NOX4调控的糖酵解在乳腺癌转移中的通路机制及分子影像学研究

Metastasis is the main cause of poor prognosis in breast cancer. Energy metabolism plays an important role in tumor growth and metastasis. Aerobic glycolysis is the unique way for tumor to obtain energy. The tumor therapy targeting glycolysis is high-profile in recent years. The microenvironment of oxidative stress is associated with glycolysis in cancer and the increase of reactive oxygen species (ROS) in tumor cells is the main manifestation of the microenvironment of oxidative stress. NADPH oxidase is a major source of ROS production in tumor cells. NOX4 is an activated subtype of NADPH oxidase. Our previous studies found that the expression of NOX4 in breast cancer is closely related to glycolysis and metastasis. However, the mechanism of glycolysis regulated by NOX4 in the metastasis of breast cancer has not been clear yet. One of the aims of this study is to observe and clarify the mechanism of glycolysis regulated by NOX4 in the metastasis of breast cancer by upregulation and downregulation of NOX4. In addition, this study will synthesize 18F-DHMT which is a molecular probe targeting ROS and establish the animal models to verify regulatory mechanism in vivo by the 18F-DHMT and 18F-FDG Micro-PET imagings. This study aim to provide evidences for establishing a new therapeutic strategy in the metastasis of breast cancer from the level of energy metabolism and establish the molecular imaging methods targeting ROS in tumor.

转移是乳腺癌预后不良的主要原因。能量代谢是肿瘤生长和转移的重要支撑，有氧糖酵解是肿瘤特有的获取能量的方式。近年，靶向肿瘤糖酵解的治疗备受关注。肿瘤氧化应激微环境与糖酵解相关，肿瘤细胞的活性氧（ROS）增加是氧化应激微环境的主要表现形式，NADPH氧化酶是肿瘤细胞产生ROS的主要来源。NADPH 氧化酶4（NOX4）是NADPH 氧化酶中的一个处于活化状态的亚型。本项目组前期研究发现，NOX4的表达与乳腺癌的糖酵解及转移密切相关。但迄今为止，NOX4调控糖酵解在乳腺癌转移中的通路机制尚不清楚。本项目拟通过上调和下调NOX4表达，观察和明确NOX4调控的乳腺癌糖酵解在转移中的通路机制；构建动物模型，合成靶向ROS的18F-DHMT分子探针，并联合18F-FDG，应用Micro-PET显像在体验证调控机制，为建立从能量代谢水平阻断乳腺癌转移的新策略提供依据和建立靶向肿瘤ROS的分子影像学方法。

能量代谢是肿瘤生长和转移的重要支撑，有氧糖酵解是肿瘤特有的获取能量的方式。近年，靶向肿瘤糖酵解的治疗备受关注。肿瘤氧化应激微环境与糖酵解相关，NADPH氧化酶是肿瘤细胞产生ROS的主要来源，NADPH 氧化酶4（NOX4）是NADPH 氧化酶中的一个处于活化状态的亚型，NOX4调控糖酵解在乳腺癌转移中的作用机制尚不清楚。课题组通过开展细胞实验和在体动物实验的研究，进一步明确了NOX4调控糖酵解在乳腺癌转移中的通路机制。研究发现NOX4在两个乳腺癌细胞系MDA-MB-231和MDA-MB-453中的表达高于乳腺正常上皮MCF10A。并通过上调和下调NOX4表达，结果显示沉默NOX4表达后，糖酵解酶的表达和ROS的含量均下降，细胞的增殖和迁移能力明显下降；增强NOX4表达，糖酵解相关酶的表达，ROS均升高细胞增殖和迁移能力明显提升。沉默和上调NOX4后，P-AKT和YAP的表达表达均下降和上升。回复实验结果显示，过表达NOX4的细胞应用NAC (ROS 抑制剂)、LY294002(PI3K/AKT抑制剂)和Peptide17(YAP抑制剂)可以逆转NOX4过表达引起的P-AKT和YAP的升高；构建乳腺癌动物模型，应用Micro-PET显像在体验证调控机制，GKT137831（NOX4抑制剂）组的18F-FDG摄取SUVMAX值明显下降；肿瘤进行免疫组化，结果显示GKT137831组糖酵解关键酶（（HK2、GLUT1、PKM2、LDHA）和通路蛋白P-AKT和YAP的表达下降。研究结果揭示NOX4在乳腺癌细胞中高表达，并且可以影响乳腺癌细胞的糖酵解。NOX4可以激活ROS/PI3K/AKT和YAP双信号通路，影响乳腺癌细胞糖酵解和侵袭。为建立从能量代谢水平阻断乳腺癌转移的新策略提供依据和方法。