BRIT1/MCPH1在肝癌发生与靶向治疗中的作用及其分子机制研究

Hepatocellular Carcinoma (HCC) is one of the most common malignancy in our country and the third most frequent cause of cancer-related death in the world. It is urgently needed to develop more effective treatments to reduce the mortality of this disease by dissecting the underlying molecular mechanism. Recently, increased evidences show that 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 cancer development. We have also identified that BRIT1 (also known as MCPH1) is a novel key regulator for DDR pathway by using our unique knockout mouse model. Our preliminary data also showed that BRIT1 is aberrantly expressed in HCC samples, and the BRTI1-deficient cells bear genomic instability due to the loss of BRIT1. Based on these data, we proposed the following specific aims in this proposal: 1) to determine the association of BRIT1 status with tumor grade, stage, metastasis, and prognosis. BRIT1 status will be deternined with its protein level examined by using immunohistochemical (IHC) staining in HCC samples; 2) to assess tumorigeneis of BRIT1-deficienct cells in the xenograft model or the liver-specific conditional knockout mouse model; 3) to disclose the alternative DNA damage response pathways employed by BRIT1-deficienct cells in HCC cell lines and samples; and 4) to screen the small molecule compounds that specifically target the BRIT1-deficient cells. These proposed studies, if sucessfully completed, will lead to: (i) reveal a novel BRIT1 deficiency-mediated mechanisms to promote HCC development, (ii) identify the alternative DDR in BRIT1-deficient HCC, and (iii) develop a novel and effective combinational therapy for HCC via targeting the alternative DDR pathways in BRIT1-deficient HCC. The success of our study will provide the immediate clinical impact toward stratifying and targeting BRIT1-deficient HCC and as a result, lead to significant reduction of HCC mortality.

我们已有研究表明BRIT1在DNA损伤修复系统中参与同源重组修复过程；在部分肝癌组织中，BRIT1表达水平降低，而且，BRIT1缺失细胞因其修复能力降低，导致基因组不稳定性。在本项目中，为了揭示BRIT1在肝癌发生过程中的作用，并探寻一种针对BRIT1缺失细胞的靶向性治疗措施，我们首先确定BRIT1表达水平与肝癌恶性度的相关性，阐释BRIT1缺失细胞的致瘤能力；然后探索BRIT1缺失细胞的代偿性DNA修复机制，特别是其它修复系统中关键性酶蛋白变化；最后，利用合成致死性原理，筛选小分子化合物库，寻找特异性针对代偿性DNA修复通路的小分子抑制剂。这样，如能获得资助并顺利完成，本项目将：1）利用我们构建的BRIT1敲除小鼠揭示肝癌发生的分子机制；2）阐释BRIT1可以作为新的肝癌发生和预后诊断标记；3）探讨基于BRIT1缺失状态的新的靶向性肝癌治疗措施，为肝癌的临床治疗提供一种新的方法。

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；在我国， 其5年生存率 不足10%。对肝癌病人来说，仅有一小部分可以接受手术治疗，且常规放疗、化疗效果较差。因此，很有必要进一步阐明肝癌发生发展机理，继而找到一种有效的治疗方法以降低肝癌患者的死亡率。近来，我们发现BRIT1参与DNA损伤修复系统，在DNA损伤修复的早期应答过程中发挥着重要作用，以维持基因组稳定性。其在肝癌标本中也呈缺失性表达，提示BRIT 1可能是一个潜在的肿瘤治疗靶点。 因此，本项目拟定目标在于阐明BRIT 1在肝癌发生发展过程中的作用，以及在BRIT1缺失条件下，利用合成致死性模式特异性地抑制代偿性的DNA修复系统，以达到特异性杀灭肝癌细胞的效果。经过项目组成员的通力合作，我们发现BRIT1通过调控DNA损伤修复缺陷，特别是参与同源重组修复机制以抑制肿瘤发生。在较大量肝癌标本中BRIT1呈低水平表达，而多聚ADP-核糖聚合酶1（PARP1）则呈高水平表达；在肝癌细胞中，BRIT1缺失易致其成瘤性增强，同时增加其对PARP1抑制剂的敏感性。在此基础上，我们发现PI3K抑制剂可以明显增强BRIT1缺失细胞对PARP1抑制剂的敏感性，呈显著的协同效应 。此外，通过对RECQL4的研究，我们发现其抑制剂也有望协同抑制肝癌细胞的增殖，从而达到特异性杀灭肝癌细胞的效果。因此，此研究或有助于为临床提供一种新的针对BRIT1缺失性肝癌的靶向性的治疗前景。