SphK2/S1P Mediates Acquired Resistance To Regorafenib Through STAT3 And NF-?B Activation In Hepatocellular Carcinoma

Background: Hepatocellular carcinoma(HCC)is a serious threat to human life and health in China.Regorafenib is a second-line drug used for HCC patients who progressed on first-line drug sorafenib treatment,which displays strong anti-tumor effects in vitro and vivo.While the survival benefit of the patients receiving this treatment is modest,which may be attributed to the acquired resistance.Therefore,it is essential to investigate the mechanisms underlying the acquired resistance to regorafenib and to further explore strategies to enhance efficacy of this drug in HCC.Recently,dysregulation of sphingolipid metabolism in tumors has been widely concerned.Sphingosine kinase 2(SphK2)is the key regulatory enzyme catalyzing the formation of sphingosine-1-phosphate(S1P).With increasing evidence of the role of SphK2 in cell survival,proliferation,apoptosis and acquired resistance,this enzyme is considered as a significant therapeutic target in various solid tumors including HCC.However,whether SphK2/S1P is involved in resistance to regorafenib in HCC and the underlying mechanisms remain unclear.We wonder whether pharmacological inhibition of SphK2 has the potential to enhance efficacy of regorafenib and even reverse resistance.Methods: The acquired resistant cell lines to regorafenib were established by treating HCC cells with stepwise increasing concentrations of this drug.The cell counting kit-8(CCK-8)assay was used to compare the sensitivity to regorafenib of resistant cells and parental cells.Subsequently,the phenotype of resistant cells was characterized by direct microscopic observation,migration and invasion assays and clone formation assay.The function of SphK2 and S1P in regorafenib-resistance of HCC cells were evaluated by CCK-8 assay,clone formation,cell cycle evaluation and Annexin V-FITC/PI double staining assays.The anti-tumor activity of combined treatment of regorafenib and the SphK2 specific inhibitor ABC294640 was examined in HCC cells in vitro and xenograft model in vivo.The molecular mechanisms of SphK2/S1P mediating regorafenib-resistance were investigated using cell line establishment and Western blot assay.Results: Compared with the parental HCC cells,the resistant cells have higher ability in migration,invasion and proliferation.SphK2 instead of SphK1 expression is upregulated in regorafenib-resistant HCC cells and is negatively associated with regorafenib sensitivity in HCC cells.The sensitivity to regorafenib of regorafenib-resistant HCC cells was restored following SphK2 knockdown or pharmacological inhibition by ABC294640.In addition,ectopic expression of SphK2 and exogenous addition of S1P decreased the sensitivity of HCC cells to regorafenib.Furthermore,the combination treatment with ABC294640 sensitized resistant tumor to regorafenib in xenograft model of HCC.The phosphorylation levels of nuclear factor ?B(NF-?B)as well as those of signal transducer and activator of transcription3(STAT3)were positively associated with SphK2 and S1P.Conclusions: SphK2/S1P mediates acquired resistance to regorafenib of HCC through STAT3 and NF-?B activation.Targeting SphK2 by ABC294640 potently reduces regorafenib resistance of HCC cells both in vitro and in vivo.The combination of ABC294640 and regorafenib could be developed as a novel potential treatment strategy for advanced HCC.