Small integrin-binding proteins and tumor progression

描述(申请人提供)：一种分泌蛋白家族，我们称之为同胞蛋白(小整合素结合配体，N-连接的糖蛋白)，含有整合素结合三肽，Rod，在骨骼中表达，并具有聚集在人类染色体4上的基因。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，这种表达与疾病严重程度有关。我们最近发现，兄弟姐妹可以结合并调节基质金属蛋白酶(MMPs)的活性。兄弟姐妹与潜伏的前MMPs结合导致催化活性，而兄弟姐妹与MMPs结合被金属蛋白酶组织抑制剂(TIMPs)或小分子量抑制剂抑制导致酶活性的恢复。我们假设，肿瘤同胞的表达通过基质金属蛋白酶的调节促进肿瘤的进展。为了验证这一假设，我们将(A)使用纯化的成分和标准序列和动力学分析来表征兄弟姐妹和基质金属蛋白酶的相互作用，以表征基质金属蛋白酶和基质金属蛋白酶抑制剂动力学的兄弟姐妹效应，并确定兄弟姐妹修饰试剂；(B)在体外细胞系统中测试兄弟姐妹修饰试剂(变体、封闭肽、抗体)，这些试剂对血管生成(内皮细胞和小管形成)和侵袭性(人类癌细胞和改良的Boyden小室分析)产生功能读数；和(C)使用兄弟姐妹修饰试剂来证明兄弟姐妹-基质金属蛋白酶相互作用在肿瘤进展中的生理学相关性(使用两个定量的鸡胚绒毛尿囊膜(CAM)系统来研究血管生成和转移)。CAM血管生成试验采用了将同胞试剂植入尼龙网内的胶原凝胶中，并对血管形成进行量化。CAM转移检测使用基于人ALU序列的聚合酶链式反应(PCR)扩增的高灵敏度检测来监测人类肿瘤细胞在鸡胚胎中的转移扩散。兄弟姐妹在调节MMPs方面的分子作用可能有助于肿瘤的进展。在人类癌症中，兄弟姐妹对基质金属蛋白酶活性的改变及其表达可能是导致基质金属蛋白酶抑制剂在临床试验中失败的原因之一。这项工作的长期目标是利用有关兄弟姐妹与基质金属蛋白酶结合的基本生化和生物学细节的知识，基于干扰或改变兄弟姐妹与基质金属蛋白酶的相互作用来开发抗肿瘤生长和进展的治疗方法。

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.