Akt2调节PTEN缺陷型乳腺癌细胞凋亡的分子机制

Breast cancer is the most common cancer among women worldwide, and it is estimated to account for 15% of new cancer cases in women in China. A detailed understanding of the signaling pathways which govern breast tumor tumorigenesis and progression is critical for the development of new targeted therapies. This project is based on the premise that the PI3K/Akt pathway is frequently deregulated in breast cancer, and our preliminary observation that Akt2 is exclusively required for the maintenance of PTEN-deficient breast tumor spheroids. These findings challenge the dogma of redundant functions of Akt isoforms in tumor growth and progression. Importantly, they point to Akt2 and its downstream substrates as novel and effective targets for PTEN-deficient breast cancer therapy. ..In this proposal, I aim at examining the function of Akt2 in breast tumor cell survival in vitro and in vivo, as well as defining the molecular mechanisms by which Akt2 regulates apoptosis. This application has three specific aims. Aim 1: We propose to identify novel Akt2-specific substrates which regulate PTEN-deficient tumor spheroid survival using a genome-wide, high-throughput proteomic screen. The role of Akt2 and the newly identified substrates in apoptosis and other cellular functions will be examined thoroughly. Aim 2: we will identify Akt2 domains that are critical for regulating breast cancer spheroid survival, using the chimeras approach. The regions on Akt2 that are required for isoform-specific substrate phosphorylation will also be determined. Aim 3: We will investigate the physiological relevance of Akt2 phosphorylation in breast tumor maintenance using xenograft models, and evaluate the co-relation of Akt2 signaling and clinical outcomes in cancer patients. In particular, we have just developed a FRET-based homogeneous time-resolved fluorescence assay to measure activity levels of individual Akt isoforms. This innovative tool will be invaluable for assessing Akt activation status in clinical tumor samples. By collaborating with a team of experts in bioinformatics and molecular profiling of breast cancer, I am confident that these proposed studies will further advance our understanding of Akt2 signaling in breast cancer tumorigenesis at the molecular, cellular and in vivo levels. They will also identify molecular determinants on Akt isoforms that confer functional specificity. Importantly, the findings will allow the development of personalized targeted therapy by exploring the Akt2 network.

乳腺癌占中国妇女所有新发癌症的15％。详细地阐明控制乳腺肿瘤发展的信号转导网络对建立新的靶向治疗法是关键的。本项目的前提条件为1）PI3K/Akt通路在乳腺癌中经常存在异常调节，2）我们的初步研究发现Akt2（区别于Akt1/3）是维持PTEN缺陷型乳腺癌球状体所必需的。 这些发现对Akt亚型在肿瘤发展是冗余的这一传统观念带来了冲击，亦突出了Akt2及其底物成为有效的靶向药物的重要性。.本项目研究的目标是检测Akt2在乳腺癌细胞存活的功能，以及确定Akt2调节细胞凋亡的分子机制。目标1：以高通量的全基因蛋白质组筛选方法，鉴定出Akt2特异性底物，并检测底物在PTEN缺陷型癌细胞凋亡的作用。目标2：用嵌合体（chimeras）研究法确定那些Akt2结构域在调节乳腺癌起决定性作用。目标3：用异种移植瘤模型研究Akt2磷酸化与维持乳腺癌的生理相关性，并评估Akt2信号和癌患者的临床预后是否相关。

乳腺癌是全球女性最常见的恶性肿瘤。详细研究和阐明控制肿瘤发生和发展的信号通路对于开发新的靶向疗法至关重要。此项目基于以下研究基础：PI3-K / Akt通路在乳腺癌中的异常活化。在此项目中，我们的主要目标是确定Akt2调节乳腺癌进展的分子机制。利用蛋白质组和转录组分析，我们得到了Akt2亚型特异性的底物数据。我们阐明了Akt2对RTK以及其他Akt底物磷酸化中发挥的特定功能。 许多三阴性乳腺癌（TNBC）缺失PTEN导致Akt异常活化,此类型的乳腺癌具有高侵袭性且预后较差的特征，以此我们将在TNBC中寻找新的Akt底物。ANLN是高表达于TNBC的基因之一，我们的研究发现其表达受超级增强子的调控。使用Crispr/Cas9技术，我们证明了ANLN是肿瘤生长的关键调节蛋白。利用Akt抑制剂和Akt敲除，我们证明了Akt2可以正调控ANLN的表达。通过分析乳腺癌样品的RNA-seq数据，我们发现，与luminal分型的乳腺癌相比，ANLN mRNA上调的病例在TNBC分型中占比更高。重要的是，ANLN在正常的乳腺组织中表达量很低，这为ANLN成为乳腺癌治疗靶点提供了可能。在接下来的研究中，我们将进一步阐明Akt2调控ANLN表达的机理。我们已经撰写了ANLN促进乳腺癌生长的论文，手稿刚被《自然通讯》杂志接收。为了研究Akt2的分子因素和细胞定位是否对其底物的选择 起决定性作用，我们利用Crispr技术在3D乳腺肿瘤球体中荧光标记Akt亚型。 这种新颖的方法将首次允许以实时方式视化内源性Akt在3D球体细胞内动态，并确定Akt2的细胞定位是否决定其功能。为阐明Akt2调节肿瘤生长的机制，我们构建了tet-on诱导型Crispr/Cas9试验系统来评估Akt2在癌症干细胞中的作用。与Akt3相反，我们发现Akt2抑制TNBC干细胞的干性。在未来的实验中，我们将进一步分析Akt2对癌症干细胞凋亡的功能。