Characterization and targeting BRIT1 deficiency in breast cancer

描述（由申请人提供）：完整有效的DNA损伤反应（DDR）对于维持基因组稳定性至关重要，并且它作为抑制肿瘤发生的关键屏障。我们最近发现BRIT 1/MCPH 1是DDR通路中一个新的关键调节因子。重要的是，我的实验室最近产生了BRIT 1敲除小鼠，并证明了BRIT 1在同源重组DNA修复和维持体内基因组稳定性中的重要作用。我们还发现BRIT 1基因拷贝数及其RNA和蛋白质表达在多个乳腺癌细胞系中显著降低。值得注意的是，我们在10个乳腺癌标本中的一个中发现了BRIT 1缺失突变，这种缺失导致BRIT 1蛋白的截短并损害了其在DNA损伤反应中的功能。因此，BRIT 1可能通过保持基因组稳定性而成为乳腺癌的一种新型抑癌基因，靶向BRIT 1缺陷可能为乳腺癌患者提供新的有效治疗方法。本研究拟从三个方面来验证这一假说：（1）利用BRIT 1基因敲除小鼠研究BRIT 1基因缺陷对乳腺肿瘤形成的影响。通过将我们独特的BRIT 1-/-小鼠与MMTV-Ras转基因小鼠杂交，我们的初步研究表明BRIT 1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/BRIT 1-/-和MMTV-Ras/BRIT 1 +/+小鼠之间的乳腺肿瘤发生率、肿瘤分级/类型和转移潜力。通过分析主要BRIT 1靶点的DNA修复功能，研究其潜在机制。此外，我们将使用BRIT 1条件性基因敲除小鼠确定BRIT 1缺陷是否加速辐射或致癌物诱导的乳腺肿瘤发展。(2)确定临床乳腺癌标本中BRIT 1的畸变。我们将从180例乳腺癌样本中鉴定BRIT 1畸变，这些样本按肿瘤分级和HER 2/ER/PR状态分层。将通过DNA测序确定编码区和外显子/内含子连接处的BRIT 1突变。还将通过免疫组织化学染色评估BRIT 1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BRIT 1缺陷是否与HER 2/ER/PR状态和患者生存相关。(3)To使用合成致死方法开发BRIT 1缺陷乳腺癌的新疗法。我们的初步研究表明，BRIT 1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靶向BRIT 1缺陷型乳腺癌的有效药物，我们将系统地评估乳腺癌细胞对细胞培养物中几种有效PARP抑制剂的反应。将使用两种携带BRIT 1缺陷型乳腺肿瘤的小鼠模型进一步评价PARP抑制剂的体内疗效。我们还将联合收割机将这些抑制剂与临床抗癌药物结合，以建立最佳的治疗方法。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学改变，并将为BRIT 1缺陷型乳腺癌的治疗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.