Molecular regulation of breast cancer metastasis

描述（由申请人提供）：我们克隆了一个基因BRMS 1，该基因可抑制六种独立来源的人和鼠细胞系的转移，但不抑制致瘤性。我们的目标是确定BRMS 1转移抑制的生化机制。 假设1 -BRMS 1抑制转移需要恢复间隙连接细胞间通讯（GJIC）。BRMS 1的转染恢复GJIC沿着连接蛋白（Cx）转录的改变，即，Cx43上调，而Cx 32下调。目的1将阐明BRMS 1表达和GJIC是否都是抑制转移所必需的。BRMS 1转染的细胞（转移抑制的）将具有使用siRNA选择性下调的BRMS 1（Aim 1a）或Cx43（Aim 1b）表达。Aim Ic将测试转移性细胞中降低的Cx 32是否降低转移潜力，而Aim Id将在BRMS 1转染的细胞中重新表达Cx 32并测试转移是否增加。将对所有转染子进行体外GJIC和体内转移检测（目的1 e）。 假设2 -BRMS 1通过与mSin 3：组蛋白脱乙酰酶（HDAC）的相互作用抑制转移。使用酵母双杂交，共IP和色谱，我们表明，BRMS 1物理相互作用的大复合物，包括mSin 3和HDAC的组件。我们将定义哪些BRMSI：HDAC：mSin 3复合物负责转移抑制。目的2a将使用FPLC、质谱、co-IP和Y2 H来确定BRMS 1复合物并鉴定BRMS 1相互作用蛋白。目标2b将绘制负责特定蛋白质相互作用的BRMS 1结构域。目的2c将测试BRMS 1途径（1）恢复GJIC、（2）调节HDAC活性和（3）抑制转移的能力。 假设3- Brmsl表达将影响原位乳腺肿瘤的转移。我们建议开发Brms 1-null和Brine 1转基因过表达小鼠，并测试内源性表达是否改变肿瘤的发展和/或转移的本地乳腺癌。目的3a将使用条件性Cre-Lox重组产生Brmsl无效小鼠，并测试当小鼠杂交时来自tg：MMTV-PyMT乳腺肿瘤的转移频率将增加的假设。目的3b将产生乳腺特异性和普遍表达的Brmsl转基因，其将导致Brmsl的高表达。将tg：Brmsl小鼠与tg：MMTV-PYMT杂交，并检验转移潜能将降低的假设。

DESCRIPTION (provided by applicant): We cloned a gene, BRMS1, which suppresses metastasis in six independently-derived human and murine cell lines without suppressing tumorigenicity. Our objective is to determine the biochemical mechanisms underlying BRMS1 metastasis suppression. Hypothesis 1 - Suppression of metastasis by BRMS1 requires restoration of gap junctional intercellular communication (GJIC). Transfection of BRMS1 restores GJIC along with altered transcription of connexins (Cx), i.e., Cx43 is up-regulated while Cx32 is down-regulated. Aim 1 will address whether BRMS1 expression and GJIC are both required to suppress metastasis. BRMSl-transfected cells (metastasis-suppressed) will have either BRMS1 (Aim la) or Cx43 (Aim lb) expression selectively down-regulated using siRNA. Aim lc will test whether decreased Cx32 in metastatic cells decreases metastatic potential while Aim ld will re-express Cx32 in BRMSl-transfected cells and test whether metastasis increases. All transfectants will be tested for GJIC in vitro and metastasis in vivo (Aim 1e). Hypothesis 2 - BRMSl suppresses metastasis via interactions with mSin3:histone deacetylase (HDAC). Using yeast two-hybrid, co-IP and chromatography, we showed that BRMS1 physically interacts with components of large complexes that include mSin3 and HDAC. We will define which BRMSI:HDAC:mSin3 complex(es) are responsible for metastasis suppression. Aim 2a will use FPLC, mass spectroscopy, co-IP and Y2H to define BRMS1 complexes and identify the BRMS1 interacting proteins. Aim 2b will map BRMS1 domains responsible for specific protein interactions. Aim 2c will test the ability of BRMS1 routto (1) restore GJIC, (2) regulate HDAC activity; and (3) suppress metastasis. Hypothesis 3- Brmsl expression will affect metastasis in autochthonous mammary tumors. We propose to develop Brmsl-null and Brine1 transgenic over-expressing mice and test whether endogenous expression alters tumor development and/or metastasis of autochthonous mammary carcinomas. Aim 3a will generate Brmsl null mice using conditional Cre-Lox recombination and test the hypothesis that the frequency of metastases from tg: MMTV-PyMT mammary tumors will increase when the mice are crossed. Aim 3b will generate mammary-specific and ubiquitous expression Brmsl transgenes that will result in high expression of Brmsl. tg:Brmsl mice will be crossed with tg: MMTV-PYMT and test the hypothesis that metastatic potential will decrease.