FSH通过PKM2上调上皮性卵巢癌细胞Warburg效应促进肿瘤增殖的机制研究

Ovarian cancer is the most lethal gynecological malignancy and epithelial ovarian cancer (EOC) is the most common pathological type of ovarian cancer with poor prognosis. Pyruvate kinase isozyme type M2(PKM2) is an important rate limiting enzyme in glycolysis, which plays an important role in the promotion of Warburg effect and cell proliferation. For the first time, we found that PKM2 was significantly expressed in EOC tissues and the expression in cancer cells was positively correlated with the glycolytic efficiency and cell proliferation. Follicle stimulating hormone(FSH) was additionally found to upregulate the expression of PKM2 in EOC and promote the tumor proliferation and the Warburg effect as well. Thus, we believe that the Warburg effect induced by PKM2 would be one of the significant mechanisms of FSH in the carcinogenesis of ovarian cancer. we attempt to verify that FSH may promote the Warburg effect and tumor proliferation afterward in EOC by upregulating PKM2 expression through PI3K/AKT/HIF-1α pathway. Furthermore, we try to testify the hypothesis that PKM2 can inhibit the degradation of HIF-1α by decreasing the expression of PHD2, which helps to promote the positive feedback regulation between HIF-1α and PKM2 and amplify the Warburg effect. The coming results of this study will prompt people to explore the occurrence of ovarian cancer by the perspective of cell metabolism and provide a potential target for ovarian cancer.

卵巢癌是死亡率最高的妇科恶性肿瘤，上皮性卵巢癌(EOC)是卵巢癌中最常见的病理类型，预后极差。糖酵解过程中重要的限速酶丙酮酸激酶M2亚型(PKM2)在促进细胞Warburg效应和肿瘤增殖中具有重要作用。我们预实验中发现PKM2在EOC中显著表达，并与肿瘤细胞有氧糖酵解以及增殖呈明显正相关，卵泡刺激素(FSH)亦能促进PKM2表达，并诱导肿瘤细胞发生Warburg效应和快速增殖。故本课题组认为卵巢癌中PKM2介导的Warburg效应是FSH重要的促瘤机制，尝试验证FSH可通过PI3K/AKT/HIF-1α途径上调PKM2介导的Warburg效应，从而促进肿瘤增殖，并进一步证明PKM2可下调脯氨酸羟化酶2(PHD2)的表达，通过抑制HIF-1α降解促进PKM2与HIF-1α正反馈调节，进而扩大Warburg效应。本课题从细胞代谢的角度审视卵巢癌发生，旨在为卵巢癌的靶向治疗提供潜在的靶点。

肿瘤细胞糖代谢异常在卵巢癌的发病及进程中起重要作用,丙酮酸激酶（Pyruvate Kinase，PK）是糖酵解过程中重要的限速酶，对肿瘤细胞代谢和增殖是必需的，但其具体机制尚不清楚。前期工作发现, PKM2在上皮性卵巢癌组织中的表达明显高于正常卵巢组织。在卵巢癌细胞株中降调PKM2 表达能显著降低卵巢癌细胞的葡萄糖摄入和乳酸生成，并抑制卵巢癌细胞的增殖转移，FSH 能上调PKM2的表达，并促进卵巢癌细胞增殖和细胞糖酵解，而这种作用一定程度上能通过下调PKM2 的表达而受到抑制。由此我们提出一个假设, FSH通过PI3K/Akt/HIF-1α途径上调PKM2 表达，诱导卵巢癌细胞发生Warburg 效应，从而促进肿瘤增殖；而PKM2及其诱导的Warburg效应能促进PKM2与HIF-1α的正反馈调节，这可能是进一步扩大Warburg效应的关键。课题组在预实验的基础上，通过免疫组化的方法扩大标本量，检测各个类型卵巢癌组织中PKM2的表达，结果发现在浆乳癌中PKM2较其他病理类型上皮来源卵巢恶性肿瘤中PKM2表达显著，并且肿瘤分化水平越低，FIGO分期越晚，淋巴结转移者PKM2的表达水平越高，接着我们研究了FSH与肿瘤细胞糖代谢和增殖的关系，我们发现，FSH能通过激活PI3K-Akt活性促进HIF-1α以及PKM2的表达。抑制PI3K-Akt信号通路后，FSH在有氧与乏氧的情况下都能促进SKOV3、OVCAR3细胞糖酵解，促进肿瘤细胞增殖。抑制PI3K-Akt通路活性，能显著抑制肿瘤细胞糖酵解，并且下游的HIF-1α、PKM2的表达受到抑制。当抑制PI3K-Akt信号通路后细胞周期阻滞在G0-G1期，凋亡早期细胞比例明显增加。最后我们利用siRNA下调PKM2表达并检测SKOV3 PHD2、HIF-1αmRNA表达情况， CoIP结果表明PKM2和HIF-1α能够结合，HIF-1a泛素化水平在PKM2敲除组增加，PHD2敲除以及DMOG处理后能减少HIF-1a的泛素化水平。我们的实验探索了FSH作用下卵巢癌细胞代谢与增殖之间的相互联系，验证FSH上调Warburg效应是FSH促瘤机制中的重要环节，PKM2与卵巢癌预后相关，同时也是介导卵巢癌异常糖酵解的关键分子, PKM2通过PHD2参与了HIF-1α的泛素化降解。这些为卵巢癌的靶向治疗提供新的视角。