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，我的实验室培育了BRIT1基因敲除小鼠，并在体内证明了BRIT1在同源重组DNA修复和维持基因组稳定性中的重要作用。有趣的是，我们最近也在我们测序的10例肝细胞癌（HCC）标本中的3例中发现了BRIT1 DNA突变。据我们所知，这是首次在HCC中发现BRIT1基因突变。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稳定性而作为HCC的新型肿瘤抑制因子，并且针对BRIT1缺陷（如使用PARP抑制剂）可能为患者提供新颖有效的靶向治疗。为了验证这一假设，我们提出了两个具体的目标：(1)评估临床HCC标本中DNA、RNA和蛋白质水平上BRIT1的改变。我们将根据肿瘤分级/分期从100个原发性HCC样本中识别BRIT1畸变。编码区和外显子/内含子连接处的BRIT1突变将通过高通量DNA测序确定。潜在的新突变将通过体外功能分析进行评估。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缺乏是否与肿瘤分级/分期和患者生存相关。(2)利用BRIT1敲除小鼠模型确定BRIT1的缺失是否以及如何促进HCC的发生和进展。我们最近建立了肝脏中BRIT1特异性缺失的条件敲除小鼠模型（BRIT1flox/flox/Alb-Cre）。为了评估BRIT1缺乏是否会在癌性应激（c-myc）下加速HCC的发生和进展，我们将通过与Alb-cMyc转基因小鼠杂交BRIT1条件敲除产生双突变小鼠（Alb- cMyc/BRIT1flox/flox/Alb- cre）。双突变体和Alb-cMyc转基因的肝肿瘤发生率和潜伏期将进行无创监测，并通过组织学分析证实。这些小鼠的肿瘤分级和肿瘤分期也将通过系统的组织学分析进行评估和比较。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。综上所述，该提案包括HCC标本中BRIT1畸变的鉴定和使用我们独特的敲除小鼠模型评估BRIT1肿瘤抑制功能，代表了一项多方面的研究，将有助于我们了解BRIT1在HCC抑制中的新作用。我们研究的成功不仅有助于揭示HCC的一种新的和必要的分子机制，而且还为分层和靶向brit1缺失型HCC提供了直接的临床影响，从而显著降低HCC死亡率。

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.