Small integrin-binding proteins and tumor progression

描述（由申请人提供）：一个分泌蛋白家族，我们称之为兄妹（小整合素结合配体，n-连接糖蛋白），包含整合素结合三肽，ROD，在骨骼中表达，基因聚集在人类4号染色体上。我们和其他人的工作表明，各种肿瘤经常表达一种或多种蛋白质，这种表达与疾病严重程度相关。我们最近发现，兄妹可以结合和调节基质金属蛋白酶（MMPs）的活性。与潜在的亲MMPs结合导致催化活性，与被金属蛋白酶组织抑制剂（TIMPs）或小分子量抑制剂抑制的MMPs结合导致酶活性恢复。我们假设肿瘤的同胞表达通过MMP调节促进肿瘤进展。为了验证这一假设，我们将(a)使用纯化组分和标准序列和动力学分析来表征兄弟姐妹和MMP相互作用，以表征MMP和MMP抑制剂的兄弟姐妹效应动力学，并确定兄弟姐妹修饰试剂；(b)在体外细胞系统中测试SIBLING-修饰试剂（变体、阻断肽、抗体），产生血管生成（内皮细胞和小管形成）和侵袭性（人类癌细胞和改良Boyden室试验）的功能读数；(c)使用弟妹，弟妹修饰试剂来证明弟妹- MMP相互作用在肿瘤进展中的生理相关性（使用两个定量鸡胚绒毛膜尿囊膜（CAM）系统来研究血管生成和转移）。CAM血管生成试验采用在尼龙网内的胶原凝胶中植入SIBLING试剂并定量血管形成。CAM转移试验采用基于人alu序列PCR扩增的高灵敏度试验，用于监测人肿瘤细胞在鸡胚中的转移传播。兄妹在调节MMPs中的分子作用可能有助于肿瘤的进展。兄妹对MMP活性的改变及其在人类癌症中的表达可能是导致MMP抑制剂在临床试验中失败的一个因素。这项工作的长期目标是利用兄弟姐妹与MMP结合的基本生化和生物学细节知识，开发基于破坏或改变兄弟姐妹- MMP相互作用的抗肿瘤生长和进展治疗。

DESCRIPTION (provided by applicant): A family of secreted proteins, which we term SIBLINGS (for Small Integrin-Binding LIgand, N-linked Glycoproteins), contain the integrin-binding tripeptide, ROD, are expressed in the skeleton and have genes clustered on human chromosome 4. Our work and that of others has shown that a variety of neoplasms frequently express one or more of the proteins and that expression correlates with disease severity. We have recently found that SIBLINGS can bind to and modulate the activity of matrix metalloproteinases (MMPs). SIBLING binding to latent pro-MMPs leads to catalytic activity, and SIBLING binding to MMPs inhibited by tissue inhibitors of metalloproteinases (TIMPs) or small molecular weight inhibitors leads to a restoration of enzymatic activity. We hypothesize that SIBLING expression by neoplasms promotes tumor progression through MMP modulation. To test this hypothesis, we will (a) characterize SIBLING and MMP interactions using purified components and standard sequence and kinetic analyses to characterize SIBLING effects of MMP and MMP inhibitor kinetics as well as identify SIBLING modifying reagents; (b) test SIBLING- modifying reagents (variants, blocking peptides, antibodies) in in vitro cell systems that yield a functional readout on angiogenesis (endothelial cells and tubule formation) and invasiveness (human cancer cells and modified Boyden chamber assays); and (c) employ SIBLINGS, SIBLING-modifying reagents to demonstrate the physiological relevance of SIBLING - MMP interactions in tumor progression (using two quantitative chick embryo chorioallantoic membrane (CAM) systems to study angiogenesis and metastasis). The CAM angiogenesis assay employs the implantation of SIBLING reagents in a collagen gel within a nylon mesh and quantifying vessel formation. The CAM metastasis assay uses a highly sensitive assay based on PCR amplification of human alu sequences for monitoring the metastatic dissemination of human tumor cells in the chick embryo. The molecular action of SIBLINGS in modulating MMPs may contribute to tumor progression. The alteration of MMP activity by SIBLINGS and their expression in human cancer may be a contributory factor to the failure of MMP inhibitors in clinical trials. It is the long term goal of this work to exploit knowledge of the basic biochemical and biological details involved in SIBLING binding to MMP to develop anti-tumor growth and progression therapy based on disrupting or altering SIBLING - MMP interactions.