Abstract A68: Sphingosine kinase 1 as a mediator and predictor of metformin's protective effect in ovarian cancer.

Background: Sphingosine kinase 1 (SPHK1) is over-expressed in multiple cancers including breast and colon cancer. SPHK1 catalyzes the phosphorylation of sphingosine to sphingosine 1-phosphate (S1P). S1P promotes tumorigenesis by inhibiting apoptosis and increasing cell proliferation and angiogenesis. Thus SPHK1 has been evaluated as a therapeutic target in cancer. The antidiabetic drug, metformin, has been reported to have protective effects in several cancers, including ovarian cancer. However, molecular mechanisms mediating the anti-tumor effects of metformin are not fully understood. In this study, we demonstrate that SPHK1 is a novel target of metformin and may predict metformin response in ovarian cancer. Methods: The S1P signaling cascade was profiled in several ovarian cancer cell lines. Four different ovarian cancer cell lines were treated with 1mM and 5mM metformin and the mRNA and protein levels of SPHK1 were measured by qRT-PCR and western blotting respectively. Next, the Tyk-nu, HeyA8, SNU119, Kuramochi cell lines were stably transfected to overexpression SPHK1 and metformin sensitivity was measured using MTT proliferation assays. Metabolomic analysis was performed on serum from ovarian cancer patients using metformin for diabetes and compared controls (IRB 13248A). Serum samples were analyzed using a Q-Exactive orbitrap mass spectrometer coupled to a Dionex ultimate UHPLC. Results: In all cell lines tested, metformin suppresses SPHK1 mRNA and protein levels. Analysis of the S1P signaling pathway showed that metformin sensitive cell lines (Tyknu and HeyA8) have high SPHK1 and low S1P lyase (SGPL1) expression, the opposite is true in metformin resistant cell lines (Kuramochi and SNU119). At the same time, S1P receptor 1 (S1PR1) is the main S1P receptor among five S1P receptors (S1PR1-S1PR5) in metformin sensitive cancer cells, however S1PR1 is not dominant in metformin resistant cancer cells. Overexpression of SPHK1 upregulates S1PR1 and increases metformin sensitivity in both metformin sensitive and resistant cell lines. The in vitro findings are augmented by preliminary metabolomic analysis of patient samples that revealed that ovarian cancer patients using metformin for diabetes have lower serum levels of S1P compared to controls. Conclusions: The findings of this study indicate that SPHK1 is a novel metformin target in ovarian cancer. Specifically, high SPHK1 expression sensitizes cells to metformin and S1P signaling profiles predicts metformin response. Based on these findings we propose that sphingolipid signaling be evaluated as a metformin-response signature in ongoing clinical trials of the drug as adjuvant treatment for ovarian cancer (NCT02122185). Citation Format: Tatsuyuki Chiyoda, Xiaojing Liu, Ernst Lengyel, Jason Locasale, Iris Romero. Sphingosine kinase 1 as a mediator and predictor of metformin9s protective effect in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A68.