NRF2-dependent redox signaling in pancreatic cancer

项目总结 胰腺导管腺癌(PDA)是一种侵袭性的恶性肿瘤，在很大程度上仍无法治愈。令人沮丧的 PDA的预后反映了其诊断时的晚期疾病和对现有疾病的深刻抵抗 治疗。KRAS癌基因在95%的PDA中发生突变，是PDA生长和 维修。致癌KRAS诱导NFE2L2转录上调，NFE2L2编码NRF2 转录因子，氧化还原稳态的主要调节者，保护细胞免受细胞毒/细胞抑制物的影响 活性氧(ROS)的影响。重要的是，我们发现NRF2的基因消融抑制了 胰腺癌细胞在体外和体内的生长情况(2)。尽管ROS水平升高通常会导致 被认为通过对大分子，特别是DNA和脂类造成不可逆转的损害而引起细胞毒性，我们 发现NRF2的遗传消融不会导致DNA或脂质损伤(2)。相反，这两种蛋白质的合成 由于半胱氨酸的可逆和选择性氧化，肿瘤细胞的适合性受到损害 蛋白质合成关键调控因子上的残留物(2)。基于这些观察，我们假设NRF2 通过调节氧化翻译后的能力促进胰腺肿瘤的发生 修改(OxPTM)。在这里，我们建议阐明这一过程背后的机制，确定 PDA中氧化还原控制的功能，并确定潜在的治疗靶点。为此，我们将使用芯片- SEQ和RNAseq以确定直接针对Nrf2的控制半胱氨酸氧化修饰的基因(目标1)。在……里面 此外，我们将使用各种生化方法来描述半胱氨酸 氧化有助于PDA中蛋白质的合成。我们将进一步定义氧化还原的特定于转录的影响- 通过核糖体图谱进行依赖的翻译调控(目标2)。除了半胱氨酸，我们最近 发现NRF2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。将采用各种生化和遗传方法来评估其功能作用。 蛋氨酸氧化在胰腺肿瘤发生中的作用(目标3)。我们预计，我们的结果将解释 PDA氧化还原动态平衡的基本方面，并将为更有效的治疗方法的开发提供信息 用于胰腺癌和其他可能由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.