BRIT1, A NOVEL HEPATOCELLULAR CARCINOMA TUMOR SUPPRESSOR

描述(由申请人提供)：完整和有效的DNA损伤反应(DDR)对于维持基因组的稳定性是必不可少的，它是抑制肿瘤发生的关键屏障。我们最近发现BRIT1/MCPH1是DDR途径中的一个新的关键调控因子。重要的是，我的实验室已经产生了BRIT1基因敲除小鼠，并证明了BRIT1在体内同源重组DNA修复和维持基因组稳定性方面的重要作用。有趣的是，我们最近还在我们测序的10个肝细胞癌(HCC)样本中发现了3个BRIT1 DNA突变。根据我们的了解，这是第一次在肝癌中发现BRIT1基因突变的研究。因此，这些有趣的数据促使我们假设BRIT1可能通过保持基因组的稳定性而作为一种新的肿瘤抑制因子在肝细胞癌中发挥作用，而针对BRIT1缺陷的治疗，如使用PARP抑制剂，可能会为患者提供新的有效的靶向治疗。提出了两个特定的目标来验证这一假设：(1)评估临床肝细胞癌标本中BRIT1在DNA、RNA和蛋白质水平的变化。我们将从100例按肿瘤分级/分期分层的原发性肝细胞癌样本中鉴定BRIT1异常。编码区和外显子/内含子连接的BRIT1突变将通过高通量DNA测序来确定。潜在的新突变将通过体外功能分析进行评估。BRIT1的蛋白表达和亚细胞定位也将通过免疫组织化学染色进行评估，其RNA水平将通过定量RT-PCR进行评估。此外，我们将确定BRIT1缺陷是否与肿瘤分级/分期和患者生存相关。(2)建立BRIT1基因敲除小鼠模型，探讨BRIT1基因缺失在肝细胞癌发生发展中的作用。我们最近建立了一种在肝脏中特异性缺失BRIT1的条件性基因敲除小鼠模型(BRIT1FLOX/FLOX/Alb-Cre)。为了评估BRIT1缺陷是否会加速癌基因应激反应(c-myc)的发生和发展，我们通过用Alb-cMyc转基因小鼠培育BRIT1条件基因敲除小鼠来产生双突变小鼠(Alb-cMyc/BRIT1Flox/Flox/Alb-Cre)。双突变和Alb-cMyc转基因的肝肿瘤发生率和潜伏期将被无创性地监测并经组织学分析证实。通过系统的组织学分析，还将对这些小鼠的肿瘤分级和肿瘤分期进行评估和比较。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。总之，这项建议包括在肝癌标本中鉴定BRIT1基因突变，并使用我们独特的基因敲除小鼠模型评估BRIT1肿瘤抑制功能，这代表了一项多方面的研究，将有助于我们理解BRIT1在肝癌抑制中的新角色S。本研究的成功不仅有助于揭示肝细胞癌发生的新的重要分子机制，而且将为BRIT1缺陷的肝细胞癌的分层和靶向治疗提供直接的临床影响，从而显著降低肝细胞癌的死亡率。 公共卫生相关性：BRIT1是同源重组(HR)的新的关键调节因子，BRIT1缺陷细胞对PARP抑制剂更敏感，PARP抑制剂是一类有希望通过合成致死性概念治疗HR缺陷癌症的新药。本研究旨在通过对肝细胞癌标本中BRIT1基因突变的分析和肝脏特异性BRIT1基因敲除小鼠的鉴定，来研究肝细胞癌中的BRIT1缺陷。因此，所有这些拟议的研究不仅将有助于了解与肝癌相关的新的关键机制，而且将加速基于PARP抑制剂的肝癌患者靶向治疗的发展。

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 generated the BRIT1 knockout mice and demonstrated the essential roles of BRIT1 in homologous recombination DNA repair and in maintaining genomic stability in vivo. Interestingly, we have also recently identified BRIT1 DNA mutations in 3 of the 10 hepatocellular carcinoma (HCC) specimens that we had sequenced. Based on our knowledge, this is the first study discovering BRIT1 gene mutations in HCC. Thus, these intriguing data trigger us to hypothesize that BRIT1 may function as a novel tumor suppressor for HCC via preserving genome stability and that targeting BRIT1 deficiency such as using PARP inhibitors may provide novel and effective targeted therapies for the patients. Two specific aims are proposed to test this hypothesis: (1) To assess BRIT1 alterations at DNA, RNA and protein levels in clinical HCC specimens. We will identify BRIT1 aberrations from100 primary HCC samples stratified by tumor grade/stage. BRIT1 mutations in the coding region and exon/intron junction will be determined by high-throughput DNA sequencing. The potential novel mutations will be assessed by in vitro functional analysis. 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 tumor grade/stage and patient survival. (2) To determine if and how the loss of BRIT1 contributes to initiation and progression of HCC using BRIT1 knockout mouse model. We have recently generated a conditional knockout mouse model with BRIT1 specifically deleted in liver (BRIT1flox/flox/Alb-Cre). To evaluate if BRIT1 deficiency may accelerate the initiation and progression of HCC in response to oncogenic stress (c-myc), we will generate the double mutant mice (Alb- cMyc/BRIT1flox/flox/Alb-Cre) by breeding the BRIT1 conditional knockout with Alb-cMyc transgenic mice. The liver tumor incidence and latency in the double mutants and Alb-cMyc transgenics will be monitored noninvasively and confirmed by histological analysis. Tumor grade and tumor stage in these mice will be also assessed and compared by systematic histological analysis. The underlying mechanisms will be investigated by analyzing the DNA repair function of major BRIT1 targets. In summary, this proposal, which includes identification of BRIT1 aberrations in HCC specimens and assessment of BRIT1 tumor suppression function using our unique knockout mouse model, represents a multifacet research that will facilitate our understanding of BRIT1's novel role in HCC suppression. The success of our study will not only contribute to revealing a novel and essential molecular mechanism for HCC but also provide the immediate clinical impact toward stratifying and targeting BRIT1-deficient HCC and as a result, lead to significant reduction of HCC mortality. PUBLIC HEALTH RELEVANCE: BRIT1 is a novel key regulator for homologous recombination (HR) and BRIT1-deficient cells are more sensitive to PARP inhibitors, a new class of promising drugs for target HR-defect cancers via synthetic lethality concept. This proposal is focused on investigation of BRIT1 deficiency in hepatocellular carcinoma (HCC) by analysis of BRIT1 aberrations in HCC specimens and by characterization of liver-specific BRIT1 knockout mice. Thus, all of these proposed studies will not only help to understand the novel key mechanism implicated in HCC but will also accelerate the development of PARP inhibitor-based targeted therapies for HCC patients.