1.18 The Roles of Sphingosine-1-phosphate Produced by Sphingosine Kinases in Tumor and Its Microenvironment

K. Miura1, M. Nagahashi1, M. Nakajima1, K. Yuza1, J. Tsuchida1, Y. Hirose1, M. Abe2, K. Sakimura2, T. Wakai1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, NIIGATA, Japan 2Brain Research Institute, Niigata University,Department Of Cellular Neurobiology,Niigata, NIIGATA, Japan

Introduction: Sphingosine-1-phosphate (S1P) is a pleiotropic lipid mediator that regulates cell survival, migration, angiogenesis and lymphangiogenesis, which are all factors involved in cancer progression. S1P is generated inside the cell by two sphingosine kinases (SphK1 and SphK2) and then exported into the tumor microenvironment. We have reported that SphK1 plays an important role in S1P secretion (J Biol Chem 2010) and cancer progression (Cancer Res 2012, J Surg Res 2016), and that SphK2 has a unique role in regulating cellular functions in the liver (Hepatology 2015). Although S1P can be secreted from both cancer cells and non-cancer components such as immune cells and vascular/lymphatic endothelial cells in the tumor microenvironment (Tumor Biology 2017), the roles of S1P produced by tumor and its microenvironment on cancer progression has not been fully investigated. The aim of this study is to investigate the role of SphKs in tumor and its microenvironment using SphK-knockout (KO) cells and SphK KO mice generated by CRISPR/Cas9 technology.

Methods:  We generated E0771 murine breast cancer cell lines with a CRISPR/Cas9 mediated targeted deletion of the SphK1 or SphK2 gene. To investigate the role of SphK1 or SphK2 in cellular proliferation, we assessed cell growth by a spectrophotometric technique using the water-soluble tetrazolium salt, WST-8. Cell migration was measured by an in vitro scratch assay. In the animal experiments, the SphK1 KO or SphK2 KO E0771 cells were injected into the subcutaneous tissue of chest of C57BL6 mice, and prognosis of C57BL/6 mice were determined.

Results: Utilizing in vitro proliferation assay of WST-8, we observed significantly less proliferation in SphK1 KO E0771 cells and more proliferation in SphK2 KO E0771 cells compared with their corresponding wild-type (WT) cells, respectively. On the other hand, there were no difference of migration between SphK1 KO and the WT or between Sph2 KO and the WT. The animal experiments showed that mice injected with SphK1 KO cells showed smaller tumor with longer survival than those injected with SphK1 WT cells. Mice injected with SphK2 KO cells also showed similar trend with smaller tumor and longer survival than those injected with SphK2 WT cells. We next implanted WT cells into SphK1 and SphK2 KO mice, and less tumors were developed in the SphK1 KO and SphK2 KO compared with the WT mice, respectively. Finally, we implanted SphK1 KO cells into SphK1 KO mice and WT mice, and found that there were much less growth of tumor of SphK1 KO cells implanted in the SphK1 KO mouse. This indicates that S1P is necessary to the tumor growth, which can be provided from cancer and tumor microenvironment.

Conclusion: Our findings indicate that S1P produced by SphKs in tumor and its microenvironment. Targeting both SphKs and S1P signaling pathways not only cancer, but also in tumor microenvironment will be a key to success to develop new targeted therapy to block S1P signaling.