Role of STEAP2 protein in hepatocarcinogenesis

我们的长期目标是揭示导致糖尿病发病率增加的分子机制。 肝细胞癌（HCC）在美国。为了了解可能导致 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， 来自局部HCC患者的配对的相邻非肿瘤肝和HCC肿瘤组织的样品。分析 配对组织之间差异表达的基因揭示了生物学特性的显著改变。 与氧化还原相关的过程和分子途径，其中涉及编码六个 前列腺跨膜上皮抗原2（STEAP 2）蛋白。我们发现STEAP 2也是 与患者的邻近非肿瘤肝组织相比， 癌症基因组图谱（TCGA）数据集以及两个最近发表的RNA-seq数据集。STEAP2 属于一个蛋白质家族，参与铁和铜离子的还原和转运， 细胞膜和各种细胞器。铁和铜离子的还原形式不仅是 不受控制的肿瘤细胞生长所需的基本元素，但也已知介导 过氧化氢产生的羟基自由基，导致DNA和蛋白质损伤以及脂质过氧化。 与STEAP 2的这些功能一致，我们发现在哺乳动物中， 肝癌组织中的癌细胞数明显多于癌旁组织。STEAP 2基因在人肝癌细胞中的表达下调 细胞系显著抑制其在软琼脂中的活力、运动性、克隆形成性和体内异种移植物的生长，沿着 应激激活的MAP激酶活性和细胞内铁和铜水平降低，而STEAP 2 过表达显示相反的效果。此外，高铁饮食显著增加HCC的发病率， 一个老鼠模型。虽然肝铜或铁超载与肝豆状核变性有关， 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 由于STEAP 2过表达导致的体内平衡可能有助于肝癌发生尚未被探索。 基于我们新的初步发现，我们假设STEAP 2的上调可以驱动HCC的发生。 通过亚铁和亚铜离子供应增加、氧化应激和脂质 过氧化导致应激激活途径的激活。在具体目标1中，我们将使用新 建立患者来源的HCC类器官培养物和HCC的自发小鼠模型，以确定 STEAP 2的表达改变是否会影响其体外和体内的恶性性质。具体目标 2，我们将首先确定STEAP 2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，并且其C-末端结构域结合HCC细胞中的血红素。 然后，我们将设计STEAP 2的定点突变，以确定其催化活性是否与基因表达相关。 其促肿瘤活性包括细胞生长和迁移以及HCC中的致瘤性是必需的 细胞在具体目标3中，我们将确定STEAP 2的表达改变是否导致不稳定铁的增加。 和铜、氧化应激、DNA损伤和蛋白加合物形成。我们将调查 STEAP 2刺激的致癌途径是否依赖于铁和铜的积累。我们还将 确定应激激活的MAP激酶是否介导STEAP 2的HCC促进活性。我们 首次显示STEAP 2在HCC中的上调和肿瘤促进功能。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， 激活致癌通路，我们提出的研究有望揭示一种新的分子机制， 有助于肝癌的发生，并为开发新的预防性药物揭示可操作的靶点。 和治疗策略。

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.