Genetic Determinants of Akt-induced Transformation

描述（由申请人提供）：蛋白激酶Akt通常在人类癌症中上调，导致存活和增殖增加，以及放松对各种细胞类型中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促进终末分化。我推测，额外的细胞因子的状态决定是否激活Akt有助于肿瘤发生或分化。由于Akt在正常细胞中的多效性作用，在肿瘤发生中与Akt合作的事件可能是比Akt本身更易处理的治疗靶点。我们已经建立了一个模型系统，其中Akt单独激活不能引起转化，但可以与其他遗传事件合作。通过在融合培养物中形成病灶的能力，可以很容易地区分携带此类事件的细胞。我们已经确定Raf的积累是一个这样的事件：Akt和Raf合作激活一些下游的Raf-dependent信号传导组件。我们将研究Akt-Raf连接的机制，以及在这个合作过程中的重要下游事件。在相同的系统中，我们也将使用一个公正的遗传筛选，以确定额外的遗传事件，可能与激活的Akt在致癌转化合作。我们已经获得了插入诱变作为基因发现项目基础的宝贵经验。我们将使用改良的逆转录病毒载体进行全基因组插入诱变，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。载体经工程改造以允许切除启动子，从而允许明确验证启动子插入是转化表型的原因。在转化中与Akt协同作用的插入事件将被鉴定，并且受影响的蛋白质在转化和分化中的作用将被进一步研究，包括与Akt和Raf途径的组分的直接相互作用的分析。我们将研究已知上调Akt的各种癌基因在转化中的新特征因子的作用。这些因素在人类癌症中的状态将使用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志物，并提供对致癌转化机制的见解。

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.