Lysyl Oxidase Propeptide: Breast Cancer Inhibitor

描述（由申请人提供）：乳腺癌是美国妇女癌症死亡的第二大原因，因此需要额外的、有效的治疗方案。赖氨酸氧化酶（LOX）是一种重要的胞外酶，控制基质沉积。LOX基因作为“ras精确”基因（rrg）在NIH 3T3细胞中被分离出来，具有抑制ras癌基因转化活性的能力。许多癌症始终显示LOX基因表达减少。LOX基因编码一个50 kDa的前酶（称为Pro-LOX），该酶分泌到细胞外环境中，在那里通过蛋白水解裂解成一个18 kDa的氨基末端前肽（LOX- pp）和一个32 kDa的功能性酶。PI的研究小组证明，rrg活性存在于LOX- pp结构域，然而，LOX酶本身已被发现在缺氧条件下促进更具侵略性的表型和肿瘤转移。重要的是，PI的研究小组表明，LOX-PP活性可以有效抑制乳腺癌细胞，这是由Her-2/neu通过Ras信号驱动的。在培养中，LOX-PP抑制PI3K/Akt和MEK/Erk通路以及NF- B。致癌性Her-2/neu诱导上皮细胞向间质转化（EMT），并通过LOX-PP恢复。LOX-PP诱导E-cadherin，降低Matrigel中Snail水平、肿瘤迁移和分支菌落形成，减少裸鼠肿瘤形成。单核苷酸多态性（SNP） （rs1800449） G473A，导致在LOX-PP结构域的高度保守区域Arg158Gln替换，在HapMap数据库中以平均24.6%的473A次要等位基因载波频率发生。然而，当对癌细胞进行检查时，G473A SNP存在于大多数细胞系中，并且LOX- pp Gln变体在体内和体外均显示出明显受损的肿瘤抑制功能，并且降低了对抗LOX致瘤作用的能力。在一项针对非裔美国女性的初步研究中，较小的473A等位基因与ER1阴性乳腺癌的风险增加有关。这些发现导致了两个相关的中心假设：1。野生型LOX-PP是一种重要的肿瘤抑制因子，其活性被精氨酸取代为谷氨酰胺而受到损害。2. LOX基因中的G473A (rs1800449) SNP会增加患侵袭性癌症的风险。在这个新的RO1应用中，我们提出：（目的1）阐明野生型LOX-PP介导其作为乳腺癌细胞中Ras信号抑制因子的作用机制，并确定这些作用如何受到Arg158Gln替代的影响；（目的2）在培养和原位乳腺脂肪垫模型中检测LOX- pp抑制LOX酶促瘤作用的能力；（目的3）使用即将准备的敲入小鼠来验证rs1800449 SNP代表更具侵袭性乳腺癌风险因素的假设。拟议的研究代表了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，我们的研究结果对由激活的Ras癌基因驱动的癌症具有更广泛的意义。

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.