p97是一种靶向泛素的ATP依赖型分离酶，调节蛋白质稳态以及其他多种细胞功能。先前的研究表明，p97通过从KEAP1-CUL3-RBX1 E3泛素连接酶复合物中提取泛素化的NRF2，促进其通过蛋白酶体的降解，从而对NRF2产生负调节作用。在本研究中，我们确定了p97作为一个包含功能性ARE的NRF2靶基因，表明存在一个NRF2-p97-NRF2的负反馈回路来维持氧化还原稳态。利用CRISPR/Cas9基因组编辑技术，我们构建了内源性p97 ARE突变的BEAS-2B细胞系。这些p97 ARE突变细胞系表现出p97和NRF2的表达改变，以及对NRF2诱导剂反应的减弱。重要的是，我们还发现NRF2的激活与人类癌症患者中p97的表达呈正相关。最后，利用慢性砷转化的细胞系，我们证明了NRF2和p97抑制在高表达NRF2和p97的癌细胞的杀伤效应中存在协同作用。我们的研究表明，在某些类型的癌症中存在NRF2和p97的双重上调，暗示同时抑制NRF2和p97可能是一种针对分层癌症患者的有前景的治疗策略。版权所有 © 2023 Elsevier B.V.发表.

p97 is a ubiquitin-targeted ATP-dependent segregase that regulates proteostasis, in addition to a variety of other cellular functions. Previously, we demonstrated that p97 negatively regulates NRF2 by extracting ubiquitylated NRF2 from the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex, facilitating proteasomal destruction. In the current study, we identified p97 as an NRF2-target gene that contains a functional ARE, indicating the presence of an NRF2-p97-NRF2 negative feedback loop that maintains redox homeostasis. Using CRISPR/Cas9 genome editing, we generated endogenous p97 ARE-mutated BEAS-2B cell lines. These p97 ARE-mutated cell lines exhibit altered expression of p97 and NRF2, as well as a compromised response to NRF2 inducers. Importantly, we also found a positive correlation between NRF2 activation and p97 expression in human cancer patients. Finally, using chronic arsenic-transformed cell lines, we demonstrated a synergistic effect of NRF2 and p97 inhibition in killing cancer cells with high NRF2 and p97 expression. Our study suggests dual upregulation of NRF2 and p97 occurs in certain types of cancers, suggesting that inhibition of both NRF2 and p97 could be a promising treatment strategy for stratified cancer patients.Copyright © 2023. Published by Elsevier B.V.