Osteopontin and Molecular Mechanisms of Bone Metastasis

描述（由申请人提供）： 在任何组织中，宿主和肿瘤之间的相互作用都不像骨转移那样明显或具有破坏性，在骨转移中，肿瘤细胞可以刺激宿主细胞破坏骨组织。在理解宿主细胞和蛋白质在这些过程中的作用时，利用许多转基因小鼠品系作为宿主将是有用的。考虑到这一点，已经开发了129品系背景下的转化鼠乳腺上皮细胞：这些细胞（r3 T）与充分表征的人MDA-MB-231细胞一样，在心内注射后形成溶骨性骨转移，但它们在近交系129宿主小鼠（一种常用于基因敲除的品系）中也是如此。将这些细胞转化为完全转移表型需要几种操作，因此在转化的中间阶段存在几种亲本细胞系，这将有助于理解正常乳腺上皮细胞转化为能够在骨中形成转移的细胞所需的分子变化。在这个提议中，这种新的细胞系统与遗传修饰的宿主小鼠一起将用于阐明肿瘤和宿主蛋白在骨转移中的作用。在此特别感兴趣的两种蛋白质是具有多种肿瘤增强作用的TGF-β和骨桥蛋白（OPN），因为它们可能作为宿主和肿瘤蛋白两者起增强肿瘤发生的作用，而骨桥蛋白是骨吸收所需的-转移性r3 T细胞是OPN缺陷的。首先，由于MDA-MB- 231细胞是研究骨转移的基准系统，因此将根据基因表达和对TGF-β的反应来表征不同的细胞系，询问这些细胞和MDA-MB-231细胞中骨转移性肿瘤生长的分子机制是否相当。然后将评价细胞在缺乏OPN或TGF-β表达降低的动物中形成溶骨性骨病变的能力，并通过比较骨和肝转移形成来评价骨特异性作用。此外，还将评估OPN对肿瘤细胞表达的影响，并通过使用蛋白质的突变形式探索OPN作用的机制。将检查转移细胞对骨细胞分化和功能的体外影响，利用转化较少的亲本细胞系来评价在转化过程中如何诱导影响骨细胞功能的活性。最后，将使用阵列分析来比较在不同转化阶段表达的基因，使用体外实验的结果来帮助鉴定可能在定义骨转移表型中重要的基因。总之，这些实验将提供重要的数据，以建立一种新的细胞系的骨转移机制，并确定宿主OPN和TGF-β作为潜在的治疗靶点的作用。

DESCRIPTION (provided by applicant): In no tissue is the interplay between host and tumor as obvious or as destructive as in bone metastases, where tumor cells can stimulate host cells to destroy bone tissue. In understanding the role of host cells and proteins in these processes, it would be useful to take advantage of the many genetically modified mouse strains as hosts. With this in mind, transformed murine mammary epithelial cells in the 129 strain background have been developed: these cells (r3T), like the well characterized human MDA-MB-231 cells, form osteolytic bone metastases after intracardiac injection, but they do so in inbred 129 host mice, a strain commonly used for gene knockouts. Transformation of these cells to the full metastatic phenotype required several manipulations, so there are several parental cell lines at intermediate stages of transformation which will be useful in understanding the molecular changes required for the conversion of normal mammary epithelial cells to those able to form metastases in the bone. In this proposal, this novel cell system together with genetically modified host mice will be used to elucidate the role of both tumor and host proteins in bone metastasis. Two proteins that are of especial interest here since they likely function to enhance tumorigenesis both as host and as tumor proteins are TGF-beta which has multiple tumor-enhancing effects, and osteopontin (OPN), which is required for bone resorption - the metastatic r3T cells are OPN deficient. First, since the MDA-MB- 231 cells are the benchmark system for studying bone metastasis, the different cell lines will be characterized in terms of gene expression and response to TGF-beta, asking if the molecular mechanism of bone metastatic tumor growth is comparable in these cells and the MDA-MB-231 cells. Then the ability of the cells to form osteolytic bone lesions in animals lacking OPN or with reduced expression of TGF-beta will be evaluated, and bone-specific effects evaluated by comparison of bone and liver metastasis formation. In addition, the effects of tumor cell expression of OPN will be evaluated, and the mechanism of OPN effects explored through the use of mutant forms of the protein. The effect of the metastatic cells on bone cell differentiation and function in vitro will be examined, making use of the less transformed parental cell lines to evaluate how activities that influence bone cell function are induced during the process of transformation. Finally, array analysis will be used to compare genes expressed at the different stages of transformation, using the results of the in vitro experiments to help identify genes that are likely to be important in defining the bone metastatic phenotype. Together, these experiments will provide important data to establish the mechanism of bone metastasis by a novel cell line and define the role of host OPN and TGF-beta as potential therapeutic targets.