Functional Analysis of Breast Cancer Susceptibility Gene

乳腺癌易感基因的功能分析

• 批准号: 7338515
• 负责人: SHYAM SHARAN
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7338515
• 关键词: Functional; Analysis; Breast; Cancer; Susceptibility

Breast cancer is the most common malignancy in women. Mutations in BRCA1 and BRCA2 genes play an important role in the development of early-onset familial breast cancer. Our goal is to carry out functional dissection of these genes using a mouse model system. Since complete loss of function mutations result in embryonic lethality, we are engaged in generating an array of specific mutations along the length of proteins encoded by these genes. To accomplish this, we have developed a transgenic mouse model system that combines the use of existing Brca1 and Brca2knockout mice and the use of bacterial artificial chromosomes (BAC) containing these genes. Desired mutations can be generated in the Brca1 or Brca2 gene in the BAC and the phenotypic effect of the mutation can be analyzed in transgenic mice that are homozygous mutant for that gene. In a continuing effort to understand the role of BRCA1 and BRCA2 in various biological processes, we have developed humanized mouse models for functional analysis of human BRCA1 and BRCA2 genes. We have shown that wild-type human BRCA1 and BRCA2 genes under the control of their own promoter present in BAC clones are fully functional in mice and can rescue the lethality associated with loss of function mutations of the respective genes in mice. The humanized mouse models provide an experimentally tractable system to generate mutations identified in human breast cancer patients and analyze how they result in tumorigenesis.We have developed a simple but powerful method to generate subtle alterations in the BACs by using recombineering in bacteria. This method is based on the generation of recombinants in the BAC DNA by using oligonucleotides as targeting vectors with as few as 70 bases of homology. We have demonstrated that this technique can be used to generate single base changes, small insertions and deletions. Due to the high efficiency of recombineering, recombinant clones can be identified by a simple PCR based screening method without the use of any selectable marker. We have used this method to generate several cancer causing missense mutations in BRCA1 and BRCA2 in the BAC and are examining their phenotypic consequences in mice in a Brca1 or Brca2 mutant background. Based on the expression of BRCA2 on meiotic chromosomes, it has been predicted to be required for normal meiotic progression. Since Brca2 null mutants are embryonic lethal, its precise function in meiosis is unknown. We have generated a mouse model to describe a role for BRCA2 in meiosis. These mice lack the endogenous Brca2 gene but lethality is rescued by a human BRCA2 transgene. However, these mice are sterile due to poor expression of the transgene in testes and ovaries. We have used these mice to describe a role for BRCA2 in meiosis that is sexually dimorphic. We have found that the BRCA2-deficient spermatocytes undergo apoptosis unlike the BRCA2-deficient breast and ovarian epithelial cells that undergo neoplastic growth. This suggests that the cellular response to DNA damage may be tissue specific, which may explain why mutations in this widely expressed gene do not result in cancer in all tissues. A mutation in an evolutionarily conserved domain of BRCA2 has revealed a novel function for the protein in alkyl-DNA repair. Mutant mice develop normally but embryonic fibroblasts derived from these mice are extremely sensitive to N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), an alkylating agent, which results in the conversion of guanines to O6-methylguanines. The major pathway of repair of this methylated guanine involves removal of a methyl group by the O6-methylguanine-methyl transferase (MGMT) enzyme. Our current research is focused on understanding the interaction between BRCA2 and MGMT function as it may open new avenues for breast cancer therapy.

乳腺癌是女性最常见的恶性肿瘤。 BRCA1和BRCA2基因的突变在早期发作家族性乳腺癌的发展中起重要作用。我们的目标是使用小鼠模型系统对这些基因进行功能解剖。由于功能突变的完全丧失导致胚胎致死性，因此我们沿着这些基因编码的蛋白质长度产生一系列特异性突变。为此，我们开发了一种转基因小鼠模型系统，该系统结合了现有BRCA1和BRCA2Knockout小鼠的使用以及使用包含这些基因的细菌人造染色体（BAC）。可以在BAC中的BRCA1或BRCA2基因中产生所需的突变，并且可以在该基因的纯合突变体的转基因小鼠中分析突变的表型效应。为了不断努力了解BRCA1和BRCA2在各种生物学过程中的作用，我们开发了人性化的小鼠模型，用于人类BRCA1和BRCA2基因的功能分析。我们已经表明，在BAC克隆中存在的启动子的控制下，野生型人BRCA1和BRCA2基因在小鼠中具有完全功能性，并且可以挽救与小鼠相应基因功能突变相关的致死性。人源化的小鼠模型提供了一种可实验性的系统，以产生在人类乳腺癌患者中鉴定出的突变并分析它们如何导致肿瘤发生。我们已经开发了一种简单但有力的方法，可以通过在细菌中使用重新结合来产生BAC中的微妙变化。该方法基于通过使用寡核苷酸作为靶向较少70个同源性碱基的向量来基于BAC DNA中的重组。我们已经证明，该技术可用于生成单基础变化，小插入和删除。由于重组的效率很高，因此可以通过简单的基于PCR的筛选方法来识别重组克隆，而无需使用任何可选标记。我们已经使用了这种方法来产生几种癌症，导致BRCA1和BRCA2中的错义突变，并正在检查BRCA1或BRCA2突变体背景中小鼠中其表型后果。基于BRCA2在减数分裂染色体上的表达，预测它是正常减数分裂进展所必需的。由于BRCA2无效突变体是胚胎致死的，因此其在减数分裂中的精确功能尚不清楚。我们已经生成了一个小鼠模型来描述BRCA2在减数分裂中的作用。这些小鼠缺乏内源性BRCA2基因，但致死性是由人类BRCA2转基因挽救的。但是，由于转基因在睾丸和卵巢中的表达不佳，这些小鼠是无菌的。我们已经使用这些小鼠来描述BRCA2在减数分裂中的作用，这是性二态性的。我们发现，与经历肿瘤生长的BRCA2缺陷乳腺和卵巢上皮细胞不同，BRCA2缺陷型精子细胞会发生凋亡。这表明细胞对DNA损伤的反应可能是组织特异性的，这可以解释为什么在该广泛表达的基因中的突变并不导致所有组织中的癌症。 BRCA2进化保守的结构域中的突变揭示了烷基-DNA修复中蛋白质的新功能。突变小鼠正常发展，但源自这些小鼠的胚胎成纤维细胞对N-甲基N'N'-硝基-N-硝基 - 瓜烷酮（MNNG）非常敏感，这是一种烷基化剂，这会导致鸟嘌呤转化为O6-甲基圭胺。这种甲基化鸟嘌呤的修复的主要途径涉及通过O6-甲基圭氨酸 - 甲基转移酶（MGMT）酶去除甲基。我们目前的研究重点是了解BRCA2和MGMT功能之间的相互作用，因为它可能为乳腺癌治疗开辟了新的途径。