The regulation and targeting of cell survival pathways in cancer

 描述（由申请人提供）：肿瘤细胞适应各种应激（包括缺血和细胞毒性化疗）的动态能力最终导致更具侵袭性的肿瘤生长和化疗耐药性。已知14-3-3 β是一种致癌的磷酸结合蛋白，在这一过程中发挥核心作用，但我们对1）14-3-3 β如何响应压力以促进细胞存活/适应的理解存在根本性差距;以及2）14-3-3 β如何靶向肿瘤细胞使其对压力敏感。在这一空白被填补之前，14-3-3 β的治疗靶向改善癌症结局将是无法实现的。长期目标是制定克服癌症耐药性和改善患者预后的策略。 本提案的总体目标是了解最近发现的14-3-3 β介导的自噬控制机制，并制定策略来抑制乳腺癌中的14-3-3 β。中心假设是缺血重排14-3-3 β相互作用组以促进ULK 1和AMPK控制的14-3-3 β与磷酸化Atg 9A的相互作用，这反过来促进三阴性乳腺癌（TNBC）中自噬介导的蒽环类抗生素抗性。此外，从治疗的角度来看，假设HDAC 6的抑制，其在关键赖氨酸残基处使14-3-3-乙酰基脱乙酰，提供了一种广泛破坏乳腺肿瘤中14-3-3-乙酰基相互作用的新策略。在强有力的初步数据的指导下，这一假设将在以下具体情况下得到检验。 目的：1）确定ULK 1和AMPK控制Atg 9A活性的机制以及破坏Atg 9A磷酸化是否克服TNBC中的化学抗性;和2）靶向14-3-3-乙酰化的机制以抑制体内14- 3 -3-乙酰化结合活性。在第一个目标中，将使用蛋白质组学、分子和显微镜方法的组合来确定AMPK和ULK 1在Atg 9A磷酸化和14-3-3 β结合的调节中的相互作用。此外，我们已经确定的Atg 9A的14-3-3 β结合缺陷磷酸突变体将用于确定该机制的消除是否阻断TNBC中的蒽环类抗生素抗性。在目标2中，将测试临床上批准的HDAC 6抑制剂在一系列患者来源的TNBC异种移植物中诱导14-3-3 β乙酰化和破坏14-3- 3 β介导的存活途径的能力。该方法是创新的，因为它利用14-3-3 β相互作用作为理解细胞存活适应机制的工具。此外，目的2中的方法采用了一种全新的策略来阻断患者来源的TNBC小鼠模型中的14-3-3 β。这项研究意义重大，因为它将从根本上促进我们对自噬的理解，自噬是一种新兴的化疗耐药机制，并可能最终产生14-3-3 β靶向策略，以改善治疗选择有限的患者人群（三阴性乳腺癌）的临床结局。

 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.