Molecular Modulators of Breast-Bone Metastasis

胸骨转移的分子调节剂

• 批准号: 6686229
• 负责人: JOHN T PRICE
• 金额: $ 10.8万
• 依托单位: ST. VINCENT'S INSTITUTE OF MEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-12 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6686229
• 关键词: antisense nucleic acid; athymic mouse; biological signal transduction; biomarker; bone neoplasms; breast neoplasms; cell adhesion; cell morphology; cell proliferation; clinical research; disease /disorder model; gene expression; genetic regulation; human tissue; immunocytochemistry; in situ hybridization; mass spectrometry; metastasis; molecular oncology; neoplasm /cancer genetics; neoplastic process; phenotype; prognosis; protein protein interaction; protein sequence; transfection /expression vector

DESCRIPTION (provided by applicant): Tumor cell metastasis is a complex, multi-step process that is a major cause of death and morbidity amongst cancer patients. Rather than being a totally random process, tumors display a preferential spread to particular organs. In breast cancer, bone metastasis occurs frequently and causes significant problems including pain, fracture, immobility, and even spinal cord compression leading to paralysis. Although bone metastasis represents a major clinical concern, the molecular mechanisms responsible for breast cancer predilection to metastasize to bone are not well understood, and few effective therapies currently exist. The major goal of this proposal is to identify novel molecular modulators of breast-bone metastasis that will ultimately result in the definition of new molecular targets to which novel therapeutics can be developed. To achieve this goal, we have utilized the intra-cardiac nude mouse model and identified sublines and subclones of the human breast cancer cell line MDA-MB-231 that display differential bone metastatic profiles. Through the use of gene array technology we have molecularly profiled and compared these sublines/subclones and identified a number of genes whose expression consistently correlates either positively or negatively with bone metastasis. We propose to define the role of two of these genes in breast-bone metastasis, one of which positively correlates with bone metastasis and the other that has been found to negatively correlate. To determine whether the genes function to enhance or reduce bone metastasis, we will employ standard molecular biology to increase and reduce the expression of the target genes in MDA-MB-231 sublines that have either a high (MDA-231Tx-SARFP) or low (MDA-231Tx-PRFP) bone metastatic phenotype (Aim 1). These sublines have previously been stably transfected with a construct that expresses red fluorescent protein (RFP) to allow full fluorescent body imaging of metastases in the in vivo bone metastatic models. Cells generated in AIM 1 will be examined for their ability to cause bone metastasis in in vivo models (intra-cardiac and intra-tibial inoculation) as well as their ability to grow at the orthotopic site (mammary-fat pad inoculation) (Aim 2). We will further examine the functional roles of the genes through a number of in vitro assays (adhesion dependent and independent proliferation, cell migration, cell invasion, cell adhesion, osteoclastogenesis assays) designed to examine the invasive and metastatic potential of cells (Aim 3). In addition we will examine a number of signal transduction pathways (EGF, IGF-I/II, TGFbeta) that have been show to be important in breast cancer progression (Aim 3). Analysis of the protein sequence of the targets reveals that each contains a number of TPR domains that are involved in protein-protein interactions. We therefore propose to identify proteins that interact with the targets to determine the possible involvement of other molecules and complexes in breast-bone metastasis (Aim 4). To determine the significance of the genes in human breast cancer we will examine their expression in archival primary and secondary breast tumors via immunohistochemistry and/or in situ hybridization to elucidate whether they may predict bone metastasis and/or survival (Aim 5). This proposal represents a unique opportunity to identify novel modulators of breast-bone metastasis.

描述（由申请人提供）： 肿瘤细胞转移是一个复杂的多步骤过程，是癌症患者中死亡和发病率的主要原因。肿瘤不是完全随机的过程，而是表现出对特定器官的优先扩散。在乳腺癌中，骨转移经常发生，并引起重大问题，包括疼痛，骨折，不动，甚至导致脊髓压缩导致麻痹。尽管骨转移是一个主要的临床问题，但尚不清楚导致乳腺癌偏爱转移到骨骼的分子机制，目前很少有有效的疗法。该提案的主要目的是确定胸骨转移的新分子调节剂，最终将导致对可以开发出新型疗法的新分子靶标的定义。为了实现这一目标，我们利用了心脏内裸小鼠模型，并确定了显示出差异骨转移性特征的人类乳腺癌细胞系MDA-MB-231的subline和子克隆。通过使用基因阵列技术，我们对分子进行了概述并比较了这些subline/subclones，并确定了许多基因的表达始终与骨转移呈正相关或负相关。我们建议定义其中两个基因在胸骨转移中的作用，其中一种与骨转移正相关，另一种与骨转移相关。为了确定基因的功能是增强或减少骨转移的功能，我们将采用标准的分子生物学来增加和减少具有高（MDA-231TX-SARFP）或低（MDA-231TX-tx-prfp）骨转移性现象的MDA-MB-231 subline中靶基因的表达。以前，这些subline已被表达红色荧光蛋白（RFP）的构建体稳定转染，以使体内骨转移模型中转移酶的全荧光体成像。将检查AIM 1中产生的细胞，以了解其在体内模型中引起骨转移的能力（内部和临时接种），以及它们在原位部位（乳腺FAT接种）中生长的能力（AIM 2）。我们将通过多种体外测定（粘附依赖性和独立的增殖，细胞迁移，细胞侵袭，细胞粘附，破骨细胞生成测定法）进一步检查基因的功能作用），旨在检查细胞的侵入性和转移性潜力（AIM 3）。此外，我们将研究许多在乳腺癌进展中很重要的信号转导途径（EGF，IGF-I/II，TGFBETA）（AIM 3）。对靶标的蛋白质序列的分析表明，每个靶标都包含许多与蛋白质 - 蛋白质相互作用有关的TPR结构域。因此，我们建议鉴定与靶标相互作用的蛋白质，以确定其他分子和复合物可能参与胸骨转移（AIM 4）。为了确定基因在人乳腺癌中的重要性，我们将通过免疫组织化学和/或原位杂交检查它们在档案中的原发性和继发性乳腺肿瘤中的表达，以阐明它们是否可以预测骨转移和/或生存（AIM 5）。该提案代表了识别胸骨转移的新型调节剂的独特机会。