Cyclin E1驱动型卵巢癌通过GCN5/PGC-1α/G6PC途径对CDK2抑制剂耐药的机制研究

High grade serous ovarian cancer (HGSOC) is a major cause of death amongst cancers in women. The Cyclin E1-driven subtype is characterized with amplification and/or constitutive up-regulation of the CCNE1 gene，high recurrence, insensitivity to platinum-based chemotherapy and worsened prognosis. Targeted therapy against such subtype with CDK2 inhibitor initially shows satisfactory effect yet soon induces resistance, which has been a major problem. Recent study has shown that shift in glucose metabolism plays an important role in drug resistance. Our previous studies have shown that HGSOC is dependent on glucose metabolism mediated by G6PC,which is closely related to CDK2 regulation. With CDK2 inhibition, activity of TCA cycle is enhanced and combined inhibition of CDK2 and glucose metabolism demonstrates obviously increased efficacy. It has been reported that TCA cycle related GCN5/PGC-1α axis, as the upstream regulator of G6PC, is mediated by Cyclins in hepatic cells. We thus speculate that GCN5/PGC-1α/G6PC axis effects as the “molecular switch” regulated by Cyclin E1-CDK2 complex. We intend to apply genetic silencing, flow cytometry, and xenograft animal models in Cyclin E1-driven HGSOC to examine the effect of PGC-1α inhibition on CDK2 inhibitor. Via immunoprecipitation, Western Blot and qPCR, we tend to unveil the mechanism of Cyclin E1-CDK2 regulating GCN5/PGC-1α/G6PC axis, holding promise to the development of targeted therapy in HGSOC with such specific genetic background.

高级别浆液性卵巢癌（HGSOC）中常见CCNE1高频扩增及高表达，这类分子亚型称为Cyclin E1驱动型卵巢癌。该病具有对化疗不敏感、易复发和预后差的特点。靶向药物CDK2抑制剂初期治疗显效良好，但后续耐药是这类肿瘤治疗中亟待解决的重要问题。前期研究中我们发现代谢通路改变是HGSOC耐药发生的重要机制。HGSOC依赖G6PC介导的糖代谢通路，同时三羧酸循环活性增强与耐药发生密切相关。故我们推测代谢通路GCN5/PGC-1α/G6PC可能作为“分子开关”接受Cyclin E1-CDK2复合体调控，介导肿瘤耐药。本研究拟在Cyclin E1驱动型HGSOC中检测抑制PGC-1α对CDK2抑制剂抑癌效果的影响，探讨 Cyclin E1-CDK2复合体控制GCN5/PGC-1α/G6PC轴的作用机制。力求对Cyclin E1驱动型HGSOC致病机理提出新观点并为克服靶向治疗耐药提供新策略。

高级别浆液性卵巢癌（HGSOC）是女性癌症的重要死亡原因。Cyclin E1驱动型卵巢癌的特征在于CCNE1基因的扩增或表达上调，该病具有对化疗不敏感、易复发和预后差的特点。靶向药物CDK2抑制剂初期治疗显效良好，但后续耐药是这类肿瘤治疗中亟待解决的重要问题。最近的研究表明，葡萄糖代谢的改变在HGSOC耐药性中起重要作用。HGSOC依赖G6PC介导的糖代谢通路，同时三羧酸循环活性增强与耐药发生密切相关。据报道，三羧酸循环相关的GCN5/PGC-1α轴作为G6PC的上游调节因子，在肝细胞中是由细胞周期蛋白介导的。因此，我们推测GCN5/PGC-1α/G6PC轴的作用是Cyclin E1-CDK2复合物调控的“分子开关”。我们通过靶向基因沉默、流式细胞仪和异种移植动物模型，以研究Cyclin E1驱动型HGSOC中GCN5抑制对CDK2抑制剂疗效的影响。通过免疫沉淀、Western Blot和qPCR研究，我们发现CCNE1扩增的HGSOC除了特征性的细胞周期改变外，还显示出明显的代谢改变。在CCNE1扩增的细胞系OVCAR-3和A2780中，我们发现CDK2和GCN5的敲低导致G6PC降低和PGC-1α水平升高，基因水平沉默以及药物（MB-3）抑制均可导致GCN5水平的显着降低，进而使PGC-1α乙酰化水平降低。CDK2的沉默也导致了PGC-1α和Rb的乙酰化水平的显着降低。GCN5的沉默显着降低了葡萄糖摄取，增加了乳酸的产生和降低了SDH活性。蛋白质印迹实验结果显示Cyclin E1-CDK2/GCN5/PGC-1α轴存在从上游到下游的调节层次。Dinaciclib的抑制作用类似于GCN5沉默，而联合治疗则进一步显着抑制肿瘤细胞增殖。在增殖、凋亡、侵袭、迁移和集落形成实验中也得到了类似的实验结果。动物移植瘤实验表明，单独靶向沉默GCN5不会改变肿瘤的生长，但是与单药相比，Dinaciclib和GCN5沉默的联合治疗具有更为明显的肿瘤生长抑制作用，且未观察到明显的生物毒性。本研究发现靶向GCN5有望增强CDK2抑制剂的治疗效果，并具有前景广阔的临床转化价值。