FGF19通过激活SOCE促进肝癌干细胞自我更新的作用及机制研究

Cancer stem cells(CSCs) are the springhead of recurrence and metastasis of HCC, and it’s self-renewal is regulated by store-operated Ca2+ entry(SOCE). However, the regulation mechanism of SOCE in liver CSCs (LCSCs) is still to be clarified. FGF19 is a driven-gene in the occurrence and metastasis of HCC, and we observed that SOCE was enhanced in the LCSCs, accompanied by up-regulation of FGF19; and blocking SOCE significantly inhibit the self-renewal of LCSCs. Moreover, over-expression of FGF19 can enhanced the self-renewal and SOCE function of HCC cells. In addition, reports showed that both PLCγ-IP3R and ERK1/2 pathway are the key upstreams of SOCE activation. However, we found that these pathways are downstreams of FGF19 signaling. According to the result above, we hypothesis that FGF19 can enhance SOCE function via activation PLCγ-IP3R and ERK1/2 pathway, and then promote the self-renewal of LCSCs. In this study, we will clarify the molecular mechanism of activating SOCE by FGF19; then explore the role of SOCE in self-renewal promoted by FGF19 via using gene editing technique, immunofluorescence and calcium imaging techniques. This study will provide a theoretical basis for the development of strategies targeting LCSCs.

肿瘤干细胞是肝癌复发和转移的根源，钙库操控钙内流（SOCE）参与调控其自我更新，但SOCE上游的调控机制有待阐明。已知FGF19在肝癌发生和转移中发挥驱动作用，我们预实验表明肝癌干细胞中SOCE增强并伴随FGF19表达上调；阻断SOCE可抑制其自我更新；而上调FGF19可促进肝癌细胞自我更新及增强SOCE。文献报道PLCγ-IP3R和ERK1/2通路是SOCE激活的关键信号；我们发现这两条通路亦可被FGF19活化。因此推测：“FGF19通过PLCγ-IP3R及ERK1/2通路协同激活肝癌干细胞SOCE促进其自我更新”。本项目拟通过基因编辑、免疫荧光及钙成像等技术，深入阐明FGF19激活SOCE的机制，并明确SOCE在FGF19促进肝癌干细胞自我更新中的作用，为靶向肝癌干细胞的治疗方法提供理论依据。

肝癌干细胞（Liver cancer stem cells, LCSCs）是肝癌复发、转移和化疗耐药的根源。在该课题的资助下，我们揭示了在肝癌干细胞中，成纤维细胞生长因子(FGF)- 19与 FGFR4 结合后通过PLCγ-IP3R通路外排内质网腔中Ca2+，并激活ERK1/2 通路促进基质相互作用分子(STIM1)磷酸化后成簇汇集并与Orai1结合，增强钙库操纵钙内流(SOCE)的通道开放而引起Ca2+内流的分子机制。我们明确了SOCE的激活介导FGF19促进的肝癌干细胞自我更新，并证实FGF19-SOCE-活化T细胞核因子(NFATc2)信号回路促进肝癌干细胞的自我更新，并且靶向阻断该回路可有效抑制肿瘤干细胞的自我更新，并提高荷瘤小鼠的生存期。该部分的研究为靶向肝癌干细胞的治疗供新的思路与策略。.此外，STIM1介导的SOCE是肿瘤细胞Ca2+内流的主要方式，然而既往STIM1对肿瘤转移影响的报道存在分歧。我们发现在肝癌发生-转移过程中，STIM1和Snail1间存在负反馈调节：在肝癌发生过程中，STIM1介导SOCE激活CaMKII/PI3K/AKT/GSK3β信号，阻断Snail1经蛋白酶体的降解；而EMT启动后，表达增强的Snail1可与STIM1的启动子结合从而转录抑制STIM1的表达，并导致SOCE减弱。过表达Snail1或敲除STIM1均显著抑制HIF-1α依赖的糖摄取、有氧糖酵解以及脂质重头合成途径，并激活LKB1/AMPK级联依赖的脂肪酸氧化途径，促进失巢凋亡耐受；而阻断脂肪酸氧化则减弱了过表达Snail1或敲除STIM1引起的失巢凋亡抵抗，这表明STIM1-Snail1负反馈回路是通过重编程糖脂代谢而促进失巢凋亡耐受。证明了肝癌转移过程中STIM1-Snail1负反馈回路重编程细胞代谢由无氧糖酵解向脂肪酸氧化转变，促进失巢凋亡抵抗及肝癌转移，为肝癌转移的精准防治提供新的思路与策略。