FGF19/SOCE/NFATc2 Signaling Circuit Facilitates The Self-renewal Of Liver Cancer Stem Cells

Background:Hepatocellular carcinoma（HCC）is one of the most lethal malignancies in the world,and its incidence keeps increasing yearly.Current therapies include surgical resection,liver transplantation,radiofrequency ablation,chemoembolization,and treatment with multiple kinase inhibitors.However,HCC has a poor prognosis owing to its high recurrence and metastasis rates.Compelling studies have shown that liver cancer stem cells（CSCs）are more likely to resistant to cancer therapies compared to non-CSCs（NCSCs）and become the significant cause for liver cancer metastasis,chemotherapy resistance and recurrence.Understanding the mechanisms underlying the self-renewal of CSCs,so as to find targets to block CSCs-induced recurrence and drug resistance,is of great significance for improving the therapeutic effect of HCC.It has been widely accepted that,calcium(Ca²⁺)participates in multiple signal pathways in cellule.The most important Ca²⁺entry in non-excited cells,store-operated Ca²⁺entry（SOCE）,is of great importance in HCC generation,development,and metastasis.SOCE-mediated Ca²⁺signaling can regulate downstream gene expression through transcription factors such as nuclear factor of activated T cells（NFAT）.Whether SOCE is involved in the self-renewal of CSCs remains unknown.FGF19,a member of the endocrine FGF family,plays a crucial role to keep the homeostasis of bile acid,glucose and lipid metabolism.In HCC,FGF19 expression is significantly upregulated and positively correlated with poor prognosis.FGFR4 is a FGF19receptor mainly expressed on the liver cell membrane,and it has been reported that FGF19/FGFR4 is related to embryonic cell stemness maintenance.Currently,pre-clinical evidence indicated that FGF19/FGFR4 activation was the driver event of HCC and the inhibitor of FGFR4 repressed the hepatocarcinogenesis.FGF19/FGFR4 pathway can lead to the activation of Ras–Raf–ERK1/2-MAPK and PI3K/AKT,which promoted the proliferation of HCC.Meanwhile,FGF19 can activate the expression of other growth factors,such as connective tissue growth factor and the EGFR ligand amphiregulin,to enhance the development of HCC.However,it remains unknown whether FGF19/FGFR4 is related to cancer stem cells.Therefore,we speculated that FGF19/FGFR4 may be related to the self-renewal characteristics of HCC stem cells.Methods:1.Effects of intervention of FGF19 expression on self-renewal ability of CSCs.1.1 Serum-free suspension of CSCs1.2 The gene expression levels of self-renewal and mature liver markers in spheroids and adherent cells were detected by RT-q PCR.FGF19 gene expressions in CSCs and NCSCs,the gene expression levels of self-renewal and mature liver marker in FGF19-deficient CSCs,the gene expression levels of self-renewal and mature hepatocytes markers in FGF19-OE FGFR4^-/-NCSCs and the gene expression levels of self-renewal and mature hepatocytes in FGF19-treated NCSCs after FGFR4 inhibitor treatment were detected by RT-q PCR.1.3 The self-renewal ability of CSCs and NCSCs,the self-renewal ability of FGF19-deficient CSCs,the self-renewal ability of FGF19-OE FGFR4^-/-NCSCs and the self-renewal ability of NCSCs treated by FGFR4 inhibitor and FGF19 were detected by sphere formation experiment.1.4 The self-renewal ability of CSCs and NCSCs,the self-renewal ability of FGF19-deficient CSCs,the self-renewal ability of FGF19-OE FGFR4^-/-NCSCs and the self-renewal ability of NCSCs treated by FGFR4 inhibitor and FGF19 were detected by clone formation experiment.1.5 The resistance of CSCs and NCSCs to sorafenib,the resistance of FGF19-deficient CSCs to sorafenib,the resistance of FGF19-OE FGFR4^-/-NCSCs to sorafenib and the resistance of NCSCs to sorafenib after the administration of FGFR4 inhibitor and FGF19were detected by CCK-8 kit.1.6 The self-renewal ability of CSCs and NCSCs,the self-renewal ability of FGF19-deficient CSCs,the self-renewal ability of FGF19-OE FGFR4^-/-NCSCs and the self-renewal ability of NCSCs treated by FGFR4 inhibitor and FGF19 were detected by tumorigenic ability experiment.1.7 The FGF19 protein expression of CSCs and NCSCs,the protein expression of FGF19while CSCs differentiation,the protein expression levels of self-renewal and mature liver markers in FGF19-deficient CSCs,the protein expression levels of self-renewal and mature liver cells in FGF19-OE FGFR4^-/-NCSCs and the protein expression levels of self-renewal and mature liver markers in NCSCs after the use of FGFR4 inhibitor and FGF19 were detected by Western Blot.1.8 The secretion of FGF19 in CSCs and NCSCs was determined by ELISA assay.1.9 The expression of FGF19 in CSCs and NCSCs transplanted tumors in nude mice and the expression of FGF19,Nanog,OCT-4 and ALB in clinical HCC tissue samples were detected by immunohistochemistry.2.Effects of FGF19 on self-renewal of NCSCs by activating SOCE.2.1 The self-renewal ability of CSCs after SOCE inhibitor and the self-renewal ability of NCSCs after treatment with SOCE inhibitor and FGF19 were detected by the sphere formation experiment.2.2 The self-renewal ability of CSCs after SOCE inhibitor and the self-renewal ability of NCSCs after treatment with SOCE inhibitor and FGF19 were detected by the clone formation experiment.2.3 The expression of mature liver markers related genes after SOCE inhibitor treatment,the self-renewal of NCSCs and the expression of related genes in mature liver markers after SOCE inhibitor and FGF19 treatment and the expression of related genes in NCSCs after FGF19 treatment were detected by RT-q PCR.2.4 The effect of SOCE inhibitor on calcium influx of NCSCs,and the changes of calcium influx of NCSCs after 3-NC,LY3214996 and FGF19 treatment were detected by calcium imaging.2.5 The resistance to sorafenib of NCSCs after treatment with SOCE inhibitor and FGF19 were detected by CCK-8 kit.2.6 The self-renewal and protein expression levels of NCSCs treated with SOCE inhibitor and FGF19,SOCE-related protein expression in NCSCs treated with FGF19,PLCγand ERK1/2 pathway protein expression after BLU9931,3-NC,LY3214996 and FGF19treatment were detected by Western Blot.2.7 STIM1 oligomerization assay was detected by laser confocal microscopy after treated with BLU-9931,3-NC,LY3214996 and FGF19.3.The role of NFATc2 in FGF19-induced self-renewal of NCSCs.3.1 The NFAT isoforms activated by FGF19 and the binding of NFAT to the reaction elements on the FGF19 promoter was detected by the dual-luciferase report assay.3.2 Knock-down of NFAT isoforms in NCSCs.3.3 The role of NFATc1,NFATc2,NFATc3 and NFATc4 in HCC was analyzed by GSEA.3.4 Kaplan-Meier analysis of NFATc2 expression and overall survival in patients with HCC in TCGA database.3.5 The expression of NFATc2 in nucleus,cytoplasm and total proteins,the expression of self-renewal and mature hepatocyte related proteins after NFATc2 knockout and FGF19treatment,the expression of NFATc2 in cytoplasm and nucleus of CSCs and NCSCs,the protein expression of FGF19 after NFATc2 knockout in NCSCs,and the protein expression of FGF19 after NFATc2 overexpression in NCSCs were detected by Western Blot.3.6 The subcellular localization of NFATc2（green）was detected in NCSCs treated with BLU9931,3-NC,LY3214996,SKF96365 and FK506 and FGF19,and the nucleus was labeled with DAPI（blue）.3.7 The expression of genes related to self-renewal and mature liver cells after NFATc2knockdown and FGF19 treatment in CSCs and NCSCs,the expression of FGF19 gene after NFATc2 knockdown in CSCs and NCSCs,and the expression of FGF19 gene after NFATc2overexpression in NCSCs were detected by RT-q PCR.3.8 The self-renewal ability of NCSCs with NFATc2 knockdown and FGF19 treatment was tested by sphere formation experiment.3.9 The self-renewal ability of NCSCs with NFATc2 knockdown and FGF19 treatment was tested by clone formation experiment.3.10 The resistance of sorafenib in NCSCs with NFATc2 knockdown and FGF19treatment was detected by CCK-8 kit.3.11 The relative relationship between NFATc2 and FGF19 expression was detected by immunohistochemistry.3.12 Chromatin immunoprecipitation was used to detect the binding of NFATc2 and FGF19 promoter.3.13 Cp G island prediction of FGF19 promoter sequence.4.Influence of targeting FGF19/NFATc2 pathway on self-renewal characteristics of CSCs.4.1 Kaplan-Meier analyzed the association between FGF19 and NFATc2 expression in the TCGA database（n=365）of HCC patients.4.2 The expressions of genes related to self-renewal and mature liver markers of CSCs after the knockdown of FGF19 and NFATc2 were detected by RT-q PCR.4.3 The expressions of self-renewal and mature hepatocyte related proteins of CSCs after the knockdown of FGF19 and NFATc2 were detected by Western Blot.4.4 The self-renewal ability of CSCs after the knockdown of FGF19 and NFATc2,and the self-renewal ability of CSCs after treated by BLU9931 and FK506 were detected by the sphere forming experiment.4.5 The self-renewal ability of CSCs after the knockdown of FGF19 and NFATc2,and the self-renewal ability of CSCs after treated by BLU9931 and FK506 were detected by the clone forming experiment.4.6 The self-renewal ability of CSCs after the knockdown of FGF19 and NFATc2,and the self-renewal ability of CSCs after treated by BLU9931 and FK506 were tested by the tumorigenesis experiment.4.7 The overall survival was determined by in situ tumor-bearing assay of nude mice after transplanted of CSCs after the knockdown of FGF19 and NFATc2.Results:1.FGF19 influence the self-renewal ability of CSCs:We enriched spheroids by serum-free suspension induction.Compared with adherent cells,the expression of stemness genes was significantly up-regulated,and the expression of marker genes in mature hepatocytes was down-regulated.In addition,the clone forming rate,sphere forming rate and tumor formation rate of nude mice were significantly increased,and the drug resistance to sorafenib was enhanced.Therefore,we successfully enriched CSCs model with serum-free induction method.The expression level and secretion of FGF19 in CSCs were significantly higher than those in NCSCs,and knockdown of FGF19down-regulated the expression of stemness gene and protein in CSCs.The CSCs clone forming rate,sphere forming rate and tumor formation rate were significantly reduced after knockdown of FGF19,and the resistance to sorafenib was weakened.Overexpression of FGF19 in NCSCs up-regulated the expression of stemness genes and proteins,and increased the clone forming rate,sphere forming rate and tumor formation rate in nude mice,and enhanced the resistance to sorafenib.At the same time,when FGFR4 was knocked out in NCSCs that overexpressed FGF19,the above changes in self-renewal characteristics disappeared.Treatment of NCSCs with FGF19 promoted the expression of stemness gene and protein.In addition,it increased the clone forming rate,sphere forming rate and tumor formation rate in nude mice,and enhanced the resistance to sorafenib,while the adding of FGFR4 inhibitor BLU9931 eliminated the above effects of FGF19.2.FGF19 activates SOCE to promote self-renewal of NCSCs:We treated CSCs with SOCE inhibitor SKF-96365 and found that the expression level of stemness genes was down-regulated,and the clone forming rate,sphere forming rate were significantly reduced.Correspondingly,treating NCSCs with FGF19 enhanced their Ca²⁺influx,which could be inhibited by SKF-96365.In combination treatment with FGF19 and SKF-96365,the expressions of stemness gene and protein in NCSCs were significantly lower than those in FGF19 alone group,and the clone forming rate,sphere forming rate,tumor formation rate in nude mice,and drug resistance to sorafenib were all lower than those in FGF19 alone group.Treatment of NCSCs with FGF19 did not significantly change the expression of STIM1,STIM2 and Orai1 gene and protein,but up-regulated the protein expression of phosphorylated PLCγand ERK1/2.The use of 3-NC,an inhibitor of PLCγ,and LY3214996,an inhibitor of ERK1/2,down-regulated the protein expression of p-PLCγand p-ERK1/2 and the increasement of Ca²⁺influx FGF19-induced in NCSCs.The number of STIM1 oligomeric spots were increased after treatment of FGF19,and BLU9931,3-NC,and LY3214996 were used to reduce the number of STIM1 oligomeric spots,while the combination of 3-NC and LY3214996 had the strongest inhibitory effect on STIM1oligomeric spots.3.FGF19/SOCE promotes NFATc2 nuclear transcription and activates dry gene expression in HCC cells:Through luciferase assay,we found that FGF19 can promote the binding of NFATc2 to NFAT-RE.In addition,enrichment analysis revealed that NFATc2 was associated with the pluripotency state of stem cells,while other NFAT subtypes were not associated with the enrichment of this pathways.Bioinformatics analysis found that patients with high NFATc2expression had shorter overall survival.We treated Huh7 NCSCs with FGF19 and found that the p-NFATc2 protein level was reduced and the NFATc2 protein in the nucleus was increased.However,the use of BLU9931,3-NC,LY3214996,SKF-96365 and FK506 all inhibited the increase of NFATc2 in the nucleus.Through confocal experiments,we found that FGF19promoted the nuclear translocation of NFATc2,and this phenomenon could be inhibited by BLU9931,3-NC,LY3214996,SKF-96365,and FK506.Knockdown of NFATc2 eliminated FGF19-induced upregulation of stemness genes and protein expression in NCSCs,as well as increased the clone forming rate,sphere forming rate,and drug resistance to sorafenib.We found that the expression level of NFATc2 in the nucleus was also higher than that in the cytoplasm in CSCs,and knockdown of NFATc2 down-regulated the gene and protein expression of FGF19 in CSCs,while overexpression of NFATc2 in NCSCs up-regulated the gene and protein expression of FGF19.Immunohistochemical results of HCC clinical samples showed that NFATc2 was positively correlated with FGF19 expression.Bioinformatics analysis showed that the sequence of the FGF19 promoter contained four NFAT-RE.Through luciferase assay and Ch IP assay,we found that NFATc2 activated the FGF19 promoter by combined with the fourth NFAT-RE.Through Cp G island analysis,we found that the fourth NFAT-RE was located in the non-methylated sequence of the FGF19 promoter.4.Targeting FGF19/NFATc2 pathway to inhibit HCC stem cell self-renewal and tumor formation rate in nude mice:Bioinformatics analysis showed that patients with both low level of FGF19 and NFATc2expression had longer overall survival compared with patients with high FGF19 or NFATc2expression.At the same time,knockdown of FGF19 and NFATc2 down-regulated the expression of stemness genes and proteins of CSCs,decreased the clone forming rate,sphere forming rate,and tumor formation rate of CSCs in nude mice,and prolonged the overall survival rate of nude mice with tumor.We found that BLU9931 and FK506 reduced the CSCs the clone forming rate,sphere forming rate,tumor formation rate in nude mice,and resistance to sorafenib.Conclusion:We found that the expression of FGF19 was up-regulated in CSCs,and FGF19 activated PLCγand ERK1/2 pathways by binding FGFR4,which enhanced SOCE levels and mediated Ca²⁺influx.Under the action of Ca N（Calcineurin）,NFATc2 dephosphorylated and translocated to nuclear to regulate the transcriptional expression of stemness genes and FGF19,forming a positive feedback signal loop that promotes self-renewal.Targeted inhibition of the FGF19 and NFATc2 pathways inhibited the self-renewal characteristics of CSCs.