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

描述(申请人提供)：PI 3-K/Akt信号轴通过促进癌细胞的存活和生长，对包括乳腺癌在内的许多癌症的发生和发展都是至关重要的。AKT过度激活在很大比例的患者中被观察到，并被认为是癌症的标志。关于控制PI3-K激活的机制，进而Akt调节和信号转导到与恶性肿瘤相关的表型，人们已经知道了很多。这些分子信息使PI 3-K和Akt抑制剂的开发能够用于癌症治疗，其中许多正在进行I期和II期试验。然而，小分子Akt抑制剂在正常体细胞中表现出副作用和毒性，反映了Akt对各种正常细胞功能是必不可少的事实。相反，不受控制的细胞分裂是另一种 人类癌症的标志，而大多数正常的体细胞处于不分裂的静止状态。因此，某些细胞周期调节因子，如细胞周期蛋白A，在癌症中经常过度表达，被认为是有希望的抗癌靶点。到目前为止，Akt过度激活和异常细胞周期调控之间的内在联系还没有确定。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，Cyclin A或CDK2的缺失显著降低了Akt的活性，这表明CDK2/Cyclin A抑制剂可能特异性地杀死乳腺癌细胞，而对正常体细胞没有影响 细胞。在目标1中，我们提出乳腺癌中CDK2/Cyclin A的异常激活与Akt的过度激活有关。我们将从机制上定义CDK2/Cyclin A如何作为一种新的上游调控机制，以细胞周期依赖的方式促进Akt的激活。我们将确定CDK2抑制剂是否能在体外和体内抑制乳腺癌细胞的生长。在目标2中，我们展示了初步证据表明，在三阴性乳腺癌细胞中，Akt的重新激活发生在PI 3-K抑制之后，并且以依赖于E3泛素连接酶Skp2的方式发生。我们将研究Skp2介导的Akt重新激活的机制基础，以驱动乳腺癌细胞中与恶性相关的表型。此外，已在乳腺癌患者中发现了E17K处Akt1和Akt2的体细胞突变。在我们的初步研究中，我们发现Akt2 E17K可以以生长因子和PI3-K非依赖性的方式促进乳腺上皮细胞的信号和增殖，而Akt1 E17K不能。我们将在体外和使用表达Akt1和Akt2 E17K的转基因小鼠来探索这种区别的机制基础，并检查Skp2在这一事件中的作用。我们在目标2中的目标是定义在乳腺癌中以PI3-K非依赖的方式控制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，这种激活对PI 3-K抑制剂没有反应。相反，我们的研究将告知使用CDK2，Skp2和Akt抑制剂进行靶向治疗，以在临床上达到最佳的治疗效果。

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.