蛋白激酶Bβ通过肿瘤M2型丙酮酸激酶调控卵巢癌增殖、转移和存活的机制研究

Protein kinase B (AKT) has been identified to be closely associated with the growth, metastases and survival of ovarian cancer, but its downstream molecular and mechanisms of its function remain to be determined. In the previous work, by using immunoprecipitation and MALDI-TOF/TOF MS and MS, we identified that tumor M2 pyruvate kinase (PKM2) is one of new substrates of protein kinase Bβ (AKT2), and further work from us exhibited that AKT2 could phosphorylate PKM2 in vitro. In this study, whether PKM2 participates in regulation of the proliferation, invasion and survival of ovarian cancer cells will be investigated through elevated or decreased the expression of PKM2. In order to understand whether silencing PKM2 will affect the function of AKT2 in ovarian cancer cells, the ovarian cancer cell lines that stably over-express AKT2 will be established and PKM2 will then be knocked down in these AKT2-over-expressed cells. In order to understand the molecular mechanisms by which AKT2 regulates ovarian cancer cells via PKM2, followed, the change of the downstream molecular of PKM2 will be detected by up or down regulation PKM2 and AKT2, respectively. Nude mice tumorigenicity and metastases assays will be used to explore the functions of AKT2 via PKM2 on affect the tumorigenicity, metastases and survival of ovarian cancer cells in vivo. At the final stage, 200 cases of clinical ovarian cancer tissue samples including those samples with the information of 5-year survival will be collected, and the expression and activity of PKM2 and AKT2 in different stages of these tissue samples will be analyzed. In order to understand the effect of the AKT2 via PKM2 on the prognosis of ovarian cancer, the expression and activity of PKM2 and AKT2 in 60 cases of ovarian cancer tissue samples from both recurrence or no recurrent patients with ovarian cancer after they had been performed surgical operation for half an year will be detected. This project will investigate the new mechanisms of AKT signaling pathways on the regulation of ovarian cancer, which will lead to provide the important evidence for development of new anti-cancer drugs.

蛋白激酶B(AKT)与卵巢癌生长、转移及存活密切相关，但其下游成分及作用机制尚未完全明确。我们前期利用免疫共沉淀和质谱等发现PKM2是AKT2的新底物，AKT2可在体外磷酸化PKM2。本项目拟通过上调或下调PKM2，检测PKM2是否在卵巢癌细胞增殖、侵袭和存活中起作用；并过表达AKT2后再下调PKM2，探索敲减PKM2后，AKT2是否还影响卵巢癌细胞增殖、侵袭和存活；然后，通过上调或下调AKT2及PKM2，观察PKM2下游分子变化情况，探索AKT2通过PKM2对卵巢癌细胞的调控机制。接下来通过裸鼠成瘤、转移及存活实验探索AKT2通过PKM2在体内对成瘤、转移和存活的影响。最后再采集200例临床卵巢癌样本包括复与未复发及已知5年存活状况的样本，检测AKT2及PKM2在不同阶段卵巢癌组织中的表达及相关性，推断AKT2通过PKM2对卵巢癌预后的影响。本课题将系统研究AKT2通过PKM2调控卵巢癌的机制。

卵巢癌是死亡率第一的妇科恶性肿瘤，其具有早期症状隐匿，易于转移和广泛播散的生物学行为，其发生及发展机制目前未明。参与卵巢癌发生发展的信号途径非常多，其中最主要的一条途径是磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号途径。AKT在卵巢癌组织和细胞中常常过表达，因此是卵巢癌发生发展的核心分子之一。卵巢癌主要存在AKT2的异常激活和过表达。AKT2在肿瘤中的调节机制以及它促进肿瘤增殖浸润的机制目前还并没有研究清楚，其发挥作用时的下游分子也远未完全明确。我们前期通过免疫共沉淀+质谱鉴定发现PKM2是未曾报道过的AKT2的下游底物。鉴于AKT2和PKM2各自在恶性肿瘤中的重要作用，如能明确AKT2调控PKM2的机制将具有重要的意义。本研究从细胞分子，动物和组织等多个水平上全面探索AKT2通过PKM2对卵巢癌细胞的调控机制。结果发现：1、卵巢癌细胞SKOV3\HEY过表达AKT2，能够引起PKM2表达量上调，而PKM2过表达并不能引起AKT2表达量上调，表明AKT2是PKM2的上游分子，这与我们前期实验结果相同。2，细胞学实验证实AKT2能够对PKM2的表达正向调控，以及对肿瘤细胞增殖、抑制凋亡增加侵袭能力具有调控作用。3、我们发现AKT2能够通过正向调控PKM2，通过影响其下游分子STAT3/NF-κB来发挥影响卵巢癌细胞的发生发展过程。4，裸鼠肺转移模型实验中，同样证实AKT2能够通过PKM2增加卵巢癌细胞的转移能力。5，临床组织样本免疫组化实验，我们发现AKT2和PKM2在卵巢癌组织中过表达，同时两者间具有统计学相关性。本研究为临床卵巢癌的发生发展机制提供了新的线索和理论依据，并为其治疗提供了潜在的干预靶标。