The regulation and targeting of cell survival pathways in cancer

 描述(申请人提供)：肿瘤细胞对各种压力的动态适应能力，包括缺血和细胞毒性化疗，最终导致更具侵袭性的肿瘤生长和化疗耐药。14-3-3ζ是一种致癌的磷酸结合蛋白，已知在这一过程中发挥核心作用，但我们对1)14-3-3ζ如何响应应激以促进细胞存活/适应；以及2)14-3-3ζ如何靶向使肿瘤细胞对应激敏感的认识上存在着根本的差距。在填补这一空白之前，以14-3-3ζ为靶点以改善癌症预后的治疗目标将是不可能实现的。长期目标是制定策略，克服癌症的化疗耐药性，改善患者预后。这项建议的总体目标是了解最近发现的14-3-3ζ介导的自噬控制机制，并开发抑制乳腺癌14-3-3ζ的策略。中心假说是，缺血重排14-3-3ζ相互作用体，促进ULK1和AMPK控制的14-3-3ζ与磷酸化ATG9A的相互作用，这反过来又促进自噬介导的三阴性乳腺癌的蒽环类药物耐药。此外，从治疗的角度来看，假设抑制HDAC6，它在关键的赖氨酸残基上对14-3-3ζ去乙酰化，提供了一种新的策略来广泛地破坏胸腺肿瘤中14-3-3ζ的相互作用。在强劲的初步数据的指导下，这一假说将在以下具体情况下进行检验 目的：1)确定ULK1和AMPK调控Atg9A活性的机制，以及阻断Atg9A的磷酸化是否会覆盖肿瘤的化疗耐药；2)靶向14-3-3ζ乙酰化的机制，在体内抑制14-3-3ζ结合活性。在第一个目标中，将结合蛋白质组学、分子和显微镜方法来确定AMPK和ULK1在调节Atg9A磷酸化和14-3-3ζ结合过程中的相互作用。此外，我们已经建立的Atg9A的14-3-3ζ结合缺陷的磷酸化突变体将被用来确定取消这一机制是否会阻止TNBC对蒽环类药物的耐药性。在目标2中，一种临床批准的HDAC6抑制剂将被测试其诱导14-3-3ζ乙酰化和干扰14-3-3ζ介导的一系列患者来源的肿瘤细胞异种移植的生存通路的能力。这种方法是创新的，因为它利用了14-3-3ζ相互作用作为一种工具来理解细胞生存的适应机制。此外，AIM 2中的方法采用了一种全新的策略来阻断患者来源的肿瘤细胞瘤小鼠模型中的14-3-3ζ。这项拟议的研究具有重要意义，因为它将从根本上帮助我们理解自噬，这是一种新的化疗耐药机制，并最终可能产生14-3-3个ζ靶向策略，以改善治疗选择有限的患者群体(三阴性乳腺癌)的临床结果。

 DESCRIPTION (provided by applicant): The dynamic ability of tumor cells to adapt to a variety of stresses, including ischemia and cytotoxic chemotherapies, ultimately leads to more aggressive tumor growth and chemoresistance. 14-3-3ζ, an oncogenic phospho-binding protein, is known to play a central role in this process, yet a fundamental gap exists in our understanding of 1) how 14-3-3ζ responds to stress to promote cell survival/adaptation; and 2) how 14-3-3ζ can be targeted to sensitize tumor cells to stress. Until this gap is filled, the therapeutic targeing of 14-3-3ζ to improve cancer outcomes will be unattainable. The long-term goal is to develop strategies to overcome chemoresistance in cancer and improve patient outcomes. The overall objective of this proposal is to understand a recently discovered 14-3-3ζ- mediated mechanism of autophagy control and develop strategies to inhibit 14-3-3ζ in breast cancer. The central hypothesis is that ischemia rearranges the 14-3-3ζ interactome to promote a ULK1- and AMPK-governed 14-3-3ζ interaction with phosphorylated Atg9A, which, in turn, promotes autophagy- mediated anthracycline resistance in triple negative breast cancer (TNBC). Additionally, from a therapeutic perspective, it is posited that inhibition of HDAC6, which deacetylates 14-3-3ζ at critical lysine residues, offers a novel strategy to broadly disrupt 14-3-3ζ interactions in breas tumors. Guided by strong preliminary data, this hypothesis will be tested in the following specific aims: 1) Determine the mechanism by which ULK1 and AMPK govern Atg9A activity and whether disrupting Atg9A phosphorylation overrides chemoresistance in TNBC; and 2) Target the mechanism of 14-3-3ζ acetylation to suppress 14-3-3ζ binding activity in vivo. In the first aim, a combination of proteomics, molecular and microscopy approaches will be used to determine the interplay between AMPK and ULK1 in the regulation of Atg9A phosphorylation and 14-3-3ζ binding. Additionally, a 14-3-3ζ-binding defective phosphomutant of Atg9A, which we have already established, will be used to determine whether abrogation of this mechanism blocks anthracycline resistance in TNBC. In aim 2, a clinically approved HDAC6 inhibitor will be tested for its ability to induce 14-3-3ζ acetylation and disrupt 14-3- 3ζ-mediated survival pathways in a series of patient derived TNBC xenografts. The approach is innovative because it has utilized 14-3-3ζ interactomics as a tool to understand adaptive mechanisms of cell survival. Moreover, the approach in aim 2 employs a completely novel strategy to block 14-3-3ζ in a patient-derived TNBC mouse model. The proposed research is significant because it will contribute fundamentally to our understanding of autophagy, an emerging mechanism of chemoresistance, and could ultimately yield 14-3-3ζ-targeted strategies to improve clinical outcomes in a patient population (triple negative breast cancer) with limited treatment options.