Disruption of the KEAP1-NRF2 pathway results in the transactivation of NRF2 target genes, consequently inducing cell proliferation and other phenotypic changes in cancer cells. Here, we demonstrated that GULP1 was a KEAP1-binding protein that maintained actin cytoskeleton architecture and helped KEAP1 to sequester NRF2 in the cytoplasm. In urothelial carcinoma of the bladder (UCB), silencing of GULP1 facilitated the nuclear accumulation of NRF2, led to constitutive activation of NRF2 signaling, and conferred resistance to the platinum drug cisplatin. Knockdown of GULP1 in UCB cells promoted tumor cell proliferation in vitro and enhanced tumor growth in vivo. In primary UCB, GULP1 silencing was more prevalent in muscle-invasive UCB compared to nonmuscle-invasive UCB. GULP1 knockdown cells showed resistance to cisplatin treatment. In parallel with decreased GULP1 expression, we observed increased expression of NRF2, HMOX1, and other candidate antioxidant genes in cisplatin-resistant cells. Furthermore, low or no expression of GULP1 was observed in most cisplatin nonresponder cases. Silencing of GULP1 was associated with GULP1 promoter hypermethylation in cell lines and primary tumors, and a high frequency of GULP1 promoter methylation was observed in multiple sets of primary clinical UCB samples. Together, our findings demonstrate that GULP1 is a KEAP1-binding protein that regulates KEAP1-NRF2 signaling in UCB and that promoter hypermethylation of GULP1 is a potential mechanism of GULP1 silencing.

KEAP1-NRF2 通路的破坏导致 NRF2 靶基因的反式激活，从而诱导癌细胞的细胞增殖和其他表型变化。在这里，我们证明 GULP1 是一种 KEAP1 结合蛋白，它维持肌动蛋白细胞骨架结构并帮助 KEAP1 将 NRF2 隔离在细胞质中。在膀胱尿路上皮癌 (UCB) 中，GULP1 的沉默促进了 NRF2 的核积累，导致 NRF2 信号的组成性激活，并赋予对铂类药物顺铂的抗性。在 UCB 细胞中敲除 GULP1 可促进体外肿瘤细胞增殖并增强体内肿瘤生长。在原发性 UCB 中，与非肌肉侵入性 UCB 相比，GULP1 沉默在肌肉侵入性 UCB 中更为普遍。GULP1 敲低细胞显示对顺铂治疗的抗性。GULP1表达，我们观察到NRF2、HMOX1和其他候选抗氧化基因在顺铂抗性细胞中的表达增加。此外，在大多数顺铂无反应病例中观察到GULP1 的低表达或无表达。GULP1 的沉默与细胞系和原发性肿瘤中的 GULP1 启动子高甲基化有关，并且在多组原发性临床 UCB 样本中观察到高频率的 GULP1 启动子甲基化。总之，我们的研究结果表明 GULP1 是一种 KEAP1 结合蛋白，可调节 UCB 中的 KEAP1-NRF2 信号传导，并且 GULP1 的启动子高甲基化是 GULP1 沉默的潜在机制。