02.04 Roles of Sphingosine-1-Phosphate Produced by Sphingosine Kinases in Pancreatic Cancer Progression

M. Nakajima1, Y. Miura1, T. Ando1, K. Yuza1, J. Tsuchida1, Y. Tajima1, M. Abe2, K. Sakimura2, T. Wakai1, M. Nagahashi1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata City, , Japan 2Brain Research Institute, Niigata University,Department Of Cellular Neurobiology,Niigata City, , Japan

Introduction: Pancreatic cancer is one of the most lethal diseases known, and it is important to develop new therapeutic agents. Sphingosine-1-phosphate (S1P) is a pleiotropic lipid mediator that regulates cell survival, migration, immmune cell recruitment, angiogenesis and lymphangiogenesis, which are all factors involved in cancer progression. S1P, which functions intra- and extracellularly, is generated inside the cell by two sphingosine kinases (SphK1 and SphK2). 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). Little is known, however, about the role of SphK1 and SphK2 in pancreatic cancer progression. The aim of this study is to investigate the role of SphK1 and SphK2 in pancreatic cancer progression using SphK-knockout (KO) cells generated by CRISPR/Cas9 technology.

Methods: We generated Pan02 murine pancreatic cancer cell lines with a CRISPR/Cas9 mediated targeted deletion of the SphK1 or SphK2 gene. Western blotting and RT-qPCR determined the expression levels of SphKs in these cell lines. 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.

Results: We confirmed the knockout of SphK1 or SphK2 in Pan02 pancreatic cancer cells by western blotting and RT-qPCR. SphK2 KO cells were significantly less proliferative than wild type (WT) cells. Unexpectedly, SphK1 KO cells were significantly more proliferative than WT cells. The in vitro scratch assay indicated that SphK2 KO cells were less migratory than WT cells, and that SphK1 KO cells had greater migratory ability than WT cells. These results indicate that S1P produced by SphK2, rather than by SphK1, may have important roles in proliferation and migratory behavior in pancreatic cancer cell lines.

Conclusion: Our findings indicate that S1P produced by SphK2, rather than by SphK1, promotes pancreatic cancer cell proliferation and migration. Targeting SphK2 may be a potential strategy for pancreatic cancer treatment. Further studies that include in vivo models are needed to explore this therapeutic possibility.