Genetic Determinants of Akt-induced Transformation

描述(申请人提供)：蛋白激酶Akt在人类癌症中普遍上调，导致生存和增殖增加，以及放松对各种细胞类型的细胞周期和基因组完整性的控制。另一方面，在一些模型系统中，Akt促进终端分化。我假设，其他细胞因子的状态决定了激活的Akt是否有助于肿瘤的发生或分化。由于Akt在正常细胞中的多效性作用，与Akt在肿瘤发生中协同作用的事件可能比Akt本身更容易成为治疗靶点。我们已经建立了一个模型系统，在这个系统中，Akt单独激活不能引起转化，但可以与其他遗传事件协同作用。通过在融合培养中形成病灶的能力，可以很容易地区分出含有这种事件的细胞。我们已经确定Raf的积累是这样一种事件：AKT和Raf合作激活Raf依赖的信号的一些下游成分。我们将研究Akt-Raf连接的机制，以及在这个合作过程中的关键下游事件。在同一系统中，我们还将使用无偏见的遗传筛选来识别可能与激活的Akt在致癌转化中合作的其他遗传事件。作为基因发现项目的基础，我们在插入突变方面获得了宝贵的经验。我们将用一种改良的逆转录病毒载体进行全基因组插入突变，该载体携带一个面向宿主DNA的启动子，驱动宿主基因的全长、截短或反义产物的产生。该载体被设计成允许切除启动子，从而允许明确地确认启动子插入是转化表型的原因。将确定与Akt在转化中协同的插入事件，并进一步研究受影响的蛋白质在转化和分化中的作用，包括分析与Akt的直接相互作用和Raf途径的组成部分。我们将研究新表征的因素在通过各种已知上调Akt的癌基因转化中的作用。这些因素在人类癌症中的地位将使用RNA和组织阵列进行研究。从长远来看，该项目将确定癌症治疗的潜在靶点和标记物，并为致癌转化的机制提供见解。

DESCRIPTION (provided by applicant): The protein kinase Akt is commonly up-regulated in human cancers, leading to increased survival and proliferation, as well as to the relaxation of controls on cell cycle and genome integrity in various cell types. On the other hand, in some model systems Akt promotes terminal differentiation. I hypothesize that the status of additional cellular factors determines whether activated Akt contributes to tumorigenesis or to differentiation. Due to the pleiotropic role of Akt in normal cells, the events that cooperate with Akt in oncogenesis are likely to be more tractable therapeutic targets than Akt itself. We have established a model system in which Akt activation alone is unable to cause transformation, but can do so in cooperation with other genetic events. The cells harboring such events could be easily distinguished by the ability to form foci in confluent cultures. We have identified the accumulation of Raf as one such event: Akt and Raf cooperate in the activation of some downstream components of Raf-dependent signaling. We will investigate the mechanism of the Akt-Raf connection, as well as the essential downstream events in this cooperative process. In the same system we will also use an unbiased genetic screen to identify additional genetic events that may cooperate with activated Akt in oncogenic transformation. We have gained valuable experience with insertional mutagenesis as a foundation for gene discovery projects. We will conduct genome-wide insertional mutagenesis with a modified retroviral vector, which carries a promoter oriented towards the host DNA, driving production of full-length, truncated or anti-sense products of host genes. The vector is engineered to permit excision of the promoter, allowing for unequivocal validation of the promoter insertion as the cause of the transformed phenotype. The insertional events cooperating with Akt in transformation will be identified and the roles of the affected proteins in transformation and differentiation will be studied further, including analysis of direct interaction with Akt and components of the Raf pathway. We will examine the roles of the newly characterized factors in transformation by various oncogenes that are known to up-regulate Akt. The status of these factors in human cancers will be studied using RNA and tissue arrays. In the long run, the project will identify potential targets and markers for cancer therapy and provide the insights into the mechanisms of oncogenic transformation.