Characterization and targeting BRIT1 deficiency in breast cancer

乳腺癌中 BRIT1 缺陷的表征和靶向治疗

基本信息

批准号：
8025736
负责人：
KAIYI LI
金额：
$ 32.47万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-05-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8025736
关键词：
8p23.1 Antineoplastic Agents BRCA1 gene BRCA2 gene Binding Proteins Biological Assay Biometry Breast Cancer Cell Breast Cancer Model Breast Cancer Treatment Cancer Patient Carcinogens Cell Culture Techniques Cell Survival Cells Chromatin Chromosomal Instability Chromosome abnormality Chromosomes Clinic Clinical Code Cytogenetics DNA Damage DNA Repair DNA Sequence Deletion Mutation Development Diagnostic Neoplasm Staging Drug Delivery Systems ERBB2 gene Exons Gene Dosage Gene Proteins Genetic Transcription Genome Stability Genotoxic Stress Human Human Genome In Vitro Incidence Introns Investigation Knockout Mice Lead Location Maintenance Malignant Neoplasms Mammary Neoplasms Mammary gland Mouse Mammary Tumor Virus Mus Mutagens Mutant Strains Mice Mutation NBS1 gene Oncogenic Pathway interactions Patients Pharmaceutical Preparations Prognostic Marker Proteins RNA Radiation Reverse Transcriptase Polymerase Chain Reaction Role Sampling Sequence Deletion Signal Transduction Site Specificity Specimen Spectral Karyotyping Staining method Stains Stress Testing Therapeutic Transgenic Mice Tumor Suppression Tumor Suppressor Proteins Tumor stage ataxia telangiectasia mutated protein cancer cell cancer therapy comparative genomic hybridization dimethylbenzanthracene effective therapy genome sequencing homologous recombination improved in vivo inhibitor/antagonist irradiation malignant breast neoplasm mouse model mutant novel protein expression ras Oncogene recombinational repair response tumor tumor xenograft tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): The intact and effective DNA damage response (DDR) is essential for the maintenance of genomic stability and it acts as a critical barrier to suppress tumorigenesis. We have recently identified BRIT1/MCPH1 as a novel key regulator in DDR pathway. Importantly, my lab has recently generated the BRIT1 knockout mice and demonstrated the essential roles of BRIT1 in homologous recombination DNA repair and in maintaining genomic stability in vivo. We also found significant decreases of BRIT1 gene copy number and its RNA and protein expression in multiple breast cancer lines. Significantly, we identified a BRIT1 deletion mutation in one of the 10 breast cancer specimens and this deletion led to truncation of BRIT1 protein and impaired its function in DNA damage response. Therefore, BRIT1 may function as a novel tumor suppressor for breast cancer via preserving genome stability, and targeting BRIT1 deficiency may provide novel and effective treatment for the breast cancer patients. Three specific aims are proposed to test this hypothesis: (1) To determine the effects of BRIT1 deficiency on mammary tumor formation using the BRIT1 knockout mouse. By crossing our unique BRIT1-/- mice with the MMTV-Ras transgenic mice, our initial study shows that loss of BRIT1 potentiates the Ras-induced mammary tumor development. We will compare the mammary tumor incidence, tumor grade/type and metastatic potential between MMTV-Ras/BRIT1-/- and MMTV-Ras/BRIT1+/+ mice by histological analysis. The underlying mechanisms will be investigated by analyzing the DNA repair function of major BRIT1 targets. In addition, we will determine if BRIT1 deficiency accelerates irradiation or carcinogen-induced mammary tumor development using BRIT1-conditional knockout mice. (2) To identify BRIT1 aberrations in clinical breast cancer specimens. We will identify BRIT1 aberrations from180 breast cancer samples stratified by tumor grade and HER2/ER/PR status. BRIT1 mutations in the coding region and exon/intron junction will be determined by DNA sequencing. The protein expression and subcellular location of BRIT1 will be also assessed by immunohistochemical staining, and its RNA level will be assessed using quantitative RT-PCR. In addition, we will determine if BRIT1 deficiency is correlated with HER2/ER/PR status and patient survival. (3)To develop novel treatment for BRIT1-deficient breast cancers using synthetic lethality approach. Our initial study shows that BRIT1-deficient cells are very sensitive to PARP inhibitors. To test if PARP inhibitors can serve as a potent drug targeting BRIT1-deficient breast cancers, we will systematically assess the response of the breast cancer cells to several potent PARP inhibitors in cell culture. Two mouse models carrying BRIT1-deficient mammary tumors will be used to further evaluate the efficacy of PARP inhibitors in vivo. We will also combine these inhibitors with clinic anti-cancer agents to establish the optimal therapeutic remedies. In summary, our study will contribute to an improved understanding of the key pathological alterations in breast cancer development and will provide the immediate clinic impact on the treatment of BRIT1-deficient breast cancer. PUBLIC HEALTH RELEVANCE: This proposal is focused on investigation of BRIT1 function in breast tumor suppression using knockout mouse models and identification of BRIT1 aberrations in breast cancer specimens. Synthetic lethality approaches will also be used to develop novel treatment for breast cancers with BRIT1 deficiency. All of these proposed studies will not only can help us to understand the potential mechanism implicated in breast cancer but also accelerate the development of novel cancer therapies targeting patients with BRIT1 deficiency.

描述（由申请人提供）：完整且有效的DNA损伤反应（DDR）对于维持基因组稳定性至关重要，并且它是抑制肿瘤发生的关键屏障。我们最近发现 BRIT1/MCPH1 是 DDR 通路中的一个新型关键调节因子。重要的是，我的实验室最近培育出了 BRIT1 基因敲除小鼠，并证明了 BRIT1 在同源重组 DNA 修复和维持体内基因组稳定性方面的重要作用。我们还发现多个乳腺癌系中 BRIT1 基因拷贝数及其 RNA 和蛋白质表达显着下降。值得注意的是，我们在 10 个乳腺癌样本之一中发现了 BRIT1 缺失突变，这种缺失导致 BRIT1 蛋白截短并损害了其在 DNA 损伤反应中的功能。因此，BRIT1可能通过保持基因组稳定性作为乳腺癌的新型肿瘤抑制因子，针对BRIT1缺陷可能为乳腺癌患者提供新颖有效的治疗方法。提出了三个具体目标来检验这一假设：(1) 使用 BRIT1 敲除小鼠确定 BRIT1 缺陷对乳腺肿瘤形成的影响。通过将我们独特的 BRIT1-/- 小鼠与 MMTV-Ras 转基因小鼠杂交，我们的初步研究表明 BRIT1 的缺失会增强 Ras 诱导的乳腺肿瘤的发展。我们将通过组织学分析比较 MMTV-Ras/BRIT1-/- 和 MMTV-Ras/BRIT1+/+ 小鼠之间的乳腺肿瘤发病率、肿瘤级别/类型和转移潜力。将通过分析主要 BRIT1 靶标的 DNA 修复功能来研究其潜在机制。此外，我们将使用 BRIT1 条件敲除小鼠确定 BRIT1 缺陷是否会加速辐射或致癌物诱导的乳腺肿瘤的发展。 (2) 鉴定临床乳腺癌标本中的BRIT1畸变。我们将从按肿瘤分级和 HER2/ER/PR 状态分层的 180 个乳腺癌样本中识别 BRIT1 畸变。编码区和外显子/内含子连接处的 BRIT1 突变将通过 DNA 测序确定。还将通过免疫组织化学染色评估 BRIT1 的蛋白表达和亚细胞定位，并使用定量 RT-PCR 评估其 RNA 水平。此外，我们将确定 BRIT1 缺陷是否与 HER2/ER/PR 状态和患者生存相关。 (3)利用综合致死方法开发针对BRIT1缺陷型乳腺癌的新疗法。我们的初步研究表明 BRIT1 缺陷细胞对 PARP 抑制剂非常敏感。为了测试 PARP 抑制剂是否可以作为针对 BRIT1 缺陷乳腺癌的有效药物，我们将系统地评估细胞培养中乳腺癌细胞对几种有效 PARP 抑制剂的反应。两种携带 BRIT1 缺陷型乳腺肿瘤的小鼠模型将用于进一步评估 PARP 抑制剂的体内功效。我们还将把这些抑制剂与临床抗癌药物结合起来，以建立最佳的治疗方案。总之，我们的研究将有助于更好地了解乳腺癌发展中的关键病理改变，并将为 BRIT1 缺陷型乳腺癌的治疗提供直接的临床影响。公共健康相关性：该提案的重点是使用基因敲除小鼠模型研究 BRIT1 在乳腺肿瘤抑制中的功能，并鉴定乳腺癌样本中的 BRIT1 畸变。合成致死方法还将用于开发针对 BRIT1 缺陷的乳腺癌的新疗法。所有这些拟议的研究不仅可以帮助我们了解乳腺癌的潜在机制，而且可以加速针对 BRIT1 缺陷患者的新型癌症疗法的开发。