M. Nakajima1, K. Yuza1, J. Tsuchida1, Y. Hirose1, K. Miura1, H. Ichikawa1, Y. Shimada1, T. Kobayashi1, J. Sakata1, H. Kameyama1, 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, NIIGATA, Japan 2Brain Research Institute, Niigata University,Department Of Cellular Neurobiology,Niigata, NIIGATA, 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, 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. 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, we assessed the prognosis of C57BL/6 mice injected with the SphK1 KO or SphK2 KO Pan02 cells described above intraperitoneally.
Results: SphK2 KO Pan02 cells were significantly less proliferative than 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. Furthermore, the animal experience showed that mice injected with SphK1 KO cells had shorter prognosis than those injected with SphK1 WT cells, while mice injected with SphK2 KO cells had longer prognosis than those injected with SphK2 WT cells. These results indicate that SphK2, rather than SphK1, may have important roles in proliferation and migratory behavior in pancreatic cancer cell lines and pancreatic cancer progression. On the other hand, SphK1 KO cells treated with gemcitabine had more survival rate than WT cells and SphK2 KO cells treated with gemcitabine had less survival rate than WT cells. These results indicate that SphK1 may have important roles in resistance against chemotherapy.
Conclusion: Our findings indicate that S1P produced by SphK1 and SphK2 may have different functions in pancreatic cancer cell. Targeting both SphK1 and SphK2 signaling pathways may be a potential strategy for pancreatic cancer treatment.