-8. Metabolic changes in SphK1KO and SphK2KO E0771 cells were analyzed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) between SphK1/SphK2KO and their corresponding control E0771 cells.
Results:
Proliferation assays revealed significantly less cell proliferation of SphK1KO E0771 cells compared to the control cells. On the other hand, SphK2KO E0771 cells showed significantly more proliferation than the control cells. CE-TOFMS analysis revealed the metabolomics profiles of both SphK1KO and SphK2KO E0771 cells, which were dramatically changed in the glycolysis pathway and tricarboxylic acid (TCA) cycle compared to the control cells. Interestingly, SphK1KO E0771 cells contained lower amount of glutathione (GSH) than control cells, while SphK2KO E0771 cells contained significantly higher amount of GSH than control cells. Considering that GSH plays roles in oxidative stress and drug resistance, our findings indicate an important role of SphK1 and SphK2 in not only cell survival, but also oxidative stress and drug resistance.
Conclusion:
Our results indicate that SphKs play pivotal roles in cancer specific metabolism, which strengthen resistance to oxidative stress and cancer cell survival. SphKs will be a promising target for patients with breast cancer.