Anti-tumor Mechanism And Combined Therapies Of SPHK2 Selective Inhibitor In Cholangiocarcinoma

Background:Cholangiocarcinoma is a highly maliganant carcinoma with a poor prognosis,which was in part due to lack of effective therapeutic agents aginast this neoplasm.Therefore,exploring new effective agents to treat this cancer is urgent and of great importance.Sphingosine kinases(SPHKs)are key kinases in the regulation of sphingolipid metabolism,responsiel for the conversion of pro-apoptotic Sphingosine(Sph)to the pro-survival Sphingosine-1-phosphate(S1P).It has been found that SPHKs are overexpressed in many tumors and can promote tumorigenesis and progression.Thus,SPHKs represent new targets for cancer treatment.SPHKs have two isomers,SPHK1 and SPHK2.It was found that the expression of SPHK2 was significantly higher than that of SPHK1 in cholangiocarcinoma cells.Meanwhile,S1 P could promote the proliferation and migration of cholangiocarcinoma cells.Therefore,the SPHK2/S1 P pathway may be a potential target for the treatment of cholangiocarcinoma.ABC294640 is the first specific small molecule inhibitor of SPHK2.It has the advantages of high oral bioavailability,low side effects and so on.ABC294640 has shown good anti-tumor effect in many tumor models.We previously have found that ABC294640 could inhibit proliferation and promote apoptosis of cholangiocarcinoma cells.In the phase I clinical trial of ABC294640,21 patients with advanced solid tumors were included,of which 3 patients with advanced cholangiocarcinoma had the best response,suggesting that ABC294640 may have unique anti-cholangiocarcinoma effect.Phase II clinical trial of ABC294640 for the treatment of cholangiocarcinoma has been initiated.However,the antitumor mechanism of ABC294640 is not very clear.Whether there are other drugs that can further enhance the anti-cholangiocarcinoma effect of ABC294640 alo worth further exploring.Therefore,this study will focus on exploring the anti-cholangiocarcinoma mechanism of ABC294640 and explore whether ABC294640 and BCL2/BCL-XL inhibitors have a synergistic antitumor effect in cholangiocarcinoma.Methods:After the treatment with ABC294640 in human cholangiocarcinoma cell lines,the expression levels of BCL2 family members were detected by Real-time PCR and estern blot.After silencing of NOXA by small interfering RNA(SiRNA),CCK-8,flow cytometry and Western blot were used to detect the effect of ABC294640 on cholangiocarcinoma.The correlation between NOXA and prognosis of cholangiocarcinoma patients was analyzed using TCGA data.After ABC294640 was combined with ABT-263 and Obatoclax,CCK-8 was used to detect cell viability.The combined index(CI)was calculated by CompuSyn software.Apoptosis was detected by flow cytometry and Caspase 3/7 activity kit.The expression and correlation of SPHK2 and MCL1 in cholangiocarcinoma were detected by immunohistochemistry.Results:ABC294640 inhibited the proliferation promoted apoptosis in HCCC9810 and RBE cells.ABC294640 significantly increased the expression of NOXA.Silencing NOXA by SiRNA can reverse the anti-cholangiocarcinoma effect of ABC294640.Analysis of TCGA data showed that patients with cholangiocarcinoma with high expression of NOXA had better prognosis.We also found that ABC294640 significantly down-regulated the expression of MCL1,but did not decrease the expression of MCL1,and did not affect the protein expression of BCL2 and BCL-XL.Pretreatment with proteasome inhibitor MG132 reversed the inhibitory effect of ABC294640 on MCL1,suggesting that ABC294640 promoted the degradation of MCL1 through proteasome pathway.Further experiments showed that silencing of NOXA by SiRNA reversed the inhibitory effect of ABC294640 on MCL1.At the same time,ABC294640 combined with ABT-263 and Obatoclax significantly inhibited the viability of cholangiocarcinoma compared to single drug alone.The combined index < 1 suggests a synergistic effect.ABC294640 combined with ABT-263 and Obatoclax also increased apoptosis.Silencing MCL1 with small interference of MCL1 can enhance the apoptotic effect of ABT-263 in cholangiocarcinoma.Immunohistochemistry showed that the expression of SPHK2 and MCL1 in cholangiocarcinoma was significantly higher than that in adjacent normal bile duct epithelium,and the expression of SPHK2 and MCL1 was positively correlated.Conclusion:ABC294640 can induce the expression of apoptotic protein NOXA,promote the degradation of anti-apoptotic protein MCL1,inhibit the proliferation ofcholangiocarcinoma cells,promote the apoptosis of cholangiocarcinoma cells,and produce synergistic anti-cholangiocarcinoma effect when combined with ABT-263 and Obatoclax.Therefore,the combination of ABC294640 and BCL2/BCL-XL inhibitors may be a new strategy for the treatment of cholangiocarcinoma.