SRSF3 Loss and Hepatocellular Carcinoma

 描述(由申请人提供)： RNA剪接是在产生最终的RNA之前从初级RNA转录本中去除内含子序列的过程。与低等生物不同，哺乳动物的绝大多数基因是拼接的。大多数基因由于不同的启动子或终止序列或使用替代外显子而产生多个mRNAs。在更广泛的临床图景中，选择性剪接是一个重要的新研究领域。现在已经在许多癌症中发现了RNA剪接的变化。由于癌症是VA人群死亡的主要原因，了解这些致癌剪接变异是如何发生的，以及它们如何促进癌症的发生，对于我们理解疾病的发病机制和发现新的治疗方法是至关重要的。我们最近在小鼠中的数据表明，消除特定的RNA剪接因子会由于肝细胞凋亡和代偿性增殖而导致慢性肝损伤。随着年龄的增长，小鼠的肝脏出现脂肪性肝炎和纤维化，最终100%的小鼠死于肝细胞癌(HCC)。剪接因子的缺失不会导致肿瘤，而是产生癌症的易感性，类似于肿瘤抑制基因。我们相信这个模型与人类肝癌有许多相似之处，可以为这种疾病的发病机制提供很好的见解，从而指导治疗开发。基于我们的剪接因子基因敲除小鼠的大量初步数据，我们提议进行一系列全面的实验，以了解肝脏中单一剪接因子的缺失如何导致转移性肝细胞癌。这些研究将解决与致癌的基本生物学过程有关的关键问题，并将把细胞和分子生物学实验与在肝脏中缺乏特定剪接因子的小鼠的生理研究结合起来。我们将通过评估SRSF3缺失在人类肝细胞癌中发生的频率，是否在所有肝细胞癌或特定亚型中发生，以及是否在非酒精性脂肪肝、非酒精性脂肪肝或肝硬变等早期肝病中发生，来建立该模型与人类肝癌的相关性。在小鼠中，SRSF3的缺失易患肝癌，但并不是每个细胞都会转化，这表明必须获得体细胞突变或表观遗传学变化才能产生癌症表型。我们将尝试通过对小鼠肿瘤中的体细胞突变进行测序，或评估差异甲基化区域中CpG甲基化的变化来确定这些继发性基因变化。我们还将测试这些基因变化是否允许肿瘤通过恢复肿瘤细胞中SRSF3的表达而独立于SRSF3状态。我们利用Clip-Seq和转录组测序以及剪接连接阵列相结合的方法研究了RNA剪接异常及其相关细胞变化的分子机制，以确定肝细胞中SRSF3的直接靶点。这些靶点将使用体外培养的人类肝癌细胞进行SRSF3依赖性测试。我们还将测试在包括肝癌在内的各种癌症中发现的一些SRSF3突变。我们将测试两种可能导致细胞凋亡、纤维化和细胞增殖的潜在机制。我们正在尝试将拼接方面的变化联系起来 纤维连接蛋白和胰岛素受体基因具有特定的中间表型，并与肿瘤生长有关。这两个基因是SRSF3的直接靶点，它们的剪接在基因敲除小鼠中发生了变化。我们将测试纤维连接蛋白基因的错误剪接是否会导致氧化应激和纤维化，以及胰岛素受体基因的错误剪接是否允许IGF2刺激肿瘤细胞的增殖和阻止正常的肝细胞末端内复制。这些实验将把新的基因敲除与药物刺激或抑制个别途径结合起来。最终，我们将测试这两个途径是否有助于肝细胞癌的发生。

 DESCRIPTION (provided by applicant): RNA splicing is the process of removal of intronic sequences from the primary RNA transcript before the final mRNA is generated. Unlike lower organisms, the vast majority of mammalian genes are spliced. Most genes give rise to multiple mRNAs resulting from differential promoters or termination sequences, or the use of alternative exons. In the broader clinical picture, alternative splicing is an important new area of investigation. Alterations in RNA splicing have now been found in many cancers. As cancer is a leading cause of death among the VA population, understanding how these oncogenic splice variants occur and how they contribute to carcinogenesis is paramount to our understanding of the pathogenesis of the disease and our discovery of novel therapeutic approaches. We have recent data in mice that eliminating a particular RNA splicing factor causes chronic liver damage due to hepatocyte apoptosis and compensatory proliferation. As the mice age, their livers show steatohepatitis and fibrosis, and eventually 100% of the mice succumb to hepatocellular carcinoma (HCC). The loss of the splicing factor does not cause tumors but rather creates a pre-disposition to cancer, similar to a tumor suppressor gene. We believe this model has many parallels to human HCC and can provide great insights into the pathogenesis of this disease that could guide therapeutic development. Based on extensive preliminary data on our splicing factor knockout mouse, we are proposing a comprehensive series of experiments to understand how loss of a single splicing factor in the liver can lead to metastatic hepatocellular carcinoma. These studies will address key questions concerning the fundamental biological process of carcinogenesis and will integrate cell and molecular biological experiments with physiological studies in mice lacking specific splicing factors in liver. We will establish the relevance of this model for human liver cancer by assessing how frequently SRSF3 loss occurs in human HCC, whether loss occurs in all HCC or particular subtypes, and whether loss occurs in early-stage liver disease such as NAFLD, NASH or cirrhosis. In the mouse, loss of SRSF3 predisposes to HCC but not every cell becomes transformed, suggesting that somatic mutations or epigenetic changes must be acquired to produce the carcinoma phenotype. We will attempt to identify these secondary genetic changes by sequencing somatic mutations in the mouse tumors, or assessing changes in CpG methylation in differentially-methylated regions. We will also test whether these genetic changes allow the tumors to become independent of SRSF3 status by restoring SRSF3 expression in the tumor cells. We investigate the molecular mechanisms underlying the aberrant RNA splicing and the associated cellular changes using a combination of CLIP-seq and transcriptome sequencing and splice junction arrays to identify the direct targets for SRSF3 in the hepatocyte. These targets will be tested for SRSF3-dependence using in vitro cultured human hepatoma cells. We will also test a number of SRSF3 mutations have been identified in a variety of cancers including HCC. We will test two potential mechanisms that may contribute to apoptosis, fibrosis, and cell proliferation. We are attempting to connect changes in splicing of the fibronectin and insulin receptor genes with specific intermediate phenotypes, and with tumor growth. These two genes are direct targets for SRSF3 and their splicing is altered in the knockout mouse. We will test whether miss-splicing of the fibronectin gene causes oxidative stress and fibrosis, and whether miss-splicing of the insulin receptor gene allows IGF2 to stimulate tumor cell proliferation and prevent the normal terminal endo-replication of the hepatocyte. These experiments will combine new genetic knockouts with pharmacological stimulation or inhibition of individual pathways. Ultimately, we will test whether these two pathways contribute to hepatocellular carcinogenesis.