Proteomic Profiling of Cancer-Related Redox Signaling Pathways

描述（由申请人提供）：广泛认识到活性氧（ROS）在癌症的发生中起主要作用，并且它们还涉及许多癌症治疗，如电离辐射、顺铂和紫杉烷。最近，已经发现癌细胞产生ROS作为促进增殖的信号分子。不幸的是，氧化还原调节如何影响细胞信号事件的分子细节还远不清楚。我们开发的新的实验和计算技术特别适合于识别由ROS修饰的分子靶点，无论是ROS损伤还是ROS信号传导。利用我们最近开发的试剂和方法，我们现在可以通过靶向独特的反应性半胱氨酸次磺酸（Cys-SOH）基团来评估细胞群的“氧化还原谱”，半胱氨酸次磺酸基团是活化的蛋白质硫醇基团与过氧化氢和过氧亚硝酸盐（可能还有其他ROS）反应后产生的初始中间体。在此R33应用中，我们的标记技术将进一步发展用于定量和多重分析，因此它将在以下方面具有广泛的适用性：1）研究ROS损伤和ROS信号传导的基本机制; 2）分子谱分析，以分层对ROS产生疗法敏感的癌症患者;和3）基于ROS依赖性增殖信号传导的抑制的新型癌症疗法的开发。提出了以下具体目标：1）开发用于样品的多重分析的额外的新的荧光标记的Cys-SOH试剂的试剂和使用方法; 2）开发定量质谱法，其具有基于凝胶的方法的一些主要优点（包括蛋白质身份的直接读出和许多翻译后修饰）; 3）利用新的定量方法检测和鉴定HEK-293细胞和卵巢癌细胞中ROS依赖性信号传导过程中产生的Cys-SOH修饰蛋白。总之，在具体目标1和2中开发的方法将为研究界提供新的工具，用于研究氧化还原调节和信号传导的机制。在特定目标3中，这些工具将用于确定ROS在细胞增殖和凋亡调节中的靶点。首先，我们将继续我们的研究NF-？HEK-293细胞中响应细胞因子（TNF-？）的B调节和肿瘤促进剂（TPA）刺激。其次，我们将使用顺铂或紫杉烷处理的卵巢癌细胞来确定哪些蛋白质氧化对调节存活和凋亡至关重要。除了提供有关氧化还原调节和信号传导机制的具体信息外，这些生物学实验还将使我们能够进一步完善我们的试剂和方法，使其对癌症生物学界最有用。这些检测细胞蛋白质的功能性氧化修饰的方法有望鉴定介导抗癌药物作用的特定蛋白质靶标，例如通过其对细胞周期停滞、细胞分裂或凋亡的影响。这些研究的结果也可能是开发新的抗癌药物和预测特定药物治疗个体患者的疗效的能力。

DESCRIPTION (provided by applicant): It is widely appreciated that reactive oxygen species (ROS) play a major role in the initiation of cancer, and they are also implicated in many cancer therapies, such as ionizing radiation, cisplatin and taxanes. More recently, it has been discovered that cancer cells produce ROS as signaling molecules that promote proliferation. Unfortunately, the molecular details of how redox regulation affects cell signaling events are far from clear. New experimental and computational technologies that we have developed are uniquely suited to identifying the molecular targets that are modified by ROS, either as a result of ROS damage or ROS signaling. With the reagents and methods that we have recently developed, we can now evaluate the "redox profile" of cell populations by targeting uniquely reactive cysteine sulfenic acid (Cys-SOH) groups, the initial intermediates generated following reaction of activated protein thiolate groups with hydrogen peroxide and peroxynitrite (and perhaps other ROS). In this R33 application, our labeling technology will be further developed for quantification and multiplex analysis, so that it will have broad applicability in: 1) the investigation of basic mechanisms of ROS damage and ROS signaling; 2) molecular profiling to stratify patients with cancers that are sensitive to ROS-generating therapies; and 3) the development of novel cancer therapies based on the inhibition of ROS-dependent proliferative signaling. The following Specific Aims are proposed: 1) to develop reagents and methods of use for additional new, multicolor fluorescently-labeled Cys-SOH reagents for multiplex analysis of samples; 2) to develop quantitative mass spectrometry methods, which have some major advantages over gel-based methods (including direct readout of protein identity and numerous posttranslational modifications); and 3) to use the new quantitative methods to detect and identify Cys-SOH modified proteins generated during ROS-dependent signaling in HEK-293 cells and ovarian cancer cells. Taken together, the approaches developed in Specific Aims 1 and 2 will provide new tools for the research community to use to study the mechanisms of redox regulation and signaling. In Specific Aim 3, these tools will be used to determine the targets of ROS in the regulation of cell proliferation and apoptosis. First we will continue our study of NF-?B regulation in HEK-293 cells in response to cytokine (TNF-?) and tumor promoter (TPA) stimulation. Second, we will use ovarian cancer cells treated with cisplatin or taxane to determine which protein oxidations are critical to regulating survival and apoptosis. Besides providing specific information about the mechanism of redox regulation and signaling, these biological experiments will allow us to further refine our reagents and methods to make them most useful to the cancer biology community. These approaches to detecting functional oxidative modifications to cellular proteins hold promise in identifying specific protein targets that mediate the actions of anticancer drugs, e.g. through their effects on cell cycle arrest, cell division or apoptosis. An outgrowth of these studies could also be the development of new anticancer drugs and the ability to predict efficacy of a given drug in the treatment of individual patients.