Lysyl Oxidase Propeptide: Breast Cancer Inhibitor

赖氨酰氧化酶前肽：乳腺癌抑制剂

• 批准号: 7767218
• 负责人: GAIL E. SONENSHEIN
• 金额: $ 52.8万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 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/7767218
• 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; Effectiveness; Elastin; Environment; Enzymes; Estrogen Receptors; 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; Lung; MEKs; Malignant Neoplasms; 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 genes; 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' s Health; Work; Xenograft Model; Yeasts; cancer cell; cohort; crosslink; effective therapy; epithelial to mesenchymal transition; extracellular; in vivo; inhibitor/antagonist; malignant breast neoplasm; malignant phenotype; matrigel; migration; mouse model; mutant; novel; pre-clinical; protein function; public health relevance; ras Oncogene; receptor; tool; transcription factor; tumor; tumor progression; tumorigenic; yeast two hybrid system

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(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)是一种控制基质沉积的重要胞外酶。该LOX基因在NIH3T3细胞中被分离为具有抑制RAS癌基因转化活性的“ras Receision”基因(RRG)。许多癌症都表现出LOX基因表达的降低。LOX基因编码一个50 kDa的前体酶(称为Pro-LOX)，它被分泌到细胞外环境中，在那里它被蛋白水解酶切割成一个18 kDa的氨基端前肽(LOX-PP)和一个32 kDa的功能性酶。PI的研究小组证明，RRG活性位于LOX-PP结构域，而LOX酶本身被发现在低氧条件下促进更具侵袭性的表型和肿瘤转移。重要的是，PI的小组显示LOX-PP活性可以有效地抑制乳腺癌细胞，这是由RAS信号转导的HER-2/neu驱动的。在培养条件下，LOX-PP可抑制PI3K/Akt和MEK/Erk信号通路及核因子-βB的表达，而致癌的HER-2/neu可诱导上皮细胞向间充质细胞转化(EMT)，LOX-PP可逆转这一过程。LOX-PP可诱导E-钙粘蛋白，降低Snail水平，减少Matrigel中肿瘤的迁移和分枝集落的形成，并减少裸鼠体内肿瘤的形成。单核苷酸多态(SNP)(Rs1800449)G473A导致LOX-PP结构域高度保守区域的Arg158Gln替换，在HapMap数据库中平均有24.6%473A微小等位基因携带者频率。然而，当检查癌细胞时，G473A SNP存在于大多数细胞系中，LOX-PP Gln变体在体外和体内显示出显著的肿瘤抑制功能，并降低了对抗LOX的促肿瘤作用的能力。在一项针对非裔美国女性的初步研究中，微小的473A等位基因与ER1阴性乳腺癌风险的增加有关。这些发现导致了两个相关的中心假设：1.野生型LOX-PP代表着一种重要的肿瘤抑制因子，其活性被Arg到Gln的替换所折衷。2.LOX基因中的G473A(Rs1800449)SNP导致更多侵袭性癌症的风险增加。在这一新的RO1应用中，我们提议：(目的1)阐明野生型LOX-PP在乳腺癌细胞中作为RAS信号抑制子的作用机制，并确定这些机制如何受到Arg158Gln替代的影响；(目的2)在培养和原位乳腺脂肪垫模型中测试LOX-PP阻止LOX酶促癌作用的能力；(目的3)使用即将准备的敲入小鼠来测试rs1800449 SNP是更具侵袭性乳腺癌的危险因素的假设。建议的研究代表了LOX-PP作为RAS信号的抑制剂和疾病进展的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，我们的发现对激活的RAS癌基因驱动的癌症具有更广泛的意义。 公共卫生相关性：总体而言，这些研究代表了乳腺癌中LOX-PP蛋白的临床前测试，LOX-PP蛋白是一种新的RAS信号抑制物。Rs1800449单核苷酸多态(SNP)导致LOX-PP中的Arg-to-Gln变异，并显著降低RAS抑制活性，增加非裔美国女性患ER1阴性乳腺癌的风险。我们建议的工作将描述LOX-PP活性的机制，Gln变体导致的变化，并准备一个敲入小鼠模型，允许测试SNP在评估乳腺癌风险方面的潜在用途。