M. Nagahashi1, J. Tsuchida1, K. Moro1, M. Ikarashi1, M. Nakajima1, M. Abe2, T. Saito3, M. Komatsu3, T. Soga4, 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 Animal Model Development,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,Shonai 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:
It is known that the unique metabolisms that cancer cells develop, such as “Warburg effect”, is linked with drug resistance. However, the underlying regulatory mechanisms of those metabolisms have not been fully understood. A pleiotropic bioactive lipid mediator, sphingosine-1-phosphate (S1P), produced by sphingosine kinases (SphK1 and SphK2), regulates many physiological and pathological processes, such as cell proliferation, migration, survival, and metabolism. Previously we have demonstrated that S1P and SphK2 play important roles in liver lipid metabolism (Hepatology 2015). We have also reported that expression of SphK1 is higher in cancer than normal breast tissue (J Surg Res 2016), and it associates with lymph node metastasis and worse prognosis in breast and gastric cancer patients (J Surg Res 2016, Surgery 2018). Based on these findings, we hypothesized that S1P and SphKs regulate cancer cell-specific metabolism, which related to the cancer cell survival and drug resistance.
Methods:
We used E0771 murine breast cancer and PAN02 murine pancreatic cancer cell lines, and SphK1 or SphK2 knock-out (KO) cells were generated with a CRISPR/Cas9 gene editing system. Metabolic changes in SphK1KO, SphK2KO, and their corresponding wild type (WT) cells were analyzed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Cytotoxic assays with chemotherapeutic drugs were performed with WST-8. Quantitative reverse transcription PCR (RT-qPCR) was used for quantification of mRNA expression in the cells.
Results:
CE-TOFMS analysis revealed that different roles of SphK1 and SphK2 in cancer cell metabolism both in E0771 and PAN02 cell lines. Metabolic pathways, including glycolysis, purine metabolism, TCA cycle, and urea cycle were altered in SphK1 or SphK2KO both in PAN02 and E0771 cells. Moreover, SphK2KO E0771 cells showed significantly higher amount of glutathione (GSH), which is known to be associated with anti-oxidative stress and drug resistance, as compared with normal control cells. Importantly, gene expression of xCT, the transporter of the cysteine, was found to be significantly higher in SphK2KO E0771 cells than in WT. Similar results were also found in SphK2KO PAN02 cells, where GSH were significantly higher than the WT cells. On the other hand, GSH was less in SphK1KO compared with WT. Finally, SphK2KO cells, which is known to have compensatory high expression of SphK1, demonstrated stronger resistance to chemotherapies compared with WT cells. Taken together, our results indicate that S1P and SphK1 are associated with drug resistance, which is mediated by xCT expression and GSH production.
Conclusion:
S1P and SphKs play an important role in regulation of cancer-specific metabolism, which strengthen resistance to oxidative stress and cancer cell survival mediated by xCT and GSH.