IC Communication in Breast Cancer Metastasis to Bone

描述（来自申请人的逐字描述）： 对乳腺癌优先转移到骨的了解很少。我们 提出间隙连接细胞间通讯（GJIC）有助于 通过促进异型细胞-细胞的乳腺癌骨转移 乳腺癌细胞和成骨细胞之间的相互作用。这些 相互作用有助于乳腺癌细胞跨成骨细胞迁移， 这是骨转移的关键步骤。长期目标是 项目是表征乳腺间隙连接的表达和功能 癌细胞（转移，转移抑制和非转移）以及如何 这有助于通过激活胞质Ca 2+动员和肌球蛋白， 轻链激酶（MLCK）活性，对乳腺癌细胞跨成骨细胞 迁移和骨转移。这些目标将通过 完成五个具体目标：（1）检测乳腺癌中的同型GJIC 癌细胞系和乳腺癌细胞之间的异型GJIC， 成骨细胞;（2）量化乳腺癌细胞接触的影响， 是否存在GJTC和胞质Ca 2+动员的抑制剂， 对成骨细胞hFOB 1.9细胞中胞浆Ca 2+浓度（[Ca +i]）的影响;（3） 定量与乳房接触后成骨细胞中的MLCK活性 癌细胞;（4）量化乳腺癌细胞的跨细胞迁移 通过成骨细胞单层;和（5）遗传改变 乳腺癌细胞中的连接蛋白，并评估体内转移潜力 骨转移模型。作为乳腺癌细胞系，我们将使用MDA-MB-23 I 和MDA-MB-435，这些细胞表达最近鉴定的转移 抑制基因BRMS 1，这些细胞表达绿色荧光蛋白（GFP） 以及适当的病媒控制。我们还将检查非转移性MDA-MB-468 和NEO 11/435乳腺癌细胞。将通过染料转移定量GJIC 技术，[Ca 2 +]，通过显微荧光光谱法，MLCK活性，通过体外 磷酸化测定和通过定量细胞的跨细胞迁移 通过基质胶迁移，利用共聚焦显微镜。骨转移将 使用GFP标记的乳腺癌细胞进行荧光测定评估。的 该项目的结果将提供深入了解的机制， 乳腺癌细胞优先转移到骨，这是乳腺癌发生的重要一步。 开发抑制骨转移的方法。

DESCRIPTION (Verbatim from the Applicant): The mechanism underlying the preferential metastasis of breast cancer to bone is poorly understood. We propose that gap junctional intercellular communication (GJIC) contributes to breast cancer metastasis to bone by facilitating heterotypic cell-cell interactions between breast cancer cells and osteoblastic cells. These interactions contribute to breast cancer cell transosteoblastic migration, which is a critical step in bone metastasis. The long-term goals of this project are to characterize gap junction expression and function in breast cancer cells (metastasizing, metastasis suppressed and non-metastatic) and how this contributes, through activation of cytosolic Ca2+ mobilization and myosin light chain kinase (MLCK) activity, to breast cancer cell transosteoblastic migration and bone metastasis. These goals will be accomplished through the completion of five Specific Aims: (1) examine homotypic GJIC among breast cancer cell lines and heterotypic GJIC between breast cancer cells and osteoblastic cells; (2) quantify the effect of breast cancer cell contact, in the presence and absence of inhibitors of GJTC and cytosolic Ca2+ mobilization, on cytosolic Ca2+ concentration ([Ca +i]) in osteoblastic hFOB 1.9 cells; (3) quantify MLCK activity in osteoblastic cells following contact with breast cancer cells; (4) quantify transcellular migration of breast cancer cells through osteoblastic monolayers; and (5) genetically alter expression of connexins in breast cancer cells and assess metastatic potential in an in vivo model of bone metastasis. As breast cancer cell lines, we will use MDA-MB-23 I and MDA-MB-435, these cells expressing the recently identified metastasis suppressing gene BRMS1, these cells expressing green fluorescent protein (GFP) and appropriate vector controls. We will also examine non-metastasic MDA-MB-468 and neol 1/435 breast cancer cells. GJIC will be quantified by dye transfer techniques, [Ca2+], by microspectrofluorometry, MLCK activity by in vitro phosphorylation assays, and transcellular migration by quantification of cell migration through Matrigel, utilizing confocal microscopy. Bone metastasis will be assessed fluorometrically utilizing GFP tagged breast cancer cells. The results of this project will provide insights into the mechanism by which breast cancer cells preferentially metastasize to bone, an important step in developing approaches to inhibit bone metastasis.