BRIT1-SerpinB2调控肝癌转移及其分子机制研究

Hepatocellular carcinoma (HCC) is one of the most common malignancies and the leading cause of cancer-related death in our country and worldwide. Most of these deaths are attributed to metastasis, which has still been a bottleneck in prognosis and treatment of HCC. Therefore, it is critical to dissect the molecular mechanisms of metastasis in order to facilitate development of predictive marker for prognosis and potential targeted therapy. Recently, BRIT1 (also known as MCPH1), a chromatin-binding protein, has been identified to be a critical player in homologous recombination repair (HR). We have previously generated the BRIT1-/- mice and demonstrated BRIT1’s essential role in tumor suppression in the mouse model. Our preliminary data also indicated that BRIT1 is aberrantly expressed in HCC specimens, and its deletion is correlated with metastasis in a small cohort. Moreover, we found that BRIT1-deficient Hep3B are prone to migration, and ectopic BRIT1 can inhibit metastatic capacity in rescue experiments. Gene expression profiling analysis showed that BRIT1 can upregulate the expression of SerpinB2 (also called plasminogen activator inhibitor 2, PAI-2), an inhibitor of serine proteases involved in remodeling of extracellular matrix and metastasis. Thus, we hypothesize that BRIT1 acts as a novel metastatic suppressor via regulating the expression of SerpinB2, and the deficiency of BRIT1-SerpinB2 can be regarded as a new predictive maker for metastasis and prognosis of HCC. To test our hypothesis, we propose the following specific aims in this proposal: 1) to determine whether BRIT1-SerpinB2 deficiency promotes migration and invasion in vitro and in vivo xenograft tumor model and liver-specific knockout mouse model. The migration and invasion capacities will be determined by using scratch-wound healing assay and Transwell migration and invasion assays. About nearly 20 HCC cells will be screened, and BRIT1-overexpressing and BRIT1-low expressing cells and SerpinB2-overexpressing and SerpinB2-knockdown cells will be established and used to confirm the findings; 2) to determine whether BRIT1-SerpinB2 level is correlated with metastatic status in a large cohort of HCC samples (n=100-150); and 3) to dissect the molecular mechanisms of BRIT1 regulating SerpinB2 during metastasis. The ChIP-seq assay will be used to determine whether BRIT1 binds the promoter region of SerpinB2 and whether BRIT1 can regulate other target genes. The protein fraction obtained during ChIP assay will be analyzed to determine the transcription factors interacted with BRIT1. The microRNA microarray will also be used to determine if BRIT1 can regulate the expression of SerpinB2 via the microRNAs. Taken together, these proposed studies, if successfully completed, will lead to: (i) reveal BRIT1 acting as a metastasis inhibitor for HCC, (ii) determine the molecular mechanism of BRIT1-SerpinB2 inhibiting metastasis in HCC, and (iii) identify BRIT1-SerpinB2 to be a novel predictive biomarker of HCC metastasis. Therefore, this project, if successfully tested, will eventually benefit patients with unresectable and metastatic HCC who has few alternative treatment choices.

我们前期研究工作表明BRIT1（也称之为MCPH1）可能与肝癌转移相关：BRIT1低水平表达与肝癌的恶性度与复发性呈正相关；小样本分析结果显示BRIT1低表达促进肝癌转移；基因表达谱分析及相关实验结果也显示，BRIT1上调转移抑制因子SerpinB2的表达。因此，在本项目中，我们提出假说：BRIT1是一种新的肝癌转移抑制因子，可通过调控SerpinB2发挥作用。为了检验此假说，我们首先阐明BRIT1-SerpinB2对肝癌细胞迁移与侵袭能力的影响，然后在大样本量临床肝癌标本中分析BRIT1-SerpinB2与转移的关系，最后探讨BRIT1调控SerpinB2表达、进而影响肝癌转移的分子机制。如能获得资助并顺利完成，本项目将：1）阐明BRIT1是否是一种新的肝癌转移抑制因子；2）确定BRIT1-SerpinB2在肝癌转移过程中的作用机制；3）提供一种新的判断肝癌转移及其预后的分子标记。

原发性肝癌（以下简称肝癌）是世界范围内常见的恶性肿瘤和致死性肿瘤，也是我国常见的恶性肿瘤之一。肝癌患者的病死率很高，主要原因就是肝内或肝外转移。为了进一步探讨肝癌转移的新的机制和分子标记，我们以BRIT1为靶点，在前期工作基础上，提出本项目的假说，即BRIT1通过调控SERPIN类相关基因的表达控制肝癌的转移过程。因此本项目拟定目标在于阐明BRIT1缺失对肝癌转移的调控机制，以及如何控制可能的下游靶基因从而达到调控转移的目的。经过项目组成员的通力合作，我们发现BRIT1过表达在细胞水平与移植瘤小鼠体内明显促进肝癌细胞的侵袭或转移潜能。在肝癌细胞中过表达BRIT1可以上调部分SERPIN基因的表达，包括SERPINB2和SERPINA5。在肝癌组织中，BRIT1蛋白与SERPINA5 蛋白的表达呈正相关性， 并与肝癌的恶性度密切相关。进一步研究发现，BRIT1蛋白通过转录因子E2F1可以调控SERPINA5的启动子区的活性，提示BRIT1与转录因子E2F1协同调节SERPINA5基因表达从而实现对肿瘤转移的调节效应。因此，我们发现BRIT1-SERPIN轴调节是肝癌转移过程调控的一个重要因素。此研究或有助于为临床提供一种新的针对BRIT1缺失性肝癌转移的分子靶标。