Metabolic Regulation of 14-3-3zeta Acetylation in Breast Cancer

描述(由申请人提供)：乳腺癌是美国最常见的癌症之一，每八名女性中就有一人在一生中患上这种疾病。除了是最常见的癌症之一，它也是最具挑战性的治疗之一，每年有超过4万名妇女死于这种疾病。乳腺癌的治疗通常使用对癌细胞造成损害的细胞毒剂，激活一种称为细胞凋亡的程序性细胞死亡形式。细胞凋亡的激活导致半胱氨酸蛋白酶的激活，半胱氨酸蛋白酶被称为半胱氨酸酶，它裂解蛋白质底物，导致细胞解体。细胞凋亡的抑制是癌症的主要特征之一，而癌细胞逃避细胞死亡的能力一直是乳腺癌治疗的主要障碍。Caspase2(C2)是乳腺癌化疗后细胞凋亡的重要启动子。在一个模型实验系统中，我们的实验室先前已经证明，C2的激活可以通过与小信号分子14-3-3？的结合而受到代谢的调节。14-3-3的释放？C2受14-3-3调控？乙酰化，这种修饰是由细胞的代谢状态控制的。随着广泛的研究表明癌细胞通常具有异常的代谢途径，我们提出乳腺癌细胞调节14-3-3？才能在基因毒性应激源中生存。为了阐明14-3-3乙酰化的改变是如何发生的？可能与乳腺癌细胞逃避细胞毒药物死亡的能力有关，我们建议确定调控14-3-3？乙酰化，代谢调控机制，14-3-3？正常和乳腺肿瘤标本中的乙酰化。此外，我们的目标是探索调节乙酰化将如何改变乳腺癌细胞的化学反应性，无论是在培养中还是在小鼠模型中。这项拟议的研究将利用已建立的不同化疗敏感性的乳腺癌细胞株，从而我们将使用特定的乙酰酶和脱乙酰酶抑制剂进行筛选，以确定控制14-3-3？乙酰化。然后将通过监测不同代谢条件下酶结合和活性的变化来评估代谢控制的机制。此外，我们还将确定14-3-3的乙酰化状态是否？通过分析正常和原发人类肿瘤样本/切片中的乙酰化14-3-3来改变乳腺癌吗？用免疫组织化学的方法。最后，为了确定调节乙酰化是否改变乳腺癌细胞的化学反应性，相关的抑制剂将与化疗药物结合，并检测它们在乳腺癌细胞株和异种移植瘤中诱导细胞死亡的能力。我们预计会增强14-3-3？乙酰化将增加乳腺癌细胞对化疗诱导的细胞凋亡的敏感性，可能导致发现一种新的治疗方法，该方法可以与标准化疗结合使用，以获得更有效的乳腺癌治疗。

DESCRIPTION (provided by applicant): Breast cancer is one of the most prevalent forms of cancer in the United States, with one in every eight women developing the disease over the course of a lifetime. Besides being one of the most prevalent forms of cancer, it is also one of the most challenging to treat, with more than 40,000 women dying from the disease each year. Breast cancer therapies often utilize cytotoxic agents that cause damage to cancer cells, activating a form of programmed cell death called apoptosis. Activation of apoptosis results in the activation of cysteine proteases, known as caspases, which cleave protein substrates and lead to a disassembly of the cell. Inhibition of apoptosis is one of the central hallmarks of cance and the ability of cancer cells to evade cell death has been a major obstacle in breast cancer treatment. Caspase 2 (C2) has been shown to be a critical initiator of apoptosis in response to breast cancer chemotherapeutics. In a model experimental system, our lab has shown previously that activation of C2 can be regulated by metabolism through binding of a small signaling molecule, 14-3-3?. The release of 14-3-3? from C2 is regulated by 14-3-3? acetylation and this modification is controlled by the metabolic status of the cell. With extensive studies showing that cancer cells commonly have abnormal metabolic pathways, we propose that breast cancer cells modulate acetylation of 14-3-3? to survive genotoxic stressors. To elucidate how alterations in acetylation of 14-3-3? may contribute to the ability of breast cancer cells to evade cell death by cytotoxic agents, we propose to determine the enzymes regulating 14-3-3? acetylation, the mechanism of metabolic control, and the status of 14-3-3? acetylation in normal and breast tumor samples. Furthermore, we aim to explore how modulating acetylation will alter the chemoresponsiveness of breast cancer cells, both in culture and mouse models. The proposed research will utilize established breast cancer cell lines of varying chemosensitivities, whereby we will screen using specific acetylase and deacetylase inhibitors to identify the enzymes controlling 14-3-3? acetylation. The mechanism of metabolic control will then be evaluated by monitoring changes in enzyme binding and activity under different metabolic conditions. In addition, we will determine if the acetylation status of 14-3-3? is altered in breas cancer by analyzing both normal and primary human tumor samples/sections for acetylated 14-3-3? using immunohistochemistry. Finally, to determine whether modulating acetylation alters the chemoresponsiveness of breast cancer cells, relevant inhibitors will be combined with chemotherapeutic agents and assayed for their ability to induce cell death in both breast cancer cell lines and xenografted tumors. We expect that enhancing 14-3-3? acetylation will increase the susceptibility of breast cancer cells to chemotherapy-induced apoptosis, potentially leading to the discovery of a new therapy that can be used in combination with standard chemotherapies for more effective breast cancer treatments.