Role of Fetuin-A in Tumor Cell Growth

描述（由申请人提供）：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）介导和调节肿瘤细胞体外和体内生长的机制。核心假设是来自血清或由肿瘤细胞合成的胎球蛋白A与其推定的细胞表面受体相互作用，触发PI 3激酶/Akt信号传导，驱动细胞生长。本研究将从以下四个方面对这一假说进行研究：（1）确定肿瘤细胞中负责生长信号的胎球蛋白-A的细胞表面受体;（2）确定肿瘤细胞中通过膜联蛋白/胎球蛋白-A相互作用调节的其他信号通路;（3）阐明异位合成的胎球蛋白-A在肿瘤细胞生长中的作用;（4）明确血清源性胎球蛋白A促肿瘤生长的特异性。 在目标1中，我们将扩展我们的研究，在有限的规模上使用LLC细胞LNCaP前列腺癌细胞系转染膜联蛋白-2-GFP。将使用共聚焦显微镜研究这些细胞中膜联蛋白2和胎球蛋白A之间的共定位和相互作用。预计膜联蛋白将作为胎球蛋白A的细胞表面受体出现，负责生长信号传导。在目标2中，Kinexus和Affyxin基因阵列将用于筛选其表达受胎球蛋白-A/膜联蛋白相互作用影响的其他信号分子。许多信号通路与主要由胎球蛋白A激活的PI 3激酶/Akt通路交叉。在目标3中，胎球蛋白A基因将被添加回不表达该基因的细胞中，以确定这是否足以使这些细胞在无血清条件下和胎球蛋白A缺失小鼠中生长。同样，LLC细胞中的胎球蛋白-A表达将通过sh-RNA方法敲低，并重新评估其在无血清条件下的生长。在目的4中，来自胎球蛋白-A野生型动物的血清将用于补充生长培养基，以确定其在各种肿瘤细胞中相对于补充有来自胎球蛋白-A缺失小鼠的血清的生长培养基的生长促进能力。在这个目标中，我们将确定是否胎球蛋白-A的独特能力，以促进细胞生长也决定了其氨基酸序列，而不仅仅是唾液酸残基的存在。该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，而不是无辜的旁观者。对这一通路的充分理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.