BRIT1, A NOVEL HEPATOCELLULAR CARCINOMA TUMOR SUPPRESSOR

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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.