NRF2-dependent redox signaling in pancreatic cancer

项目摘要 胰腺导管腺癌（PDA）是一种侵袭性的恶性肿瘤，在很大程度上仍然无法治愈。的惨淡 PDA的预后反映了其诊断时的晚期疾病阶段及其对现有治疗的深刻抵抗。 治疗KRAS癌基因在95%的PDA中发生突变，并作为PDA生长的有力驱动因素， 上维护致癌性KRAS诱导编码NRF 2的NFE 2L 2的转录上调 转录因子，氧化还原稳态的主要调节剂，保护细胞免受细胞毒性/细胞生长抑制 活性氧簇（ROS）的影响。重要的是，我们发现NRF 2的基因切除抑制了 胰腺癌细胞在体外和体内的生长（2）。虽然ROS水平的升高通常 据信通过对大分子，特别是DNA和脂质的不可逆损伤诱导细胞毒性， 发现NRF 2的基因切除不会导致DNA或脂质损伤（2）。相反，蛋白质合成 半胱氨酸的可逆性和选择性氧化损害了肿瘤细胞的适应性 蛋白质合成关键调节因子上的残基（2）。基于这些观察，我们假设NRF 2 促进胰腺肿瘤的发生，通过其调节氧化翻译后的能力， 修饰（oxPTM）。在此，我们建议阐明这一过程的机制，确定 氧化还原调控在PDA中的作用，并确定潜在的治疗靶点。为此，我们将使用ChIP- seq和RNAseq来鉴定控制半胱氨酸氧化修饰的直接Nrf 2靶向基因（Aim 1）。在 此外，我们将使用各种生物化学方法来描述半胱氨酸的机制， 氧化有助于PDA中的蛋白质合成。我们将进一步定义氧化还原的转录特异性效应- 通过核糖体分析依赖翻译调控（Aim 2）。除了半胱氨酸，我们最近 发现NRF 2活性的扰动也会导致其他含硫化合物的可逆氧化。 氨基酸，蛋氨酸。将采取各种生物化学和遗传学方法来评估其功能作用 蛋氨酸氧化在胰腺肿瘤发生（目的3）。我们希望我们的结果能解释 PDA氧化还原稳态的基本方面，并将为更有效的治疗方法的发展提供信息 胰腺癌和其他潜在的KRAS驱动的恶性肿瘤。

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy that remains largely incurable. The dismal prognosis of PDA reflects its advanced disease stage at diagnosis and its profound resistance to existing therapies. The KRAS oncogene is mutated in 95% of PDAs and acts as a potent driver of PDA growth and maintenance. Oncogenic KRAS induces transcriptional upregulation of NFE2L2, which encodes the NRF2 transcription factor, a master regulator of redox homeostasis that protects cells from the cytotoxic/cytostatic effects of reactive oxygen species (ROS). Importantly, we found that genetic ablation of NRF2 suppresses the growth of pancreatic cancer cells both in vitro and in vivo (2). Although elevated levels of ROS are generally believed to induce cytotoxicity through irreversible damage to macromolecules, particularly DNA and lipids, we discovered that genetic ablation of NRF2 did not lead to DNA or lipid damage (2). Instead, both protein synthesis and tumor cell fitness were compromised as a consequence of reversible and selective oxidation of cysteine residues on key regulators of protein synthesis (2). Based on these observations, we hypothesize that NRF2 promotes pancreatic tumorigenesis through its ability to regulate oxidative post-translational modifications (oxPTM). Herein, we propose to elucidate the mechanisms underlying this process, determine the functions of redox control in PDA, and identify potential therapeutic targets. To this end, we will use ChIP- seq and RNAseq to identify direct Nrf2 targeting genes that govern cysteine oxidative modification (Aim 1). In addition, we will use a variety of biochemical approaches to delineate the mechanisms through which cysteine oxidation contributes to protein synthesis in PDA. We will further define transcript-specific effects of redox- dependent translation regulation through ribosome profiling (Aim 2). In addition to cysteine, we recently discovered that perturbation of NRF2 activity also leads to the reversible oxidation of the other sulfur-containing amino acid, methionine. Various biochemical and genetic approaches will be taken to assess the functional role of methionine oxidation in pancreatic tumorigenesis (Aim 3). We anticipate that our results will explain fundamental aspects of redox homeostasis in PDA and will inform the development of more effective therapies for pancreatic cancer and potentially other KRAS-driven malignancies.