An inducible kras(V12) transgenic zebrafish model for liver tumorigenesis and chemical drug screening.
An inducible kras(V12) transgenic zebrafish model for liver tumorigenesis and chemical drug screening.
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DOI：
10.1242/dmm.008367
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发表时间：
2012-01
影响因子：
4.3
通讯作者：
Gong Z
中科院分区：
文献类型：
--
作者：
Nguyen AT;Emelyanov A;Koh CH;Spitsbergen JM;Parinov S;Gong Z
由于Ras信号经常被主要的肝细胞癌病因激活，我们的实验室先前产生了在肝脏中组成型表达krasV 12癌基因的转基因斑马鱼。尽管该模型描述并揭示了斑马鱼和人类肝脏肿瘤发生之间的保守性，但低肿瘤发生率和早期死亡率限制了其用于肿瘤进展和抑制的进一步研究。在这里，我们采用了米非司酮诱导型转基因系统，以实现诱导型krasV 12在肝脏中的表达。该系统由两个转基因株系组成：肝脏驱动株系具有肝脏特异性fabp 10启动子以产生LexPR嵌合反式激活因子，Ras效应株系含有LexA结合位点以控制EGFP-krasV 12表达。在双转基因斑马鱼（驱动器-效应器）胚胎和成人中，我们证明了米非司酮诱导的EGFP-krasV 12在肝脏中的表达。在诱导1个月后，在100%的双转基因鱼中产生了稳健且均质的肝肿瘤，并且肿瘤从治疗后1周（wpt）的增生进展到4 wpt的癌。引人注目的是，肝脏肿瘤发生被发现是“上瘾”的Ras信号的肿瘤维持，因为米非司酮撤回导致肿瘤消退通过细胞死亡转基因鱼。我们进一步证明了透明EGFP-krasV 12幼虫在抑制剂治疗中的潜在用途，以通过靶向其下游效应物（包括Raf-MEK-ERK和PI 3 K-AKT-mTOR途径）来抑制Ras驱动的肝脏肿瘤发生。总的来说，这种米非司酮诱导和可逆的krasV 12转基因系统提供了一种新的模型，用于了解肝癌的发生和抗癌药物的高通量筛选平台。
Because Ras signaling is frequently activated by major hepatocellular carcinoma etiological factors, a transgenic zebrafish constitutively expressing the krasV12 oncogene in the liver was previously generated by our laboratory. Although this model depicted and uncovered the conservation between zebrafish and human liver tumorigenesis, the low tumor incidence and early mortality limit its use for further studies of tumor progression and inhibition. Here, we employed a mifepristone-inducible transgenic system to achieve inducible krasV12 expression in the liver. The system consisted of two transgenic lines: the liver-driver line had a liver-specific fabp10 promoter to produce the LexPR chimeric transactivator, and the Ras-effector line contained a LexA-binding site to control EGFP-krasV12 expression. In double-transgenic zebrafish (driver-effector) embryos and adults, we demonstrated mifepristone-inducible EGFP-krasV12 expression in the liver. Robust and homogeneous liver tumors developed in 100% of double-transgenic fish after 1 month of induction and the tumors progressed from hyperplasia by 1 week post-treatment (wpt) to carcinoma by 4 wpt. Strikingly, liver tumorigenesis was found to be ‘addicted’ to Ras signaling for tumor maintenance, because mifepristone withdrawal led to tumor regression via cell death in transgenic fish. We further demonstrated the potential use of the transparent EGFP-krasV12 larvae in inhibitor treatments to suppress Ras-driven liver tumorigenesis by targeting its downstream effectors, including the Raf-MEK-ERK and PI3K-AKT-mTOR pathways. Collectively, this mifepristone-inducible and reversible krasV12 transgenic system offers a novel model for understanding hepatocarcinogenesis and a high-throughput screening platform for anti-cancer drugs.