Role of STEAP2 protein in hepatocarcinogenesis

STEAP2蛋白在肝癌发生中的作用

• 批准号: 10183205
• 负责人: LUZHE SUN
• 金额: $ 43.81万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 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/10183205
• 关键词: 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; Heat shock proteins; 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 Tissue; Up-Regulation; Validation; Western Blotting; Xenograft procedure; adduct; base; cell growth; cell motility; cuprous ion; differential expression; hepatocellular carcinoma cell line; in vivo; knock-down; 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

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.

我们的长期目标是发现有助于增加的发病率的分子机制 美国的肝细胞癌（HCC）。为了了解可能的分子机制 驱动HCC的发展和进展，我们使用总RNA进行了整个基因组RNA测序 来自局部HCC患者的相邻非肿瘤肝脏和HCC肿瘤组织的样品。分析 配对组织之间差异表达的基因显示生物学的显着改变 与还原氧化相关的过程和分子途径，涉及编码六个的基因 前列腺2（STEAP2）蛋白的跨膜上皮抗原。我们发现STEAP2也是 与患者的相邻非肿瘤肝组织相比，肿瘤中显着上调 癌症基因组图集（TCGA）数据集以及最近发布的RNA-Seq数据集中。 STEAP2 属于参与铁和铜离子横穿铁和铜离子的蛋白质家族 细胞和各种细胞细胞器的膜。铁和铜离子的减少形式不仅是 不受控制的肿瘤细胞生长所需的基本要素，但也已知可以介导 来自过氧化氢的羟基自由基，导致DNA和蛋白质损伤和脂质过氧化。 与STEAP2的这些功能一致，我们发现总铜的总水平明显更高 HCC组织比在相邻的非肿瘤组织中。人类HCC细胞中STEAP2表达的敲低 线可以显着抑制其生存力，运动性，软琼脂中的克隆性和沿体内异种移植的生长 随着应力激活的MAP激酶活性以及细胞内铁和铜水平的降低，而STEAP2 过表达显示相反的影响。此外，高铁饮食显着增加了HCC的发生率 鼠标模型。虽然与威尔逊氏病有关的肝铜或铁超负荷 血色素症分别是HCC的已知危险因素，无论铜和铁的失调是否失调 由于STEAP2过表达引起的稳态可能尚未探索肝癌发生。 根据我们新颖的初步发现，我们假设STEAP2上调可以驱动HCC 通过增加亚铁和乡土离子的供应，氧化应激和脂质的发展和进展 过氧化导致应力激活途径的激活。在特定的目标1中，我们将使用新的 建立的患者衍生的HCC器官培养物和HCC的自发小鼠模型 STEAP2表达的改变是否会在体外和体内影响其恶性特性。在特定目标中 2，我们将首先确定STEAP2是否具有金属果酶催化活性以及其N-是否具有 末端结构域具有明确的NADPH结合位点，其C末端结构域在HCC细胞中结合血红素。 然后，我们将设计以STEAP2的位置定向突变，以确定其催化活性是否为 其肿瘤促进活性所需的必要 细胞。在特定目标3中，我们将确定STEAP2表达的改变是否导致不稳定铁 铜，氧化应激，DNA损伤和蛋白质加合物在HCC细胞中的形成。我们将调查 STEAP2刺激的致癌途径是否取决于铁和铜的积累。我们也会 确定应力激活的MAP激酶是否介导STEAP2的HCC促进活动。我们是 第一个显示STEAP2在HCC中的上调和肿瘤功能的上调和肿瘤功能。鉴于它在 促进癌细胞生长和细胞内铁/铜的积累，导致氧化应激和 致癌途径的激活，我们提出的研究有望发现一种新型分子机制 有助于肝癌发生，并揭示可行的靶标，以开发新的预防性 和治疗策略。