Glutaredoxin, Glutathione Metabolism and Lung Cancer

项目摘要 谷胱甘肽（GSH）是一种小的巯基抗氧化剂，在维持氧化还原稳态中至关重要，并增加 谷胱甘肽在KRAS突变型肺腺癌中存在。谷胱甘肽也可导致S-谷胱甘肽化 （PSSG），GSH与蛋白质中的反应性半胱氨酸的缀合，其可以通过脱- 谷胱甘肽酶，谷氧还蛋白（GLRX）。虽然谷氧还蛋白/PSSG系统已经成为一个关键的调节因子， 氧化还原信号，很少有人知道它在肺腺癌（LUAD）的中断。我们创造了令人兴奋的 发现GLRX表达在人LUAD中降低，并且KrasG 12 D诱导的肺肿瘤发生是 在缺乏Glycogen的小鼠中增强。在氧化还原蛋白质组学筛选中，我们发现， 卵巢肿瘤去泛素化酶OTUB 1，最近被鉴定为XC-系统的调节剂。系统XC-是 一种胱氨酸/谷氨酸反向转运蛋白，输出谷氨酸并输入胱氨酸，胱氨酸反过来在细胞内被还原 半胱氨酸，谷胱甘肽合成中的限速氨基酸。系统XC-由SLC 7A 11和 SLC3A2。OTUB 1与SLC 7A 11结合以防止其泛素化和蛋白酶体降解。我们取得了 令人兴奋的发现是OTUB 1的S-谷胱甘肽化在SLC 7A 11的稳定中是重要的，导致 GSH的增加。与邻近的对照组织相比，OTUB 1和SLC 7A 11的表达增加， LUAD。这些令人兴奋的发现使我们假设，谷氧还蛋白表达的减少， KRAS驱动的肺腺癌通过S- 卵巢肿瘤去泛素化酶1（OTUB 1）的谷胱甘肽化，增加谷胱甘肽并促进 肺癌细胞的存活。此外，我们假设增加谷氧还蛋白的途径将减少， GSH水平导致顺铂诱导的肺癌细胞杀伤效力增加。具体目标1， 将确定GLRX状态是否控制KrasG 12 D诱导的肿瘤发生。具体目标2建议， 解决OTUB 1 S-谷胱甘肽化和系统XC-在KrasG 12 D中增加GSH的重要性- 而在具体目标3中，我们旨在解决AAV介导的GLRX转导对肿瘤的影响。 顺铂诱导的小鼠原发性LUAD肿瘤类器官和KrasG 12 D驱动的肿瘤中的肿瘤杀伤。完成 这一建议将提供新的见解GLRX-蛋白S-谷胱甘肽氧化还原系统的作用， LUAD的发病机制，并提供了一个理由，增加GLRX在肺肿瘤，以促进杀伤。 GLRX的药物开发有可能提供新的治疗策略来对抗LUAD， 与标准护理疗法相结合。

PROJECT SUMMARY Glutathione (GSH) is a small thiol antioxidant that is critical in maintaining redox homeostasis, and increases in glutathione occur in KRAS-mutant lung adenocarcinoma. Glutathione can also lead to S-glutathionylation (PSSG), the conjugation of GSH to reactive cysteines in proteins, which can be reversed by the de- glutathionylase, glutaredoxin (GLRX). While the glutaredoxin/PSSG system has emerged as a key regulator of redox signaling, little is known about its disruption in lung adenocarcinoma (LUAD). We have made the exciting discovery that GLRX expression is decreased in human LUAD and that KrasG12D-induced lung tumorigenesis is enhanced in mice lacking Glrx. In a redox proteomics screen we revealed increases in S-glutathionylation of ovarian tumor deubiquitinase, OTUB1, which was recently identified as a regulator of System XC-. System XC- is a cystine/glutamate antiporter that exports glutamate and imports cystine, which is in turn reduced intracellularly to cysteine, the rate limiting amino acid in glutathione synthesis. System XC- is composed of SLC7A11 and SLC3A2. OTUB1 binds to SLC7A11 to prevent its ubiquitination and proteasomal degradation. We have made the exciting discovery that S-glutathionylation of OTUB1 is important in the stabilization of SLC7A11, leading to increases in GSH. Compared to adjacent control tissue, expression of OTUB1 and SLC7A11 are increased in LUAD. These exciting findings led us to hypothesize that decreases in glutaredoxin expression in mutant KRAS-driven lung adenocarcinoma cause increases in system XC- activity through the S- glutathionylation of ovarian tumor deubiquitinase 1 (OTUB1), augmenting glutathione and promoting survival of lung cancer cells. Furthermore, we hypothesize that avenues to augment glutaredoxin will diminish GSH levels leading to increased potency of cisplatin-induced killing of lung cancer cells. In Specific Aim 1 we will determine whether GLRX status controls KrasG12D-induced tumorigenesis. Specific Aim 2 proposes to address the importance of OTUB1 S-glutathionylation and System XC- in the augmentation of GSH in KrasG12D- induced tumors while in Specific Aim 3 we aim to address the impact of AAV-mediated transduction of GLRX on cisplatin-induced tumor killing in primary LUAD tumor organoids and KrasG12D-driven tumors in mice. Completion of this proposal will provide new insights into the role of the GLRX-protein S-glutathionylation redox system in the pathogenesis of LUAD and offers a rationale for augmenting GLRX in lung tumors in order to promote killing. The pharmaceutical development of GLRX has the potential to offer new therapeutic strategies to combat LUAD, in combination with standard of care therapy.