MOLECULAR REGULATION OF BREAST CANCER METASTASIS

描述：(改编自研究人员的摘要)我们鉴定、克隆 并定位了一个新的人类基因BRMS1(乳腺转移抑制因子-1)，该基因 将其导入两个无关的人乳房可抑制肿瘤转移 癌症。然而，转染体的致瘤性并未受到抑制。这些 特征定义了转移抑制基因。新的BRMS1基因图谱 到11q13.1-q13.2，这是一个在晚期常见的染色体改变区域， 转移性乳腺癌。因为它的体积相对较小，核 定位，存在盘绕和亮氨酸拉链图案，我们假设 BRMS1是参与转录的多聚体蛋白复合体的一部分 监管。目前提案的目标是确定机制 BRMS1通过其抑制乳腺癌转移。 对于特定的目标1，潜在的假设是精确地扰乱 BRMS1蛋白会导致无法抑制转移 表型。遵循标准的生化特征(亚细胞 本地化，翻译后修饰)，BRMS1突变体将被产生 通过缺失和定点突变，导入转移性人 乳腺癌细胞(不表达内源性BRMS1)及其评价 突变体对肿瘤转移的影响。初步研究将扰乱 假定的核定位信号以确定核定位是否 是BRMS1功能的关键。引入突变以删除或修改 然后将完成特定的域，以评估哪些是关键的。后来， 错义突变(例如，关键氨基酸的丙氨酸扫描)将导致 更明确的突变。在一项补充研究中，BRMS1相互作用的蛋白质 将通过共沉淀和酵母双杂交分析进行鉴定。突变 导致蛋白质-蛋白质相互作用的中断和转移 电势将确定负责的关键氨基酸。使用这个 结合方法，可以开发出结构-活性关系 BRMS1. 特定目的2将产生纯合子缺失brms1小鼠。这一目标将考验 Brms1基因缺失是否会导致原发灶转移增加 小鼠乳腺癌。使用Cre-Lox的条件淘汰法将 被用来克服与致命的 突变。将Lox侧翼BRMS1引入生殖系将完成， 然后在MMTV和WAP启动子的控制下使用Cre进行重组 (分别给出了广泛性缺失和乳腺组织特异性缺失)。 Brms1缺失的小鼠将被培育成常规发育乳房的转基因小鼠。 确定肿瘤和转移的发生率和频率。假说 缺乏brms1的小鼠会比小鼠在更多的部位发生转移 这些基因仍然表达brms1。

DESCRIPTION: (Adapted from the investigator's abstract) We identified, cloned and mapped a novel human gene, BRMS1 (Breast Metastasis Suppressor-1) which suppresses metastasis when transfected into two unrelated human breast carcinomas. Yet, tumorigenicity was not suppressed in the transfectants. These characteristics define a metastasis-suppressor gene. The novel BRMS1 gene maps to 11q13.1-q13.2, a chromosomal region commonly altered in late-stage, metastatic breast carcinoma. Because of its relatively small size, nuclear localization, presence of coiled-coil and leucine zipper motifs, we hypothesize that BRMS1 is part of a multimeric protein complex involved in transcriptional regulation. The objective of the current proposal is to determine the mechanism by which BRMS1 suppresses breast carcinoma metastasis. For Specific Aim 1, the underlying hypothesis is that precise disruption of the BRMS1 protein will result in the inability to suppress the metastatic phenotype. Following standard biochemical characterization (subcellular localization, post-translational modification), BRMS1 mutants will be generated by deletion and site-directed mutagenesis, transfected into metastatic human breast carcinoma cells (which do not express endogenous BRMS1) and evaluated for the mutants' effects o metastasis. Initial studies will disrupt the putative nuclear localization signals to determine whether nuclear localization is critical for BRMS1 function. Introduction of mutations to delete or modify specific domains will then be done to assess which are critical. Subsequently, missense mutations (e.g., alanine scan of critical amino acids) will introduce more defined mutations. In a complementary study, BRMS1-interacting proteins will be identified by co-precipitation and yeast two-hybrid analyses. Mutations which result in disruption of protein-protein interactions and metastatic potential will identify the critical amino acids responsible. Using this combined approach, a structure-activity relationship can be developed for BRMS1. Specific Aim 2 will generate homozygous null brms1 mice. This aim will test whether genetic loss of brms1 will result in increased metastasis by primary murine mammary carcinomas. A conditional knockout approach using Cre-Lox will be used in order to overcome the complications associated with a lethal mutation. Introduction of Lox-flanked brms1 into the germline will be done, followed by recombination using Cre under control of the MMTV and WAP promoters (giving wisespread and mammary-tissue-specific deletion, respectively). brms1-null mice will be bred to transgenic mice which routinely develop mammary tumors and the incidence and frequency of metastasis determined. The hypothesis is that mice lacking brms1 will develop more metastasis in more sites than mice which still express brms1.