Targeting Sphingosine Kinase 2 As A Novel Therapeutic Strategy In Cholangiocarcinoma

Backgroud and ObjectCholangiocarcinoma(CCA)is a highly malignant adenocarcinoma arising from the bile duct epithelial cells.CCA has a poor prognosis in part due to luck of effective drugs.Thus,there is an urgent need to develop novel effective therapeutics against this neoplasm.Sphingosine kinases(SphK)are lipid kinases that regulate the sphingolipid metabolic pathway.Sphk isoforms(Sphkl and Sphk2)catalyze the conversion of the pro-apoptotic sphingosine to the mitogenic lipid sphingosine-1-phosphate(SIP).It has been shown that Sphks are overexpreesed in many cancers and have been proposed to contribute to cancer development and progression.SphK therefore represents a potential novel target for cancer therapeutics.One recent study suggests that SlP,probably generated from Sphk2,may promote CCA cell proliferation and migration,suggesting Sphk2 and SlP as potential therapeutic targets in CCA.ABC294640 is a novel first-in-class Sphk2 specific inhibitor,with high specificity for Sphk2,excellent oral bioavailability and low side effects.It shows high efficacy in several preclinical models of cancer and has entered Phase ? clinical trial aginst solid tumors.ABC294640 has previously been shown to induce cancer cell death by both apoptotic and autophagic pathways.However,emerging evidence suggests that autophagy can also enable cell survival and lead to treatment resistance.ABC294640 has been shown to have an additive to synergistic effect with sorafenib ininhibiting tumor growth in hepatocellular carcinoma and renal carcinoma cells.Sorafenib also has a tumor suppression role in CCA.To date,the role of Sphk2 and the antitumor activity of its inhibitor ABC294640 are not known in CCA.Therefore,the current study is to determine whether Sphk2 is overexpressed in CCA cells and to investigate the effect of ABC294640 on proliferation,apoptosis and autophagy of human CCA cells.Furthermore,wheather autophagy inhibitors can enhance the anticancer efffect of ABC294640 and wheather sorafenib has a synergistic effect with ABC294640 in CCA cells will be investigated.MethodsReal-time PCR was used to determine the expression level of Sphk2 mRNA in different CCA cell lines(WITT,HuCCTl,EGI-1,OZ,HuH28 and LIV27)and normal human cholangiocytes(H69).After the treatment with sphingosine kinase 2 inhibitor ABC294640,BrdU ELISA assay and colony formation assay were used to access cell proliferation,DAPI staining and Annexin V-FITC-PI staining were used to access apoptosis,Western Blotting was used to detect the apoptosis markers PARP,Caspase 3,Caspase 8 and Caspase 9 and STAT3 phosphorylation.After the treatment with ABC294640,Western Blotting and immunofluorescence were used to detect autophagy maker LC3-II,transmission electron microscopy was used to monitor the formation of autophagosomes and autophagolysosomes.BrdU ELISA,colony formation assay and DAPI staining were used to determine the combination effect of ABC294640 and autophagy inhibitors(bafilomycin A1 and chloroquine)in CCA cells.For analysis of synergy between ABC294640 and sorafenib,cells were exposed to different fixed-ratio combinations of ABC294640(dose range,10-50 ?M)and sorafenib(dose range,1.5-7.5 ?M)and cell proliferation was assessed by the BrdU ELISA assay after 72 h.The combination index(CI)of ABC294640 and sorafenib was determined using CompuSyn software,based on the median-effect model of Chou-Talalay.Western Blotting was used to determine the STAT3 phosphorylation in WITT and HuCCTl cells exposed to the ABC294640 and sorafenib alone or in combination.ResultsSphk2 is overexpressed in six human cholangiocarcinoma cell lines(WITT,HuCCTl,EGI-1,OZ,HuH28 and LIV27)compared to H69 normal cholangiocytes(P<0.05).BrdU ELSIA assay showed that inhibition of Sphk2 by ABC294640 dose-dependently inhibited the proliferation of six human cholangiocarcinoma cell lines with an IC50 between 36.3 ?M and 48.6 ?M at 72 h.ABC294640 dose-dependently induced caspase cleavage and apoptosis.Pretreatment with a pan-caspase inhibitor Z-VAD-FMK inhibited ABC294640-induced apoptotis.Furthermore,we found that ABC294640 inhibited STAT3 phosphorylation,one of the key signaling pathways regulating cholangiocarcinoma cell proliferation and survival.ABC294640 also significantly induced autophagy in CCA cells.Autophagy inhibitors bafilomycin A1 or chloroquine significantly potentiated ABC294640-induced cytotoxicity and apoptosis(P<0.05).ABC294640 in combination with sorafenib synergistically inhibited cell proliferation of CCA cells(CI<1).Strong decreases in STAT3 phosphorylation were observed in WITT and HuCCTl cells exposed to the ABC294640 and sorafenib combination.ConclusionsSphk2 is overexpressed in CCA cells.Inhibition of Sphk2 by the novel inhibitor ABC294640 inhibits CCA cell proliferation and induces apoptosis.In addition,ABC294640 inhibits STAT3 signaling pathway.Furthermore,ABC294640 induces a protective autophagy in CCA cells.Autophagy inhibitors bafilomycin Al and chloroquine enhance the sensitivity of CCA cells to ABC294640.ABC294640 in combination with sorafenib synergistically inhibit cell proliferation of CCA cells,which may be due to strong decreases in STAT3 phosphorylation exposed to the ABC294640 and sorafenib combination.Therefore,combinations of ABC294640 with sorafenib and/or autophagy inhibitors may provide novel and promising strategies to improve the treatment of cholangiocarcinoma.