Elucidating the Role of Akt and Keratins in Autophagy and Tumorigenesis

描述（由申请人提供）：NIH K08导师职业发展奖为我提供了必要的基础，以实现我的长期职业目标，成为一名独立资助的转化研究者，专注于自噬在癌症治疗中的治疗操作。导师奖还将帮助我实现我的直接目标，即获得自噬研究和非黑色素瘤皮肤癌管理方面的专业知识。在达拉斯德克萨斯大学西南医学中心的支持下，我希望通过以下方式实现这些目标：1)国际公认的自噬、皮肤病学和癌症生物学专家的职业指导；2)结构化阅读和课程形式的具体指导，我将把这些指导应用于实验和患者护理。我指导的研究项目集中在Akt和角蛋白中间丝调节自噬的新模型上，并测试该模型对肿瘤发生的影响；我们的模型挑战了当前的范式，即自噬的调节主要通过mTOR发生。解决自噬如何被调节的问题不仅对解释正在进行的临床试验的结果至关重要，而且对改善自噬在未来癌症治疗中的操纵方式也至关重要。目的1验证了我们的假设，即Akt和Beclin 1的磷酸化直接调节自噬。首先，我们将验证我们的假设，即Akt可以通过磷酸化Beclin 1独立于mTOR调节自噬。接下来，我们将确定癌细胞中PI3K-Akt的激活状态如何影响Beclin 1的磷酸化。最后，我们将讨论Beclin 1磷酸化对癌细胞系自噬和细胞增殖的影响。Aim 2将测试上皮角蛋白磷酸化是否在体外调节自噬，以及已知的致病性角蛋白突变是否影响自噬和细胞增殖。首先，在已建立的体外实验中，检测突变的中间纤维（IF）蛋白和缺乏特定IF细胞的细胞对自噬的影响。接下来，我们将确定这种自噬抑制是否会导致体外细胞过度增殖。最后，我们将使用角蛋白缺陷（缺失和突变）的小鼠模型来测试受损的自噬是否有助于角蛋白病和肿瘤的发生。目的3将在体外实验和体内小鼠模型中测试预测复合物的破坏对肿瘤发生的影响。IF突变对肿瘤发生的影响将在体外确定。接下来，我们将测试化学诱导自噬或基因诱导自噬是否可以通过破坏所提出的调节复合体来阻止体内肿瘤的发生。这些研究将采用一个已建立的模型，即在皮肤中诱导PTEN缺失的非黑色素瘤皮肤癌形成。这些研究将改变我们目前对自噬如何被调节的理解，并影响在未来的临床试验中如何操纵自噬来治疗癌症。

DESCRIPTION (provided by applicant): The NIH K08 mentored career development award provides the necessary foundation for me to fulfill my long-term career goals of being an independently-funded translational investigator focusing on the therapeutic manipulation of autophagy in the treatment of cancer. The mentored award will also help me achieve my immediate goals of obtaining expertise both in autophagy research and in the management of non- melanoma skin cancer. Under the auspices of the University of Texas Southwestern Medical Center at Dallas, I hope to attain these objectives through the following: 1) career mentorship from internationally recognized physicians and scientists who are experts in autophagy, dermatology, and cancer biology, 2) specific guidance in the form of structured reading and courses, which I will apply to experiments and patient care. My mentored research project centers on a novel model for the regulation of autophagy by Akt and keratin intermediate filaments and tests this model for its effects on tumorigenesis; our model challenges the current paradigm that the regulation of autophagy occurs primarily through mTOR. Addressing the question of how autophagy is regulated will be essential not only for interpreting the results of ongoing clinical trials but also for improving the way autophagy is manipulated in future cancer therapies. Aim 1 tests our hypothesis that Akt and phosphorylation of Beclin 1 directly regulate autophagy. First, we will test our hypothesis that Akt can regulate autophagy independently of mTOR through phosphorylation of Beclin 1. Next, we will determine how the status of PI3K-Akt activation in cancer cells affects the phosphorylation of Beclin 1. Finally, we will address what effects the phosphorylation of Beclin 1 has on autophagy and cell proliferation in cancer cell lines. Aim 2 will test whether the phosphorylation of epithelial keratins regulates autophagy in vitro and whether known pathogenic keratin mutations affect autophagy and cell proliferation. First, mutant intermediate filament (IF) proteins and cells deficient for specific IFs cells will b tested for their effects on autophagy in established in vitro assays. Next, we will determine whether this inhibition of autophagy results in excess cell proliferation in vitro. Finally, we wil use mouse models with keratin defects (both deletions and mutations) to test whether impaired autophagy contributes to keratinopathies and tumorigenesis. Aim 3 will test what effects the disruption of the predicted complex has on tumorigenesis in in vitro assays and in vivo mouse models. The effects of IF mutations on tumorigenesis will be determined in vitro. Next, we will test whether the chemical induction of autophagy or genetic induction of autophagy through disruption of proposed regulatory complex can prevent tumorigenesis in vivo. These studies will employ an established model of non-melanoma skin cancer formation in skin with an inducible deletion of PTEN. These studies will transform our current understanding of how autophagy is regulated and impact how autophagy is manipulated to treat cancer in future clinical trials.