AKT激酶磷酸化多梳蛋白Mel-18调控染色质修饰促进乳腺癌恶性演进的分子机制及其临床意义

Aberrant AKT activation is highly related to tumor malignant progression and treatment resistance. Understanding the AKT signaling network is a key to improve the clinical therapeutic efficacy of AKT inhibition. Mel-18 is a member of the polycomb group (PcG) proteins, which suppresses gene expression by modulating the chromatin status of gene promoter region. In our preliminary study, we identified that Mel-18 contains a consensus substrate motif of AKT kinase. AKT interacts with and phosphorylates Mel-18 at Thr334, leading to specific downregulation of ubiquitination status at histone H2A lysine 119. In this proposal, we will focus on the biological and pathological effects of this novel regulatory mechanism. We will investigate the roles of AKT-mediated Mel-18 phosphorylation on the breast cancer cell proliferation, invasion and migration, and maintenance of cancer stem cell activity. We will also evaluate whether Mel-18 phosphorylation can serve as a cancer biomarker by analyzing the correlation of Mel-18 phosphorylation with breast cancer progression, prognosis and treatment response in breast cancer patients. Moreover, we will use ChIP-Seq to perform a comprehensive analysis of the effect of AKT-mediated chromatin modification on Mel-18 target gene profile. Combined with TCGA data, we will dig out potential therapeutic targets for breast cancer patients with the aberrant AKT activation by using Gene prioritization method. Finally, we will propose potential combinational therapeutic strategies by combining AKT inhibitors with inhibitors of these new targets, and validate their synergistic anti-tumor efficacy in breast cancer animal models. Overall, this study will improve our understanding of the molecular regulation of AKT network and provide new combinational strategies for breast cancer treatment.

AKT的异常激活与乳腺癌的进展及治疗抵抗密切相关，对其分子调控网络的深入理解是提高此类抑制剂疗效的关键。多梳蛋白Mel-18是一个通过染色质修饰调控靶基因的转录抑制因子。我们研究发现AKT能与Mel-18结合，磷酸化其T334位点，并负性调控组蛋白H2A的泛素化水平。本课题拟通过研究AKT磷酸化Mel-18的T334调控组蛋白泛素化修饰的分子机制及其对乳腺癌细胞增殖、侵袭转移及肿瘤“干性”的影响，探讨这种磷酸化对乳腺癌患者病程进展、预后及治疗反应的预测作用。通过ChIP-Seq分析AKT介导的染色质修饰对Mel-18与靶基因DNA交互作用的影响，结合TCGA数据，采用基因优化法，找出AKT通路异常激活的乳腺癌患者新的潜在的治疗靶点；从这些靶基因中寻找合理的与AKT抑制剂的联用方案，并在乳腺癌动物模型上进行验证。本研究为深入理解AKT的分子调控网络及其在乳腺癌发生发展和诊治中的作用提供新策略

AKT激酶的异常激活与乳腺癌的恶性进展及治疗抵抗密切相关，对其分子调控网络的深入理解是提高此类抑制剂疗效的关键。在本项目中我们重点探讨了 AKT激酶磷酸化多梳基因家族蛋白Mel-18调控染色质修饰促进乳腺癌恶行演进的分子机制及其意义。重要结果及关键数据如下: ①获得AKT1激酶与Mel-18 蛋白结合并磷酸化其T334位点的证据：发现具有激酶活性的AKT1能与Mel-18蛋白在细胞内外相互结合；Mel-18蛋白具有AKT1激酶磷酸化底物的共同保守序列，结合质谱磷酸化位点检测及制备特异性磷酸化抗体，发现AKT1激酶能磷酸化Mel-18蛋白的T334位点；②明确AKT1激酶磷酸化Mel-18对乳腺癌细胞恶性表型的促进作用：发现AKT1磷酸化Mel-18的T334位点能促进乳腺癌细胞的增殖和成瘤能力，促进乳腺癌细胞的侵袭和迁移能力，促进乳腺癌细胞转移至骨和脑的能力，并使小鼠的生存期显著缩短；③阐明AKT1通过磷酸化Mel-18影响PRC1复合体形成并调控H2AK119ub表达的分子机制：发现AKT1激酶磷酸化Mel-18后，Mel-18与PRC1复合体的其他成分如RING1B、CBX7、CBX8、PHC1的结合减弱，从而抑制melPRC1复合体的形成，使组蛋白H2A-K119泛素化水平下降；高通量ChIP-Seq和RNA-seq分析显示AKT1激酶磷酸化Mel-18影响Mel-18与靶基因DNA交互作用，进而解除Mel-18对致癌靶基因的转录抑制作用，并促进乳腺癌细胞的恶性表型；④明确AKT1激酶磷酸化Mel-18的对乳腺癌的临床指导意义：发现浸润性乳腺癌组织中p-AKT1和p-Mel-18(T334)的表达呈正相关，且两者高表达能成为乳腺癌患者疾病进展，预后差及治疗反应欠佳的预测指标。铁死亡诱导剂Erastin能增强高表达p-AKT1和p-Mel-18(T334)的乳腺癌细胞对AKT抑制剂的敏感性。本研究发现AKT1信号途径下游一个新的重要的受AKT1磷酸化调控的靶蛋白Mel-18，进一步完善了AKT1的分子调控网络；首次阐明AKT1对Mel-18蛋白T334位点的磷酸化调控在乳腺癌恶性进展中的重要的生物学意义，为乳腺癌患者的疾病进展、预后及治疗反应提供了新的预测指标。