DDR1 controls tumor homing in breast to bone metastasis

描述(由申请人提供)： 这项拟议研究的总体目标是阐明Discoidin结构域受体1(DDR1)在乳腺肿瘤细胞扩散到骨骼中的作用。与其他受体酪氨酸激酶不同，DDR1不是由可溶性生长因子激活，而是由纤维和基底膜胶原蛋白激活。DDR1在正常乳腺发育过程中有表达，在乳腺导管癌中高表达。缺乏DDR1的基因敲除小鼠无法发育出功能性产乳组织。到目前为止，我们已经鉴定了5种不同的DDR1亚型，它们起源于膜旁区域的选择性剪接。假设如下：1)DDR1在乳腺-骨转移中起归巢受体的作用。2)如果不存在DDR1a和DDR1b两个全长异构体，乳腺肿瘤细胞侵袭性地定植于骨组织。验证这些假说的具体目的是：1)在高转移的人乳腺癌细胞系MDA-MB 231中重新表达DDR1a和-b，该细胞系缺乏内源性的激酶活性DDR1的表达。这些细胞将被结构性或诱导性地导入全长DDR1a和-b。在体外，我们将比较DDR1转基因细胞在骨基质上黏附和降解细胞外基质的能力。由于以前的工作表明DDR1会触发包括基质金属蛋白酶MMP3和MMP10在内的蛋白水解酶的释放，我们将特别关注这些MMPs的表达，以进一步表征导致基底膜和骨组织破坏的途径。2)将高表达全长DDR 1的MDA-MB 231细胞注入裸小鼠左心内，研究乳腺到骨的体内转移。与对照组动物相比，我们希望观察到DDR1过表达细胞的动物骨转移形成减少。通过将GFP导入MDA-MB 231细胞，我们将监测DDR1信号在组织切片或活体动物的渗出和转移进展过程中的作用。3)利用实验室以前建立的一套微阵列芯片，专门研究人类或小鼠组织中细胞外基质相关基因的调控。这些阵列将被用来识别骨肿瘤病变中上调或下调的基因，这些基因来自注射了高表达全长DDR1的MDA-MB 231的小鼠。我们将能够使用人类阵列选择性地描述ECM相关基因在肿瘤中的表达，并使用小鼠阵列进行宿主特异性反应。通过这项拟议的研究，我们希望评估DDR1作为迄今尚未认识的但重要的治疗靶点在骨转移中的作用。CRISP数据库的一项调查显示，美国国立卫生研究院目前没有为DDR1的其他研究提供资金。

DESCRIPTION (provided by applicant): The overall objective of the proposed research is to elucidate the role of Discoidin Domain Receptor 1 (DDR1) in the spread of breast tumor cells to the bone. Unlike other receptor tyrosine kinases, DDR1 is not activated by soluble growth factors but by fibrillar and basement membrane collagens. DDR1 is expressed during normal mammary gland development and overexpressed in primary ductal breast carcinoma. Knockout mice lacking DDR1 fail to develop functional lactogenic tissue. So far, we have identified 5 different isoforms of DDR1, which originate from alternative splicing in the juxtamembrane region. The hypotheses are: 1) DDR1 functions as a homing receptor in breast to bone metastasis. 2) Breast tumor cells aggressively colonize bone tissue, if the two full-length isoforms DDRla and DDRlb are not present. The specific aims to test these hypotheses are: 1) To re-express DDRla and -b in the highly metastatic human breast tumor cell line MDA-MB 231, which lacks endogenous expression of kinase-active DDR1. These cells will be either constitutively or inducibly transfected with full length DDR1 a and -b. The potential of DDRl-transfected cells to adhere to bone matrix and to degrade ECM in vitro will be compared to control cells. Because previous work showed that DDR1 triggers the release of proteolytic enzymes, including matrix metalloproteinase MMP3 and MMP10, we will particularly focus on the expression of these MMPs in order to further characterize pathways leading to destruction of basement membranes and bone tissue. 2) To study breast to bone metastasis in vivo by injecting MDA-MB 231 cells overexpressing full length DDR 1 into the left cardiac ventricle of nude mice. Compared to control animals, we expect to observe reduced formation of bone metastasis in animals with DDRl-overexpressing cells. By introducing GFP into MDA-MB 231 cells, we will monitor effects of DDR1 signaling during extravasation and metastatic progression in tissue sections or live animals. 3) To employ a set of microarray chips previously established in my laboratory, which are designed to specifically study the regulation of ECM-related genes in either human or mouse tissues. These arrays will be used to identify genes up- or down-regulated in bone tumor lesions from mice injected with MDA-MB 231 overexpressing full-length DDR1. We will be able to selectively profile the expression of ECM-related genes in the tumor using human arrays and host-specific responses using the mouse arrays. With the proposed research we want to evaluate the role of DDR1 as a so far unrecognized, but important therapeutic target in bone metastasis. A survey of the CRISP database showed that the NIH currently funds no other research on DDR1.