MICAL2磷酸化修饰通过诱导细胞骨架重排促进头颈部鳞癌进展的机制研究

Actin cytoskeleton rearrangement is essential for proliferation, invasion and metastasis of cancer cells. We found that silencing MICAL2 significantly attenuates F-actin depolymerization, proliferation, migration and invasion of head and neck squamous cell carcinoma(HNSCC). There is reason to think that phosphorylation modification is vital for activation of MICALs according to the existing literature. It was found that MICAL2 interacts with ARG kinase and the phosphorylation level of MICAL2 significantly decreases when ARG silenced in our earlier work. Therefore, we are hypothesizing that MICAL2 is phosphorylated by ARG and promotes HNSCC tumorigenesis by regulating actin skeletal rearrangement. Firstly, we will propose to use plenty of in vitro and in vivo assays to explore the impacts on HNSCC actin skeletal rearrangement, proliferation, migration and invasion of MICAL2 expression. Secondly, Co-IP, GST-pulldown, in vitro phosphorylation assay and other experiments will be done to illuminate specific molecular mechanism. Finally, the co-expression of MICAL2 and ARG and their correlation with prognosis and other clinicopathological factors will be verified by clinical samples. Our study, for the first time, will illustrate the mechanism of how MICAL2 phosphorylation modification impacts actin skeletal rearrangement and tumor progression in HNSCC and make actin skeletal rearrangement inhibition a possible treatment for HNSCC cancer.

肿瘤的微丝细胞骨架重排是恶性肿瘤实现恶性增殖和侵袭转移的先决条件。前期体内和体外研究结果显示，降表达单氧酶MICAL2后，头颈部鳞癌细胞的F-actin解聚能力、增殖能力和侵袭迁移能力减弱。磷酸化修饰对MICAL2的活化起到重要的开关作用。前期工作显示，MICAL2与激酶蛋白ARG相互作用；降表达ARG后，MICAL2的酪氨酸磷酸化水平显著降低。因此本课题提出假设：MICAL2受到激酶ARG磷酸化修饰从而通过诱导细胞骨架重排促进头颈部鳞癌进展。为验证这一假设，本项目拟首先通过体内外实验论证MICAL2的表达变化对微丝骨架重排、增殖和侵袭转移能力的影响；其次通过Co-IP、体外磷酸化等实验阐明ARG磷酸化底物蛋白MICAL2的具体机制；最后利用临床样本证实MICAL2和ARG的共表达关系，以及二者与预后等临床病理因素的相关性。本课题将为头颈部鳞癌的研究和治疗提供新的思路和理论支撑。

微丝细胞骨架为细胞的基本生物学行为提供了结构基础。在肿瘤细胞中，F-actin骨架重排是实现恶性增殖和侵袭转移的先决条件。然而，头颈部鳞癌中F-actin骨架重排的具体机制尚不明确。MICAL2是能够氧化F-actin的M44和M47位点以促进其解聚的单氧酶。我们发现其在头颈部鳞癌组织中显著高表达，并且其高表达与患者预后显著负相关。通过功能学实验发现，MICAL2能够促进头颈部鳞癌的增殖和侵袭转移。进一步研究发现，MICAL2与激酶蛋白ARG相互作用，并且ARG蛋白也在头颈部鳞癌组织中高表达并与预后显著负相关。我们通过体外磷酸化实验等一系列深入研究，证实激酶ARG能够直接磷酸化底物蛋白MICAL2的Y445、Y463和Y488位点。将纯化的重组MICAL2-redox的Y445和Y463位点替换为苯丙氨酸后，ARG无法再增强MICAL2介导F-actin解聚的能力。相应地，在敲低MICAL2的头颈部鳞癌细胞中回复表达MICAL2Y445F和MICAL2Y463F，无法挽救敲低MICAL2导致的增殖减慢表型，而回复表达野生型MICAL2或MICAL2Y488F则能够挽救表型。此外，我们还发现MICAL2的抑制剂CCG-1423，能够显著抑制头颈部鳞癌细胞的增殖、侵袭和转移。本课题的实验结果证实MICAL2在头颈部鳞癌中是一重要的促癌基因,其可能成为头颈部鳞癌治疗及预后判断潜在的新分子靶点，本研究的结果为阐明头颈部鳞癌细胞骨架重排的相关机制提供了新的理论依据。