Lysyl Oxidase Propeptide: Breast Cancer Inhibitor

赖氨酰氧化酶前肽：乳腺癌抑制剂

基本信息

批准号：
8211091
负责人：
GAIL E. SONENSHEIN
金额：
$ 50.56万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-12 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8211091
关键词：
1-Phosphatidylinositol 3-Kinase Affect African American Agar Alleles American Anabolism Anchorage-Independent Growth Anthracenes Aromatic Polycyclic Hydrocarbons Bioluminescence Boston Breast Cancer Cell Cancer Etiology Cancer cell line Carcinogen exposure Carcinoma Case-Control Studies Cell Line Cell Proliferation Cells Cessation of life Co-Immunoprecipitations Collagen Data Databases Deposition Development Disease Progression E-Cadherin ERBB2 gene Effectiveness Elastin Environment Enzymes Estrogen Receptors Estrogen receptor negative Exposure to Extracellular Matrix Fatty acid glycerol esters Fibroblasts Frequencies Gene Expression Genes Glutamine Growth HRAS gene Heat shock proteins Hypoxia Image In Vitro Investigation Knock-in Mouse Lead Length MEKs Malignant Neoplasms Malignant neoplasm of lung Malignant neoplasm of pancreas Mammary gland Mass Spectrum Analysis Mediating Minor Modeling Mutation NIH 3T3 Cells Neoplasm Metastasis Normal Cell Nude Mice Oncogenic Oxidases Pancreatic carcinoma Participant Pathway Analysis Pathway interactions Phenotype Pilot Projects Protein Tyrosine Phosphatase Protein-Lysine 6-Oxidase Proteins Proteolytic Processing Ras Inhibitor Risk Risk Assessment Risk Factors Role Screening procedure Signal Pathway Signal Transduction Single Nucleotide Polymorphism Snails TP53 gene Testing Tissues Treatment Protocols Tumor Suppressor Proteins United States National Institutes of Health Universities Up-Regulation Variant Vimentin Woman Women&apos s Health Work Xenograft Model Yeasts cancer cell cohort crosslink dimethylbenzanthracene effective therapy epithelial to mesenchymal transition extracellular in vivo inhibitor/antagonist lung Carcinoma malignant breast neoplasm malignant phenotype matrigel migration mouse model mutant novel pancreatic cancer cells protein function public health relevance ras Oncogene receptor research clinical testing tool transcription factor tumor tumor progression tumorigenic yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Breast cancer is the second leading cause of cancer deaths among American women, and thus additional, effective treatment protocols are needed. Lysyl oxidase (LOX) is an essential extracellular enzyme that controls matrix deposition. The LOX gene was isolated as the "ras recision" gene (rrg) with an ability to inhibit the transforming activity of the Ras oncogene in NIH 3T3 cells. Consistently many cancers display reduced LOX gene expression. The LOX gene encodes a 50 kDa pro-enzyme (termed Pro-LOX), which is secreted into the extracellular environment where it is processed by proteolytic cleavage to an 18 kDa amino terminal propeptide (LOX-PP) and a functional 32 kDa enzyme. The PI's group demonstrated that the rrg activity resides in the LOX-PP domain, whereas, the LOX enzyme itself has been found to promote a more aggressive phenotype and tumor metastasis under hypoxic conditions. Importantly, LOX-PP activity was shown by the PI's group to effectively inhibit breast cancer cells, driven by Her-2/neu which signals via Ras. In culture, LOX-PP suppressed the PI3K/Akt and MEK/Erk pathways and NF-?B. Oncogenic Her-2/neu induces epithelial to mesenchymal transition (EMT) and this was reverted by LOX-PP. LOX-PP induced E-cadherin, decreased levels of Snail, tumor migration and formation of branching colonies in Matrigel, and reduced tumor formation in nude mice. A single nucleotide polymorphism (SNP) (rs1800449) G473A, resulting in an Arg158Gln substitution in a highly conserved region in the LOX-PP domain, occurs with an average 24.6% 473A minor allele carrier frequency in the HapMap database. However when cancer cells were examined, the G473A SNP was present in the majority of lines and the LOX-PP Gln variant displayed substantially impaired tumor suppressor function in vitro and in vivo, and a reduced ability to oppose the pro-tumorigenic effects of LOX. In a pilot study of African-American women, the minor 473A allele was associated with increased risk of ER1 negative breast cancer. These findings lead to two related central hypotheses: 1. Wildtype LOX-PP represents an important tumor suppressor, the activity of which is compromised by an Arg to Gln substitution. 2. A G473A (rs1800449) SNP in the LOX gene leads to increased risk of more invasive cancers. In this new RO1 application, we propose to: (Aim 1) Elucidate the mechanisms whereby wildtype LOX-PP mediates its action as a suppressor of Ras signaling in breast cancer cells and determine how these are affected by the Arg158Gln substitution; (Aim 2) Test the ability of LOX-PP to impede the tumor promoting effects of the LOX enzyme in culture and in an orthotopic mammary fat pad model; (Aim 3) Test the hypothesis that the rs1800449 SNP represents a risk factor for more invasive breast cancer using a knock-in mouse that will be prepared. The proposed studies represent pre-clinical testing of LOX-PP as an inhibitor of Ras signaling and as a risk factor of disease progression. As the LOX-PP activity was also effective against lung and pancreatic cancers, our findings have broader implications for carcinomas driven by an activated Ras oncogene. PUBLIC HEALTH RELEVANCE: Overall, these studies represent pre-clinical testing of the LOX-PP protein, a novel suppressor of Ras signaling, in breast cancer. The rs1800449 single nucleotide polymorphism (SNP) results in an Arg-to-Gln variant in LOX-PP and in substantially reduced Ras suppressing activity and increased risk of ER1 negative breast cancer in African-American women. Our proposed work will delineate the mechanism of LOX-PP activity, the changes that result from the Gln variant and prepare a knock-in mouse model that allows for testing of the potential use of SNP in assessment of risk for breast cancer.

描述（由申请人提供）：乳腺癌是美国女性癌症死亡的第二大原因，因此需要额外的、有效的治疗方案。赖氨酰氧化酶 (LOX) 是一种重要的细胞外酶，可控制基质沉积。 LOX基因被分离为“ras切除”基因(rrg)，能够抑制NIH 3T3细胞中Ras癌基因的转化活性。许多癌症始终表现出 LOX 基因表达降低。 LOX 基因编码 50 kDa 的酶原（称为 Pro-LOX），它被分泌到细胞外环境中，在细胞外环境中通过蛋白水解裂解成 18 kDa 氨基末端前肽 (LOX-PP) 和功能性 32 kDa 酶。 PI团队证明rrg活性存在于LOX-PP结构域中，而LOX酶本身被发现在缺氧条件下促进更具侵袭性的表型和肿瘤转移。重要的是，PI 团队证明 LOX-PP 活性可有效抑制乳腺癌细胞，其由通过 Ras 发出信号的 Her-2/neu 驱动。在培养物中，LOX-PP 抑制 PI3K/Akt 和 MEK/Erk 通路以及 NF-κB。致癌性 Her-2/neu 诱导上皮细胞向间质细胞转化 (EMT)，而这一现象可被 LOX-PP 逆转。 LOX-PP诱导E-钙粘蛋白，降低Snail的水平，降低Matrigel中肿瘤迁移和分支集落的形成，并减少裸鼠中的肿瘤形成。单核苷酸多态性 (SNP) (rs1800449) G473A 导致 LOX-PP 结构域中高度保守区域中的 Arg158Gln 取代，在 HapMap 数据库中，473A 次要等位基因携带频率平均为 24.6%。然而，当检查癌细胞时，大多数细胞系中都存在 G473A SNP，而 LOX-PP Gln 变体在体外和体内表现出显着受损的肿瘤抑制功能，并且抵抗 LOX 促肿瘤作用的能力降低。在一项针对非裔美国女性的试点研究中，次要 473A 等位基因与 ER1 阴性乳腺癌风险增加相关。这些发现引出了两个相关的中心假设： 1. 野生型 LOX-PP 代表一种重要的肿瘤抑制因子，其活性因 Arg 到 Gln 的取代而受到损害。 2. LOX 基因中的 G473A (rs1800449) SNP 会导致罹患更具侵袭性的癌症的风险增加。在这个新的 RO1 应用中，我们建议：（目标 1）阐明野生型 LOX-PP 在乳腺癌细胞中介导其作为 Ras 信号传导抑制剂的作用的机制，并确定这些机制如何受到 Arg158Gln 替代的影响；（目标 2）测试 LOX-PP 在培养物和原位乳腺脂肪垫模型中阻碍 LOX 酶促肿瘤作用的能力；（目标 3）使用即将准备的敲入小鼠测试 rs1800449 SNP 代表更具侵袭性乳腺癌的危险因素的假设。拟议的研究代表了 LOX-PP 作为 Ras 信号传导抑制剂和疾病进展危险因素的临床前测试。由于 LOX-PP 活性对肺癌和胰腺癌也有效，因此我们的研究结果对由激活的 Ras 癌基因驱动的癌症具有更广泛的影响。公共健康相关性：总体而言，这些研究代表了乳腺癌中 LOX-PP 蛋白（一种新型 Ras 信号传导抑制剂）的临床前测试。 rs1800449 单核苷酸多态性 (SNP) 导致 LOX-PP 中的 Arg 到 Gln 变异，并显着降低 Ras 抑制活性，并增加非洲裔美国女性患 ER1 阴性乳腺癌的风险。我们提出的工作将描述 LOX-PP 活性的机制、Gln 变体引起的变化，并准备一个敲入小鼠模型，以测试 SNP 在评估乳腺癌风险中的潜在用途。