Background & Aims

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects children and young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1–PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1–Prkaca fusion and monitored the mice for liver tumor development.

Methods

We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1–Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8-week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1–Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing.

Results

Livers from 12 of the 15 mice given the vectors to induce the Dnajb1–Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1–Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by the Dnajb1–Prkaca fusion revealed a lack of mutations in genes commonly associated with liver cancers, as observed in human FL-HCC.

Conclusions

Using CRISPR/Cas9 technology, we found generation of the Dnajb1–Prkaca fusion gene in wild-type mice to be sufficient to initiate formation of tumors that have many features of human FL-HCC. Strategies to block DNAJB1–PRKACA might be developed as therapeutics for this form of liver cancer.

中文翻译：

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成年小鼠肝脏的CRISPR / Cas9工程表明Dnajb1 – Prkaca基因融合足以诱导类似于纤维状肝细胞癌的肿瘤

背景与目标

纤维状肝细胞癌（FL-HCC）是原发性肝癌，主要影响没有基础肝病的儿童和年轻人。在FL患者中已反复鉴定出19号染色体上的400 Kb体细胞缺失，该缺失融合了DnaJ热休克蛋白家族（Hsp40）成员B1基因（DNAJB1）的一部分与蛋白激酶cAMP激活的催化亚基α基因（PRKACA）的融合。 -HCC。但是，DNAJB1 – PRKACA基因融合尚无诱导肝肿瘤发生的证据。我们使用CRISPR / Cas9技术在小鼠中删除了第8号染色体上的同义区域，从而创建了Dnajb1–Prkaca融合体，并监测了小鼠肝肿瘤的发展。

方法

我们递送设计成的幻影长矛外显子1 CRISPR / Cas9矢量Dnajb1与外显子2 PRKACA创建 Dnajb1-PRKACA用FL-HCC，或控制Cas9向量相关联的基因融合，经由液力尾静脉注射到8周龄的肝脏雌性FVB / N小鼠。这些小鼠没有任何其他工程改造的遗传改变，也没有暴露于肝毒素或致癌物。分娩后14个月收集肝组织。通过PCR分析基因组DNA，以检测Dnajb1-Prkaca融合蛋白，并通过组织学，免疫组织化学，RNA测序和全外显子组测序对组织进行表征。

结果

15只小鼠中有12只给了载体以诱导Dnajb1-Prkaca基因融合，但11只小鼠没有得到控制。肿瘤包含Dnajb1-Prkaca基因融合，并具有人类FL-HCC的组织学和细胞学特征：具有颗粒，嗜酸性和线粒体富集的大型多角形细胞，突出的核仁以及肝细胞和胆管细胞的标志物。在比较小鼠肿瘤和非肿瘤肝细胞之间基因的表达水平时，我们发现了与人类FL-HCC中检测到的变化相似的变化，其中包括影响细胞周期和有丝分裂调控的基因。Dnajb1–Prkaca诱导的小鼠肿瘤的基因组分析 正如人类FL-HCC所观察到的，融合显示出通常与肝癌相关的基因中缺乏突变。

结论

使用CRISPR / Cas9技术，我们发现在野生型小鼠中Dnajb1-Prkaca融合基因的产生足以引发具有人类FL-HCC许多特征的肿瘤的形成。阻断DNAJB1-PRKACA的策略可能被开发为这种肝癌的治疗方法。