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SIBLINGS（小整合素结合配体，N-连接糖蛋白），含有整合素结合三肽ROD，在骨架中表达，并具有在人4号染色体上聚集的基因。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，并且表达与疾病的严重程度相关。我们最近发现，SIBLINGS可以结合并调节基质金属蛋白酶（MMPs）的活性。SIBLING与潜在的pro-MMP的结合导致催化活性，并且SIBLING与MMP的结合被金属蛋白酶的组织抑制剂（TIMP）或小分子量抑制剂抑制导致酶活性的恢复。我们假设肿瘤表达SIBLING通过MMP调节促进肿瘤进展。为了检验这一假设，我们将（a）使用纯化的组分和标准序列和动力学分析来表征SIBLING和MMP相互作用，以表征MMP的SIBLING效应和MMP抑制剂动力学以及鉴定SIBLING修饰剂;（B）测试同胞修饰试剂（变体，封闭肽，抗体）在体外细胞系统中产生关于血管生成的功能读数（内皮细胞和小管形成）和侵袭性（人癌细胞和改良的Boyden室测定）;及（c）雇用兄弟姊妹，SIBLING修饰试剂用于证明SIBLING-MMP相互作用在肿瘤进展中的生理相关性（使用两种定量鸡胚绒毛尿囊膜（CAM）系统来研究血管生成和转移）。CAM血管生成测定采用将SIBLING试剂植入尼龙网内的胶原凝胶中并定量血管形成。CAM转移测定使用基于人alu序列的PCR扩增的高灵敏度测定，用于监测人肿瘤细胞在鸡胚中的转移性播散。SIBLINGS在调节MMP中的分子作用可能有助于肿瘤进展。SIBLINGS对MMP活性的改变及其在人类癌症中的表达可能是MMP抑制剂在临床试验中失败的一个促成因素。这项工作的长期目标是利用SIBLING与MMP结合所涉及的基本生物化学和生物学细节的知识，以开发基于破坏或改变SIBLING-MMP相互作用的抗肿瘤生长和进展疗法。

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.