Role of Fetuin-A in Tumor Cell Growth

描述(申请人提供)：我们的长期目标是阐明胎球蛋白-A，一种由肝脏合成的血清糖蛋白，调节和调节肿瘤细胞的体外和体内生长的机制。中心假设是来源于血清或由肿瘤细胞合成的胎球蛋白-A与其假定的细胞表面受体相互作用，触发PI3激酶/Akt信号，从而驱动细胞的生长。这一假说将通过四个特定的目标进行研究：(1)确定肿瘤细胞中负责生长信号的细胞表面胎儿蛋白-A的受体；(2)确定在肿瘤细胞中受膜联蛋白/胎儿蛋白-A相互作用调节的其他信号通路；(3)阐明异位合成的胎儿蛋白-A在肿瘤细胞生长中的作用；以及(4)确定血清衍生的胎儿蛋白-A在促进肿瘤生长中的特异性。 在目标1中，我们将使用LLC细胞进行的有限规模的研究扩展到转Annexin-2-GFP的LNCaP前列腺癌细胞系。将使用共聚焦显微镜研究膜联蛋白-2和胎球蛋白-A在这些细胞中的共定位和相互作用。预计膜联蛋白将成为胎球蛋白-A的细胞表面受体，负责生长信号。在Aim 2中，Kinexus和Affymetrix基因阵列将用于筛选其他信号分子，这些分子的表达受到胎球蛋白-A/膜联蛋白相互作用的影响。许多信号通路与主要由胎球蛋白-A激活的PI3激酶/Akt通路相互作用。在目标3中，胎球蛋白-A基因将被重新添加到不表达它的细胞中，以确定这是否足以让这些细胞在无血清条件下和在胎球蛋白-A基因缺失的小鼠中生长。同样，胎球蛋白-A在LLC细胞中的表达将被sh-RNA方法下调，并重新评估其在无血清条件下的生长。在目的4中，将来自野生动物的胎球蛋白-A血清用于补充生长介质，以测定其在多种肿瘤细胞与添加胎球蛋白-A基因缺失小鼠血清的生长介质中的促生长能力。为此，我们将确定胎球蛋白-A促进细胞生长的独特能力是否也由其氨基酸序列决定，而不仅仅是唾液酸残基的存在。这项提案旨在证明，胎球蛋白-A是肿瘤细胞体内生长的重要燃料，不是无辜的旁观者。对这一途径的充分了解对大多数癌症类型的管理和治疗策略具有广泛的意义。

DESCRIPTION (provided by applicant): Our long-term objectives are to elucidate the mechanisms by which fetuin-A, a serum glycoprotein synthesized by the liver, mediates and regulates the in vitro and in vivo growth of tumor cells. The central hypothesis is that fetuin-A derived from the serum or synthesized by the tumor cells interacts with its putative cell surface receptors, triggering PI3 kinase/Akt signaling that drives the growth of the cells. The hypothesis will be investigated through 4 specific aims: (1) To define the cell surface receptors for fetuin-A in tumor cells responsible for growth signaling; (2) To determine other signaling pathways which are modulated in tumor cells by the annexin/fetuin-A interaction; (3) To elucidate the role of ectopically synthesized fetuin-A in tumor cell growth; and (4) To define the specificity of serum derived fetuin-A in tumor growth promotion. In aim 1, we will extend our studies that were done on a limited scale using LLC cells to LNCaP prostate cancer cell line transfected with annexin-2-GFP. The co-localization and interaction between annexin-2 and fetuin-A in these cells will be studied using confocal microscopy. It is expected that annexins will emerge as the cell surface receptors for fetuin-A responsible for growth signaling. In aim 2, Kinexus and Affymetrix gene arrays will be used to screen for other signaling molecules whose expression is affected by the fetuin-A/annexin interaction. A number of signaling pathways cross-talk with the PI3 kinase/Akt pathway that is predominantly activated by fetuin-A. In aim 3, fetuin-A gene will be added back to cells that do not express it to determine if this is sufficient to allow these cells to grow under serum-free conditions and in fetuin-A null mice. Likewise, fetuin-A expression in LLC cells will be knocked down by sh-RNA approach and their growth under serum-free conditions re-evaluated. In aim 4, the serum from fetuin-A wild-type animals will be used to supplement growth medium to determine its growth promoting abilities in a variety of tumor cells vis-a-vis growth medium supplemented with serum from fetuin-A null mice. In this aim, we will determine whether the unique ability of fetuin-A to promote cell growth is also dictated by its amino acid sequence and not merely the presence of sialic acid residues. This proposal seeks to demonstrate that fetuin-A is an important fuel for the in vivo growth of tumor cells and is not an innocent bystander. The full understanding of this pathway has broad implications in the management and treatment strategies for majority of cancer types.