Zhang Zhenzhen, Luan Qiu, Hao Wanting, Cui Yan, Li Yaming, Li Xuena
Department of Nuclear Medicine, The First Hospital of China Medical University, Shengyang, China.
J Cancer. 2023 Aug 21;14(13):2562-2573. doi: 10.7150/jca.81099. eCollection 2023.
NOX4 is highly expressed in breast cancer and is closely associated with cell invasion and metastasis. The involvement of NOX4 in glycolysis in breast cancer remains unclear. The aim of this study was to investigate the role and mechanism of NOX4 in glycolysis in breast cancer. NOX4 expression in breast cancer cells was detected by qRT-PCR and western blotting. siRNAs and plasmids were used to silence or enhance the expression of NOX4. The mRNA and protein expression of HK2, GLUT1, PKM2, LDHA, and YAP was detected by qRT-PCR and western blotting, and the F-FDG uptake rate was detected by γ-radiometer. Detection of reactive oxygen species (ROS) in cells was performed using a commercial ROS kit. After transfection, CCK8, EDU and Transwell experiments were conducted to detect cell proliferation and migration ability. MicroPET imaging was used to detect the effects of NOX4 on tumor metabolism. Immunohistochemistry was used to detect the expression of NOX4, glycolytic enzymes HK2, GLUT1, PKM2, LDHA, the proliferation index KI67, and the activation of YAP pathway molecule. In this study, the expression of NOX4 in MDA-MB-231 and MDA-MB-453 was higher than in MCF10A. qRT-PCR and western blotting experiments showed that NOX4 downregulation decreased the expression of glycolytic enzymes HK2, GLUT1, PKM2, LDHA, and 18F-FDG uptake. Conversely, the overexpression of NOX4 enhanced the expression of HK2, GLUT1, PKM2, LDHA, and 18F-FDG uptake. Proliferation and migration experiments showed that after down-regulation of NOX4, cell proliferation and migration ability decreased, while NOX4 overexpression promoted cell proliferation and migration ability. Additionally, ROS content and YAP expression decreased after NOX4 down-regulation, while ROS content and YAP expression increased following NOX4 overexpression, which was reversed by N-acetyl cysteine (NAC), a ROS inhibitor. Furthermore, exposure to NAC and Peptide17, a YAP inhibitor, blocked the increase in glycolytic enzyme expression, and the enhancement of proliferation and migration caused by NOX4 overexpression. In addition, in animal experiments, the results of the MicroPET imaging showed that the glucose metabolism rate of the NOX4 inhibitor group was significantly lower than that of the control group. ROS levels in the NOX4 inhibitor group was lower than that in the control group. Immunohistochemistry showed that the expression of HK2, GLUT1, PKM2, LDHA, KI67, and YAP in the NOX4 knock-down group were decreased. NOX4 affects breast cancer glycolysis through ROS-induced activation of the YAP pathway, further promoting the proliferation and migration of breast cancer cells.
NOX4在乳腺癌中高表达，且与细胞侵袭和转移密切相关。NOX4在乳腺癌糖酵解中的作用仍不清楚。本研究旨在探讨NOX4在乳腺癌糖酵解中的作用及机制。通过qRT-PCR和蛋白质免疫印迹法检测乳腺癌细胞中NOX4的表达。使用小干扰RNA（siRNAs）和质粒来沉默或增强NOX4的表达。通过qRT-PCR和蛋白质免疫印迹法检测己糖激酶2（HK2）、葡萄糖转运蛋白1（GLUT1）、丙酮酸激酶M2（PKM2）、乳酸脱氢酶A（LDHA）和Yes相关蛋白（YAP）的mRNA和蛋白质表达，并用γ射线探测仪检测F-FDG摄取率。使用商业活性氧（ROS）检测试剂盒检测细胞中的ROS。转染后，进行CCK8、EDU和Transwell实验以检测细胞增殖和迁移能力。使用小动物正电子发射断层显像（MicroPET）成像检测NOX4对肿瘤代谢的影响。采用免疫组织化学法检测NOX4、糖酵解酶HK2、GLUT1、PKM2、LDHA、增殖指数KI67的表达以及YAP信号通路分子的激活情况。在本研究中，MDA-MB-231和MDA-MB-453中NOX4的表达高于MCF10A。qRT-PCR和蛋白质免疫印迹实验表明，下调NOX4可降低糖酵解酶HK2、GLUT1、PKM2、LDHA的表达以及18F-FDG摄取。相反，过表达NOX4可增强HK2、GLUT1、PKM2、LDHA的表达以及18F-FDG摄取。增殖和迁移实验表明，下调NOX4后，细胞增殖和迁移能力下降，而过表达NOX4则促进细胞增殖和迁移能力。此外，下调NOX4后ROS含量和YAP表达降低，而过表达NOX4后ROS含量和YAP表达增加，这一现象可被ROS抑制剂N-乙酰半胱氨酸（NAC）逆转。此外，使用NAC和YAP抑制剂Peptide17可阻断因NOX4过表达导致的糖酵解酶表达增加以及增殖和迁移增强。另外，在动物实验中，MicroPET成像结果显示，NOX4抑制剂组的葡萄糖代谢率显著低于对照组。NOX4抑制剂组的ROS水平低于对照组。免疫组织化学显示，NOX4敲低组中HK2、GLUT1、PKM2、LDHA、KI67和YAP的表达均降低。NOX4通过ROS诱导的YAP信号通路激活影响乳腺癌糖酵解，进而促进乳腺癌细胞的增殖和迁移。
