Novel regulation of PI3K/Akt to direct targeted breast cancer therapies

描述（由申请人提供）：PI 3-K/Akt信号传导轴通过促进癌细胞存活和生长对许多癌症（包括乳腺癌）的起始和进展至关重要。Akt过度活化在大部分患者中观察到，并且被认为是癌症的标志。关于控制PI 3- K活化的机制，以及反过来Akt调节和与恶性肿瘤相关的表型的信号传导，已经知道很多。这些分子信息使得能够开发用于癌症治疗的PI 3-K和Akt抑制剂，其中许多处于I期和II期试验中。然而，小分子Akt抑制剂在正常体细胞中显示出毒性的副作用，反映了Akt对于各种正常细胞功能是必需的这一事实。相反，不受控制的细胞分裂是另一个 体细胞是人类癌症的标志，而大多数正常的体细胞处于非分裂的静止状态。因此，某些细胞周期调节因子，如细胞周期蛋白A，经常在癌症中过表达，被认为是有前途的抗癌目标。迄今为止，Akt过度活化和异常细胞周期调控之间的内在联系尚未确定。我们的初步研究表明Akt活性在细胞周期中波动，并反映了Cyclin A的表达。此外，细胞周期蛋白A或Cdk 2的耗竭显著降低Akt活性，表明Cdk 2/细胞周期蛋白A抑制剂可以特异性地杀死乳腺癌细胞，而对正常体细胞没有影响。 细胞在目的1中，我们提出乳腺癌中Cdk 2/Cyclin A的异常激活有助于Akt的过度激活。我们将机械地定义Cdk 2/Cyclin A作为一种新的上游调节机制如何以细胞周期依赖性方式促进Akt活化。我们将确定Cdk 2抑制剂是否可以在体外和体内抑制乳腺癌细胞的生长。在目标2中，我们显示了初步证据，即在三阴性乳腺癌细胞的亚组中，Akt的再活化发生在PI 3-K抑制之后，并且以依赖于E3泛素连接酶Skp 2的方式发生。我们将研究Skp 2介导的Akt再激活驱动乳腺癌细胞恶性相关表型的机制基础。此外，已在乳腺癌患者中鉴定出Akt 1和Akt 2在E17 K处的体细胞突变。在我们的初步研究中，我们表明，Akt 2 E17 K可以促进信号和增殖的乳腺上皮细胞在生长因子和PI 3-K-独立的方式，而Akt 1 E17 K不能。我们将在体外和使用表达Akt 1和Akt 2 E17 K的转基因小鼠探索这种区别的机制基础，并检查Skp 2在此事件中的贡献。我们在目标2中的目标是确定在乳腺癌中以PI 3-K非依赖性方式控制Akt激活的精确机制。总体而言，我们相信我们提出的研究将为乳腺肿瘤中Akt的激活提供新的分子见解，这将对PI 3-K抑制剂无反应。相反，我们的研究将为Cdk 2、Skp 2和Akt抑制剂的靶向治疗提供信息，以在临床上实现最佳治疗效果。

DESCRIPTION (provided by applicant): The PI 3-K/Akt signaling axis is critical for both the initiation and progression of many cancers, including breast cancer, by promoting cancer cell survival and growth. Akt hyperactivation is observed in a large proportion of patients and is considered a hallmark of cancer. Much is known concerning the mechanisms that govern PI 3- K activation, and in turn Akt regulation and signaling to phenotypes associated with malignancy. This molecular information has enabled the development of PI 3-K and Akt inhibitors for cancer therapy, and many of these are in phase I and II trials. However, small molecule Akt inhibitors have shown side effects with toxicity in normal somatic cells, reflecting the fact that Akt is essential for various normal cellular functions. In contrast, uncontrolled cell division is another hallmark of human cancer, whereas most normal somatic cells are in a non-dividing, quiescent state. Consequently, certain cell cycle regulators such as Cyclin A, frequently overexpressed in cancer, are considered promising anti-cancer targets. To date, an intrinsic connection between Akt hyperactivation and aberrant cell cycle regulation has not been defined. Our preliminary studies show that Akt activity fluctuates during cell cycle and mirrors Cyclin A expression. Moreover, depletion of Cyclin A or Cdk2 dramatically reduces Akt activity, suggesting that Cdk2/Cyclin A inhibitors may specifically kill breast cancer cells with no effect of normal somatic cells. In Aim 1, we propose that aberrant activation of Cdk2/Cyclin A in breast cancer contributes to hyperactivation of Akt. We will mechanistically define how Cdk2/Cyclin A functions as a novel upstream regulatory mechanism to promote Akt activation in a cell cycle- dependent manner. We will determine whether Cdk2 inhibitors can suppress breast cancer cell growth in vitro and in vivo. In Aim 2, we show preliminary evidence that in a subset of triple-negative breast cancer cells, reactivation of Akt occurs subsequent to PI 3-K inhibition, and in a manner dependent on the E3 ubiquitin ligase Skp2. We will investigate the mechanistic basis for Skp2-mediated reactivation of Akt to drive phenotypes associated with malignancy in breast cancer cells. Furthermore, somatic mutations in Akt1 and Akt2 at E17K have been identified in breast cancer patients. In our preliminary studies, we show that Akt2 E17K can promote signaling and proliferation of breast epithelial cells in a growth factor and PI 3-K- independent manner, whereas Akt1 E17K cannot. We will explore the mechanistic basis for this distinction in vitro and using transgenic mice expressing Akt1 and Akt2 E17K, and examine the contribution of Skp2 in this event. Our goal in Aim 2 is to define the precise mechanisms that govern Akt activation in a PI 3-K- independent manner in breast cancer. Overall, we believe that our proposed studies will provide new molecular insights into the activation of Akt in breast tumors, that would be unresponsive to PI 3-K inhibitors. Instead, our studies will inform the use of Cdk2, Skp2 and Akt inhibitors for targeted therapy to achieve optimal treatment efficacy in the clinic.