Lysyl Oxidase Propeptide: Breast Cancer Inhibitor

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治疗方案。赖氨酰氧化酶（LOX）是一种重要的细胞外酶，控制基质沉积。LOX基因被分离为具有抑制NIH 3 T3细胞中Ras癌基因的转化活性的能力的“ras recision”基因（rrg）。一致地，许多癌症显示LOX基因表达降低。LOX基因编码50 kDa的酶原（称为Pro-LOX），其分泌到细胞外环境中，在细胞外环境中通过蛋白水解切割加工成18 kDa的氨基末端前肽（LOX-PP）和功能性32 kDa酶。PI的小组证明rrg活性存在于LOX-PP结构域中，然而，已经发现LOX酶本身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的小组显示LOX-PP活性有效地抑制乳腺癌细胞，其由通过Ras发出信号的Her-2/neu驱动。在文化，LOX-PP抑制PI 3 K/Akt和MEK/ERK途径和NF-？B。致癌性Her-2/neu诱导上皮向间充质转化（EMT），并且这被LOX-PP逆转。LOX-PP诱导E-钙粘蛋白，降低Snail水平，降低Matrigel中的肿瘤迁移和分支集落形成，并减少裸鼠中的肿瘤形成。单核苷酸多态性（SNP）（rs 1800449）G473 A导致LOX-PP结构域中高度保守区域的Arg 158 Gln取代，在HapMap数据库中以平均24.6%的473 A次要等位基因携带频率发生。然而，当检查癌细胞时，G473 A SNP存在于大多数细胞系中，并且LOX-PP Gln变体在体外和体内显示出显著受损的肿瘤抑制功能，以及对抗LOX的促肿瘤发生作用的能力降低。在一项针对非裔美国女性的初步研究中，次要473 A等位基因与ER 1阴性乳腺癌风险增加相关。这些发现导致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，其活性受到Arg至Gln取代的损害。2. LOX基因中的G473 A（rs 1800449）SNP导致更侵袭性癌症的风险增加。在这种新的RO 1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信号传导抑制剂的作用的机制，并确定Arg 158 Gln取代如何影响这些作用;（目的2）在培养物和原位乳腺脂肪垫模型中测试LOX-PP阻碍LOX酶的肿瘤促进作用的能力;（目的3）使用将制备的基因敲入小鼠检验rs 1800449 SNP代表更侵袭性乳腺癌的风险因素的假设。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风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，因此我们的发现对由激活的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.