Molecular regulation of breast cancer metastasis

我们克隆了一个名为BRMS1的基因，它抑制了六个独立来源的人和小鼠细胞系的转移 而不会抑制致瘤性。我们的目标是确定BRMS1背后的生化机制 转移抑制。 假设I-BRMS1抑制转移需要细胞间缝隙连接的恢复 通信(GJIC)。BRMS1的转染会恢复GJIC，并改变连接蛋白(Cx)的转录，即， Cx43表达上调，而Cx32表达下调。目标1将解决BRMS1表达和GJIC是否都是 用来抑制肿瘤转移。转导BRMS1的细胞(转移抑制)将有BRMS1(Aim 1a)或 利用siRNA选择性下调Cx43(Aim Lb)的表达。AIM LC将测试Cx32是否在 转移细胞降低了转移潜能，而Aim ld将在BRMS1细胞中重新表达Cx32并检测 转移是否增加。将对所有转染体进行体外GJIC和体内转移试验(AIM LE)， 假设2-BRMS1通过与mSin3：组蛋白脱乙酰酶(HDAC)相互作用抑制肿瘤转移。 通过酵母双杂交、共IP和层析，我们证明了BRMS1与大分子的 包括mSin3和HDAC的复合体。我们将定义哪个BRMSI：HDAC：mSin3复合体负责 转移抑制。AIM 2a将使用FPLC、质谱学、共IP和Y2H来定义BRMS1络合物和 鉴定BRMS1相互作用蛋白。AIM 2b将绘制负责特定蛋白质相互作用的BRMS1结构域。 目的2c将测试BRMS1通路(1)恢复GJIC的能力，(2)调节HDAC活性的能力，(3)抑制转移的能力。 假设3-Brms1的表达将影响原发乳腺肿瘤的转移。 我们建议建立Brms1基因缺失和Brine1转基因过表达的小鼠，并测试内源性表达 改变原发乳腺癌的肿瘤发展和/或转移。AIM 3a将生成Brmsl空 小鼠使用条件性Cre-Lox重组，并检验以下假设：来自TG： 当小鼠杂交时，MMTV-PYMT乳腺肿瘤会增加。AIM 3b将生成特定于乳房的 无处不在的表达Brms1的转基因会导致Brms1的高表达。TG：Brms1小鼠将与 TG：MMTV-PYMT，并检验转移潜能将下降的假设。

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 I - 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 le), 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.