运用荧光共振能量转移(FRET)技术观察鼻咽癌细胞EMT及血管侵袭进程中的钙动力学改变

Previously, we showed that Epstein–Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) promoted the EGF-induced cell migration and switch to mesenchymal morphology by amplifying the store-operated Ca2+ entry (SOCE)-mediated Ca2+ signaling in nasopharyngeal carcinoma (NPC) cells, and blockage of SOCE suppressed vasculature-invasion and distant metastatic colonization in the cells expressing LMP1. However, how LMP1-boosted Ca2+ signaling modulates epithelial-mesenchymal transition (EMT) and affects vasculature-invasion still remains unclear. The proposed study is to establish the NPC cell lines constantly expressing fluorescent Ca2+ probe based on fluorescence resonance energy transfer (FRET) technique, clarify how LMP1-boosted Ca2+ signaling promotes the EGF-induced EMT through continuous observation of changes in Ca2+ dynamics during the EMT process in NPC cells. Utilizing the transgenic zebrafish hematogenous metastatic model established in our earlier works, we plan to monitor the cytosolic Ca2+ signal transduction and Ca2+ distribution in the hematogenous disseminated NPC cells during vasculature-invasion process, to determine the role of the LMP1-boosted Ca2+ signaling plays in vasculature-invasion. Our proposed study aims to set up a novel system that can be used for in vitro and in vivo recording of intracellular Ca2+ dynamics in cancer cells, by which hopefully reveal the underlying mechanism through which EBV contributes to the invasive and metastatic biological behaviors in NPC cells.

我们前期发现EB病毒编码的潜伏膜蛋白1(LMP1)通过放大钙池操纵钙内流(store-operated Ca2+ entry, SOCE)所介导的钙信号促进EGF诱导下鼻咽癌细胞迁徙及间质化形态转变，阻断SOCE则可抑制表达LMP1细胞的血管侵袭及远处转移定植，但LMP1所放大的钙信号如何调控EMT及影响血管侵袭进程尚不清晰。本研究拟构建可稳定表达基于荧光共振能量转移(FRET)技术的钙离子探针的鼻咽癌细胞株，通过观察EGF所诱导的EMT进程中鼻咽癌细胞钙动力学的动态改变，并利用我们前期建立的转基因斑马鱼血行转移模型，记录血行播散的鼻咽癌细胞在血管侵袭过程中的胞內钙信号传导及钙分布信息，阐明LMP1放大钙信号如何参与鼻咽癌细胞EMT及血管侵袭。本研究将建立起一套可在体外体内记录肿瘤细胞内钙动力学信息的全新研究体系，有望从中进一步揭开EB病毒引起鼻咽癌细胞的侵袭转移生物学行为形成的潜在机制。

鼻咽癌是一种与EB病毒感染密切相关，具有独特地理及种族分布，同时具有局部淋巴结侵袭及远处转移倾向的人类肿瘤。然而鼻咽癌高侵袭转移的恶性生物学行为的形成机制尚未完全清晰。细胞内钙离子浓度变化是一种由多种细胞器协同操控、高度动态的细胞生命活动形式。通过激活不同下游的钙离子依赖通路(Ca2+-dependent pathway)，迅速而又精确地调控着许多重要的细胞生命活动以适应外周局部微环境的改变，如增殖与凋亡，兴奋性，细胞外分泌，运动性及可塑性，基因转录活性、蛋白结构转换等等。根据我们的前期工作基础及同行的研究结果，我们提出假说：EBV通过增强钙池操纵钙离子内流(SOCE)放大了局部肿瘤微环境中的信号因子(如EGF, TGF-β)的胞內信号强度，激活/促进了EMT的进程。本课题通过运用基于荧光共振能量转移(FRET)技术的细胞自表达荧光探针观察鼻咽癌细胞EMT进程中的钙动力学动态改变，阐明EBV所放大的SOCE钙信号在EMT进程中所扮演的角色与作用。进一步利用转基因斑马鱼鼻咽癌异体移植瘤模型，在单细胞水平上实现对体内鼻咽癌细胞侵袭的动态观察。通过项目实施我们发现：（1）EBV通过促进STIM1蛋白分子的胞内动员聚集以实现对EGF唤起钙信号传导的重塑；（2）EBV通过增强STIM1蛋白所介导的钙信号传导以促进鼻咽癌细胞的EMT进程；（3）阻断STIM1介导的钙信号可明显降低EBV阳性鼻咽癌细胞的侵袭转移能力；（4）临床随访提示合并乙肝病毒 (HBV) 感染及同步放化疗中应用抗生素可增加鼻咽癌远处转移的风险，同时STIM1是潜在的可预测转移风险的独立因子。本项目的实施为鼻咽癌的侵袭转移及肿瘤细胞钙动力学的研究提供了崭新的体外及体内模型，进一步揭开了EBV如何参与鼻咽癌细胞转移侵袭恶性生物学行为形成的潜在机制。

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