Role of STEAP2 protein in hepatocarcinogenesis

STEAP2蛋白在肝癌发生中的作用

基本信息

批准号：
10632092
负责人：
LUZHE SUN
金额：
$ 42.98万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10632092
关键词：
Acceleration Affect Agar Automobile Driving Binding Binding Sites Biochemical Biological Assay Biological Process Biophysics C-terminal Cancer Cell Growth Cancer Etiology Cells Cessation of life Code Copper DNA DNA Damage Data Set Development Diet Elements Engineering Family Family member Flavins Genes Goals Growth Heme Hemochromatosis Hepatic Hepatocarcinogenesis Hepatolenticular Degeneration Homeostasis Human Hydrogen Peroxide Hydroxyl Radical In Vitro Incidence Intake Ions Iron Iron Overload Lipid Peroxidation Liver MAPK8 gene Malignant - descriptor Malignant Epithelial Cell Malignant Neoplasms Mediating Membrane Metals Mitogen-Activated Protein Kinases Molecular Mutation N-terminal NADP NADPH Oxidase Oncogenic Organelles Organoids Oxidation-Reduction Oxidative Stress Oxidoreductase Pathway interactions Patients Preventive Primary carcinoma of the liver cells Production Property Protein Family Proteins Publishing RNA Research Reverse Transcriptase Polymerase Chain Reaction Risk Factors Role Sampling Site Stains Stress Testing The Cancer Genome Atlas Therapeutic Time Tissues Tumor Promotion Tumor Tissue Up-Regulation Validation Western Blotting Xenograft procedure adduct cell growth cell motility cuprous ion differential expression hepatocellular carcinoma cell line in vivo knock-down liver cancer model mouse model neoplastic cell novel overexpression p38 Mitogen Activated Protein Kinase six transmembrane epithelial antigen of the prostate 2 transcriptome sequencing tumor tumorigenesis tumorigenic whole genome

项目摘要

Our long-term goal is to uncover molecular mechanisms that contribute to the increased incidence of hepatocellular carcinoma (HCC) in the US. In order to understand potential molecular mechanisms that may drive HCC development and progression, we performed whole genome RNA sequencing using total RNA samples from paired adjacent non-tumor liver and HCC tumor tissues of local HCC patients. Analysis of the differentially expressed genes between the paired tissues revealed significant alterations of Biological Processes and Molecular Pathways associated with oxidation reduction, which involves a gene coding for Six Transmembrane Epithelial Antigen of Prostate 2 (STEAP2) protein. We found that STEAP2 was also significantly upregulated in the tumors in comparison to adjacent non-tumor liver tissues of the patients in The Cancer Genome Atlas (TCGA) dataset as well as in two recently published RNA-seq datasets. STEAP2 belongs to a family of proteins involved in the reduction and transport of iron and copper ions across membranes of a cell and various cellular organelles. The reduced forms of iron and copper ions are not only essential elements needed for uncontrolled tumor cell growth, but also known to mediate the production of hydroxyl radicals from hydrogen peroxide, which cause DNA and protein damage and lipid peroxidation. Consistent with these functions of STEAP2, we found total levels of copper were significantly higher in the HCC tissues than in the adjacent non-tumor tissues. Knockdown of STEAP2 expression in human HCC cell lines significantly inhibited their viability, motility, clonogenicity in soft agar, and xenograft growth in vivo along with decreased stress-activated MAP kinase activity and intracellular iron and copper levels, whereas STEAP2 overexpression showed opposite effects. Furthermore, a high iron diet significantly increased HCC incidence in a mouse model. Although hepatic copper or iron overload associated with Wilson's disease and hemochromatosis respectively is a known risk factor for HCC, whether dysregulation of copper and iron homeostasis due to STEAP2 overexpression may contribute to hepatocarcinogenesis has not been explored. On the basis of our novel preliminary findings, we hypothesize that STEAP2 upregulation can drive HCC development and progression via increased supply of ferrous and cuprous ions, oxidative stress, and lipid peroxidation resulting in the activation of stress-activated pathways. In specific aim 1, we will use newly established patient-derived HCC organoid cultures and a spontaneous mouse model of HCC to determine whether altered expression of STEAP2 will affect their malignant properties in vitro and in vivo. In specific aim 2, we will first determine whether STEAP2 possesses metalloreductase catalytic activity and whether its N- terminal domain has a well-defined NADPH binding site and its C-terminal domain binds heme in HCC cells. We will then engineer site-directed mutations of STEAP2 to determine whether its catalytic activity is necessary for its tumor-promoting activity including cell growth and migration, and tumorigenic property in HCC cells. In specific aim 3, we will determine whether altered expression of STEAP2 results in increased labile iron and copper, oxidative stress, DNA damage, and protein adduct formation in HCC cells. We will investigate whether STEAP2-stimulated oncogenic pathways is dependent on iron and copper accumulation. We will also determine whether stress-activated MAP kinases mediate the HCC-promoting activities of STEAP2. We are the first to show the upregulation and tumor-promoting functions of STEAP2 in HCC. Given its role in promoting cancer cell growth and intracellular iron/copper accumulation leading to oxidative stress and activation of oncogenic pathways, our proposed research promises to uncover a novel molecular mechanism contributing to hepatocarcinogenesis and to reveal actionable targets for the development of novel preventive and therapeutic strategies.

我们的长期目标是揭示导致发病率增加的分子机制美国的肝细胞癌（HCC）。为了了解可能的潜在分子机制为了推动 HCC 的发展和进展，我们使用总 RNA 进行了全基因组 RNA 测序来自当地 HCC 患者的配对相邻非肿瘤肝脏和 HCC 肿瘤组织的样本。分析配对组织之间差异表达的基因揭示了生物特征的显着改变与氧化还原相关的过程和分子途径，其中涉及编码六的基因前列腺跨膜上皮抗原 2 (STEAP2) 蛋白。我们发现 STEAP2 也是与患者邻近的非肿瘤肝组织相比，肿瘤中的表达显着上调癌症基因组图谱 (TCGA) 数据集以及最近发布的两个 RNA-seq 数据集。阶段2 属于参与铁离子和铜离子的还原和转运的蛋白质家族细胞膜和各种细胞器。铁离子和铜离子的还原形式不仅不受控制的肿瘤细胞生长所需的必需元素，但也已知可介导过氧化氢产生的羟基自由基，会导致 DNA 和蛋白质损伤以及脂质过氧化。与 STEAP2 的这些功能一致，我们发现铜的总水平明显更高 HCC组织高于邻近的非肿瘤组织。人 HCC 细胞中 STEAP2 表达的敲低品系显着抑制其在软琼脂中的活力、运动性、克隆形成性以及体内异种移植物的生长应激激活的 MAP 激酶活性和细胞内铁和铜水平降低，而 STEAP2 过度表达表现出相反的效果。此外，高铁饮食显着增加了肝癌的发病率鼠标模型。尽管肝铜或铁超载与威尔逊病和血色素沉着症分别是 HCC 的已知危险因素，无论是铜和铁的失调 STEAP2 过度表达导致的稳态可能导致肝癌发生尚未得到探索。根据我们新的初步发现，我们假设 STEAP2 上调可以驱动 HCC 通过增加亚铁离子和亚铜离子、氧化应激和脂质的供应来促进发育和进展过氧化导致应激激活途径的激活。在具体目标1中，我们将新使用建立源自患者的 HCC 类器官培养物和自发性 HCC 小鼠模型，以确定 STEAP2表达的改变是否会影响其体外和体内的恶性特性。在特定目标下 2、首先确定STEAP2是否具有金属还原酶催化活性以及其N- 末端结构域具有明确的 NADPH 结合位点，其 C 末端结构域结合 HCC 细胞中的血红素。然后我们将设计 STEAP2 的定点突变以确定其催化活性是否其促进肿瘤的活性（包括细胞生长和迁移）以及 HCC 的致瘤特性是必需的细胞。在具体目标 3 中，我们将确定 STEAP2 表达的改变是否会导致不稳定铁增加铜、氧化应激、DNA 损伤和 HCC 细胞中蛋白质加合物的形成。我们将调查 STEAP2 刺激的致癌途径是否依赖于铁和铜的积累。我们也会确定应激激活的 MAP 激酶是否介导 STEAP2 的 HCC 促进活性。我们是首次展示 STEAP2 在 HCC 中的上调和促癌功能。鉴于其在促进癌细胞生长和细胞内铁/铜积累，导致氧化应激和激活致癌途径，我们提出的研究有望揭示一种新的分子机制促进肝癌发生并揭示开发新型预防药物的可行目标和治疗策略。