TBC1D8通过调控PKM2聚合促使卵巢癌有氧糖酵解的分子机制研究

Ovarian cancer (OCa) is the most lethal of gynecological maligant tumor, despite great efforts in clinical and basic research, little is known about the mechanism of OCa development and metastasis. In our preliminary experiments, we established two ovarian cell sublines OVCAR-3high and SK-OV-3high with higher malignant degree. We used SILAC based quantitative proteomics to analyze the proteins level changes between OVCAR-3 and OVCAR-3high. TBC1D8 was found up-regulated in OVCAR-3high cell line. A higher TBC1D8 mRNA and protein levels were further validated in high- maligant OCa cells and OCa tissues compared with low- maligant OCa cells and normal ovarian tissues, respectively. And silencing of TBC1D8 inhibited OCa cells proliferation, migration and invasion, colony formation in vitro, suggesting that TBC1D8 may be a new candidate oncogene. To discover the cue of TBC1D8 induce OCa cells malignant behavior, a known protein PKM2 with cancer glucose metabolism is preliminarily found to be interacted with TBC1D8 by using co-immunoprecipitation together with proteomics technology. We futher demonstrate this interaction contributes to form more dimeric PKM2 and hinder PKM2 tetramerization. Therefore, a scientific hypothesis in which TBC1D8 promotes OCa progression and metastasis by inhibiting PKM2 polymerization and subsequently up-regulating OCa aerobilc glycolysis is here provided..In this project, based on the novel finding above, the correlactions between the expression of TBC1D8 and features will be investigated, and the correlation of TBC1D8 with a poor prognosis in patients with OCa will be analyzed. The project will demonstrate that TBC1D8 promotes OCa progression and metastasis by preventing dimer-to-tetramer transition of PKM2, and blocking PKM2 pyruvate kinase activity in vivo and in vitro. The TBC1D8 domain that interacts with PKM2 will be discovered and validated. Whether or not can TBC1D8 influence translocation of PKM2 into the nucleus will be analyzed. The molecular mechanism in which TBC1D8 regulates PKM2 polymerization, blocks PKM2 pyruvate kinase activity, up-regulates OCa aerobilc glycolysis, will be fully elucidated in this project..In conclusion, the functional roles and molecular mechanism of TBC1D8 in OCa tumorigenesis and progression will be fully elucidated in the project. The study will be helpful for us to discover novel progonisis biomakers and drug targets for OCa.

卵巢癌是女性生殖系统最致命的恶性肿瘤，但其发病机制尚不明确。前期我们筛选建立了同一来源不同恶性程度的卵巢癌细胞亚系模型，并用此模型采用蛋白质组学技术筛选发现了一个新的卵巢癌相关蛋白TBC1D8，但其在肿瘤中的功能机制未知。我们前期预实验发现TBC1D8在高恶性卵巢癌细胞和卵巢癌组织中表达上调，TBC1D8沉默抑制了卵巢癌细胞的增殖、克隆形成和侵袭转移。进一步筛选发现TBC1D8可能与PKM2存在相互作用，并发现TBC1D8可能调控了PKM2的聚合。据此我们提出科学问题：TBC1D8通过与PKM2相互作用，抑制PKM2的聚合和代谢酶活性，介导肿瘤细胞有氧糖酵解和PKM2移位入核，从而促使卵巢癌发生发展和侵袭转移。本项目将在细胞模型、动物实验和临床样品中，证实此科学问题。最终阐明TBC1D8调控卵巢癌发生发展和侵袭转移的功能和作用机制，为发现卵巢癌预后标记物和药物干预靶标提供理论依据。

卵巢癌是死亡率最高的妇科恶性肿瘤，其发病和转移机理尚不明确。前期我们筛选发现了一个新的卵巢癌相关蛋白TBC1D8，但其具体作用机制还不清楚。本研究中，我们阐明了TBC1D8调控卵巢癌发生发展和侵袭转移的功能和作用机制。体外细胞模型和体内动物实验证实了TBC1D8 促使卵巢癌发生发展和侵袭转移的功能。进一步发现TBC1D8通过Rab-GAP TBC域与PKM2结合。TBC1D8通过与PKM2结合，阻碍PKM2四聚体化，抑制PK活性促进有氧糖酵解，但不影响PKM2的乙酰化和磷酸化修饰。TBC1D8在体外和体内促进卵巢癌的发生和有氧糖酵解的方式不依赖GAP活性。此外，TBC1D8还刺激解聚的PKM2易位入核，并诱导核内参与葡萄糖代谢和细胞周期基因的表达。我们在临床样品中确认TBC1D8高的卵巢癌患者表现出更恶性的临床病理特征和较差的预后。总的来说，TBC1D8通过阻止PKM2四聚体化来促进卵巢癌的发生和代谢重编程。