DDR1 controls tumor homing in breast to bone metastasis

描述（由申请人提供）： 这项研究的总体目标是阐明盘状结构域受体1（DDR 1）在乳腺肿瘤细胞向骨扩散中的作用。与其他受体酪氨酸激酶不同，DDR 1不被可溶性生长因子激活，而是被纤维和基底膜胶原激活。DDR 1在正常乳腺发育过程中表达，在原发性乳腺导管癌中过表达。缺乏DDR 1的基因敲除小鼠不能发育功能性催乳组织。到目前为止，我们已经鉴定了5种不同的DDR 1亚型，它们起源于质膜区域的选择性剪接。假设：1）DDR 1在乳腺骨转移中起归巢受体的作用。2)如果两种全长同种型DDRla和DDRlb不存在，则乳腺肿瘤细胞侵袭性地定殖于骨组织。检验这些假设的具体目的是：1）在高转移性人乳腺肿瘤细胞系MDA-MB 231中重新表达DDRla和-B，其缺乏激酶活性DDR 1的内源性表达。这些细胞将用全长DDR 1 a和-B组成型或诱导型转染。将DDR 1转染的细胞粘附于骨基质和体外降解ECM的潜力与对照细胞进行比较。由于以前的工作表明，DDR 1触发蛋白水解酶的释放，包括基质金属蛋白酶MMP 3和MMP 10，我们将特别关注这些MMP的表达，以进一步表征导致基底膜和骨组织破坏的途径。2)通过将过表达全长DDR 1的MDA-MB 231细胞注射到裸鼠左心室中，研究体内乳腺向骨的转移。与对照动物相比，我们预期在具有DDR 1过表达细胞的动物中观察到骨转移的形成减少。通过将GFP引入MDA-MB 231细胞，我们将监测DDR 1信号传导在组织切片或活体动物中外渗和转移进展期间的影响。3)利用本实验室建立的一套微阵列芯片，专门研究人或小鼠组织中ECM相关基因的调控。这些阵列将用于鉴定注射过表达全长DDR 1的MDA-MB 231的小鼠骨肿瘤病变中上调或下调的基因。我们将能够使用人类阵列和使用小鼠阵列的宿主特异性反应来选择性地描绘ECM相关基因在肿瘤中的表达。通过这项研究，我们希望评估DDR 1作为骨转移中迄今尚未认识到但重要的治疗靶点的作用。对CRISP数据库的一项调查显示，NIH目前没有资助DDR 1的其他研究。

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.