BRIT1, A NOVEL HEPATOCELLULAR CARCINOMA TUMOR SUPPRESSOR

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，我的实验室已经产生了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。有趣的是，我们最近还在我们测序的10个肝细胞癌（HCC）标本中的3个中发现了BRIT 1 DNA突变。根据我们的知识，这是第一个发现肝癌中BRIT 1基因突变的研究。因此，这些有趣的数据促使我们假设BRIT 1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，并且靶向BRIT 1缺陷（例如使用PARP抑制剂）可能为患者提供新型有效的靶向治疗。我们提出了两个具体的目标来验证这一假设：（1）评估BRIT 1在临床HCC标本中的DNA，RNA和蛋白质水平的改变。我们将从100例原发性肝细胞癌样本中鉴定BRIT 1畸变，这些样本按肿瘤分级/分期分层。将通过高通量DNA测序确定编码区和外显子/内含子连接处的BRIT 1突变。将通过体外功能分析评估潜在的新突变。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缺陷是否与肿瘤分级/分期和患者生存相关。(2)利用BRIT 1基因敲除小鼠模型研究BRIT 1基因缺失是否以及如何促进肝癌的发生和发展。我们最近建立了一种条件性基因敲除小鼠模型，其中BRIT 1在肝脏中特异性缺失（BRIT 1flox/flox/Alb-Cre）。为了评估BRIT 1缺陷是否可能加速HCC对致癌应激（c-myc）的响应的起始和进展，我们将通过将BRIT 1条件性敲除与Alb-cMyc转基因小鼠繁殖来产生双突变小鼠（Alb-cMyc/BRIT 1flox/flox/Alb-Cre）。将非侵入性地监测双突变体和Alb-cMyc转基因体中的肝肿瘤发生率和潜伏期，并通过组织学分析确认。还将通过系统性组织学分析评估和比较这些小鼠中的肿瘤等级和肿瘤分期。通过分析主要BRIT 1靶点的DNA修复功能，研究其潜在机制。总之，这项提案，其中包括识别肝癌标本中的BRIT 1畸变和评估BRIT 1肿瘤抑制功能，使用我们独特的敲除小鼠模型，代表了一个多方面的研究，这将有助于我们理解BRIT 1的新作用，在肝癌抑制。我们研究的成功不仅有助于揭示HCC的新的和重要的分子机制，而且还为分层和靶向BRIT 1缺陷型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.