c-Met激酶磷酸化Fis1蛋白调控线粒体裂变促进肝癌转移的分子机制及其临床意义

Hepatocellular carcinoma (HCC) is a significant cause of cancer-related morbidity and mortality worldwide. Metastasis and invasion are the basic characteristics of HCC. Therefore, it is important to identify key molecular mechanisms driving HCCs metastasis for the development of more efficacious therapies. It has been reported that mitochondrial fission is frequently upregulated in HCC cells and significantly contributes to poor patient prognosis. But it is not clear whether the fission can promote the metastasis of HCC. In our preliminary study, we found that mitochondria were more fragmented in metastatic hepatocellular carcinoma cells. Knockout of mitochondrial fission-related protein Fis1 attenuated the ability of migration and lung-metastasis of HCC cells. We also found that HGF/c-met pathway was involved in the regulation of mitochondrial fission to promote of HCC metastasis, and the mechanism may be related to c-Met-mediated Fis1 phosphorylation. In this proposal, we will discuss the effect of mitochondrial fission on regulation of HCC metastasis in animal models , and identify a clear correlation between mitochondrial fission and metastasis of HCC in tissue specimens. We will further explore the mechanism how the c-Met-mediated Fis1 phosphorylation(Y38) recruits the mitochondrial fission protein to the mitochondrial division point and promotes mitochondrial fission. We will also investigate the roles of c-met-mediated Fis1 phosphorylation on the HCC cells lamellipodia formation, invasion and metastasis. We will also evaluate whether Fis1 phosphorylation can serve as a cancer biomarker by analyzing the correlation of c-met phosphorylation with metastasis, prognosis and treatment response in HCC patients. Finally, we will propose potential combinational therapeutic strategies by using mitochondrial fission inhibitor and/or inhibitors targeting c-met kinase, and validate their synergistic anti-tumor efficacy in HCC metastasis animal models and patient-derived liver cancer xenograft models. Overall, this study will improve our understanding of mitochondrial fission and provide new strategies for HCC metastasis treatment.

肝癌易转移预后差，缺乏有效治疗手段。肝癌组织中细胞线粒体裂变增强，但裂变是否能促进肝癌转移及其调控机制尚不清楚。我们发现迁移能力高的肝癌细胞线粒体裂变能力增强，敲除线粒体裂变相关蛋白表达能抑制裸鼠肺转移；发现HGF/c-met通路参与调控线粒体裂变并促进肝癌转移，具体机制可能与c-met磷酸化线粒体外膜蛋白Fis1相关。本课题拟以肝癌为研究对象，进一步在动物转移模型上探讨抑制线粒体裂变对肝癌转移的调控作用，并在组织标本中明确线粒体裂变与肝癌转移的相关性；研究c-met通过磷酸化Fis1的Y38位点，促进Fis1与线粒体裂变动力蛋白结合并将其募集到线粒体分裂点从而介导裂变的分子机制，及其对细胞伪足形成、侵袭转移的影响，明确这种磷酸化对肝癌患者转移、预后及治疗反应的预测作用；最后在裸鼠转移模型和肝癌PDX模型上明确线粒体抑制剂和c-Met抑制剂的单药/联合用药作用，为肝癌转移的防治提供新策略。

肝癌易转移预后差，缺乏有效治疗手段。肝癌组织中细胞线粒体裂变增强，但裂变是否能促进肝癌转移及其调控机制尚不清楚，对这一科学问题的探讨，为肝癌转移的诊治提供新的策略。重要结果及关键数据如下: ①明确线粒体裂变对肝癌细胞转移的调控作用：发现与迁移能力相对低的肝癌细胞相比，迁移能力高的肝癌细胞线粒体裂变能力增强，敲除线粒体裂变相关蛋白Fis1 的表达后，肝癌细胞的侵袭迁移能力及转移至肺的能力明显减弱；②明确c-Met激酶对线粒体裂变的调控作用：通过大规模筛选，发现c-Met激酶的高亲和性配体HGF显著促进肝癌细胞线粒体发生裂变，c-Met激酶能依赖其激酶活性定位于线粒体上并调控线粒体裂变促进肝癌细胞侵袭迁移；③获得c-Met激酶与Fis1蛋白结合并磷酸化其Y38位点的证据：发现具有激酶活性的c-Met能与Fis1蛋白在细胞内外相互结合并磷酸化其Y38位点；④阐明c-Met通过磷酸化Fis1 Y38位点介导线粒体裂变的分子机制：发现c-Met激酶磷酸化Fis1后，能够促进Fis1与Drp1的结合，将Drp1募集至线粒体裂变点，促进线粒体裂变；⑤探讨c-Met激酶磷酸化Fis1对肝癌转移的影响：发现c-Met磷酸化Fis1后，促使裂变后线粒体聚集于伪足区域，促进细胞伪足形成，进而增强肝癌细胞的侵袭迁移能力以及转移至肺的能力；⑥探讨c-Met激酶磷酸化Fis1对肝癌临床诊治的意义：发现肝癌组织中HGF和p-Fis1 (Y38)的表达呈正相关，二者高表达能成为乳腺癌患者疾病进展及预后差的预测指标。联合应用c-Met激酶抑制剂crizotinib与线粒体裂变抑制剂Mdivi-1能够抑制肝癌细胞迁移和侵袭，可以成为抑制肝癌转移的新策略。本研究首次阐明HGF/c-Met途径是参与肝癌线粒体裂变的重要信号通路，发现c-Met信号途径下游一个新的重要的靶蛋白Fis1，进一步完善了 c-Met 的分子调控网络；首次阐明c-Met对Fis1蛋白Y38位点的磷酸化调控在肝癌转移中的生物学意义，为肝癌患者的疾病进展、预后及治疗反应提供了新的预测指标； 首次从线粒体裂变的角度提出干预肝癌转移的形成，通过联用线粒体裂变抑制剂和c-Met激酶抑制剂阻断转移病灶的形成，为肝癌转移的防治提供新的治疗策略和理论基础。