Characterization and targeting BRIT1 deficiency in breast cancer

描述（由申请人提供）：完整和有效的DNA损伤反应（DDR）对于维持基因组稳定性至关重要，它是抑制肿瘤发生的关键屏障。我们最近发现BRIT1/MCPH1是DDR通路的一个新的关键调节因子。重要的是，我的实验室最近培育了BRIT1敲除小鼠，并证明了BRIT1在体内同源重组DNA修复和维持基因组稳定性中的重要作用。我们还发现，在多种乳腺癌细胞系中，BRIT1基因拷贝数及其RNA和蛋白表达显著降低。值得注意的是，我们在10个乳腺癌标本中的一个中发现了BRIT1缺失突变，这种缺失导致BRIT1蛋白的截断，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的稳定性作为一种新的乳腺癌肿瘤抑制因子，靶向BRIT1缺失可能为乳腺癌患者提供新的有效的治疗方法。为了验证这一假设，我们提出了三个具体的目的：(1)利用BRIT1敲除小鼠来确定BRIT1缺乏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的缺失增强了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+/+小鼠的乳腺肿瘤发生率、肿瘤分级/类型和转移潜能。我们将通过分析BRIT1主要靶点的DNA修复功能来研究其潜在的机制。此外，我们将使用BRIT1条件敲除小鼠来确定BRIT1缺乏是否会加速辐照或致癌物质诱导的乳腺肿瘤发展。(2)在临床乳腺癌标本中发现BRIT1畸变。我们将从按肿瘤分级和HER2/ER/PR状态分层的180例乳腺癌样本中识别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。免疫组织化学染色检测BRIT1蛋白表达和亚细胞定位，定量RT-PCR检测其RNA水平。此外，我们将确定BRIT1缺乏是否与HER2/ER/PR状态和患者生存相关。(3)利用合成致死性方法开发治疗brit1缺陷乳腺癌的新方法。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，我们将在细胞培养中系统地评估乳腺癌细胞对几种有效PARP抑制剂的反应。我们将使用两种携带brit1缺陷乳腺肿瘤的小鼠模型来进一步评估PARP抑制剂在体内的疗效。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法。总之，我们的研究将有助于提高对乳腺癌发展过程中关键病理改变的理解，并将为治疗brit1缺失型乳腺癌提供直接的临床影响。

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.