Inducible Intestine-Specific Expression of kras Triggers Intestinal Tumorigenesis In Transgenic Zebrafish

Abstract

KRAS mutations are a major risk factor in colorectal cancers. In particular, a point mutation of KRAS of amino acid 12, such as KRASV12, renders it stable activity in oncogenesis. We found that krasV12 promotes intestinal carcinogenesis by generating a transgenic zebrafish line with inducible krasV12 expression in the intestine, Tg(ifabp:EGFP-krasV12). The transgenic fish generated exhibited significant increases in the rates of intestinal epithelial outgrowth, proliferation, and cross talk in the active Ras signaling pathway involving in epithelial-mesenchymal transition (EMT). These results provide in vivo evidence of Ras pathway activation via krasV12 overexpression. Long-term transgenic expression of krasV12 resulted in enteritis, epithelial hyperplasia, and tubular adenoma in adult fish. This was accompanied by increased levels of the signaling proteins p-Erk and p-Akt and by downregulation of the EMT marker E-cadherin. Furthermore, we also observed a synergistic effect of krasV12 expression and dextran sodium sulfate treatment to enhance intestinal tumor in zebrafish. Our results demonstrate that krasV12 overexpression induces intestinal tumorigenesis in zebrafish, which mimics intestinal tumor formation in humans. Thus, our transgenic zebrafish may provide a valuable in vivo platform that can be used to investigate tumor initiation and anticancer drugs for gastrointestinal cancers.

Inflammatory Responses during Tumour Initiation: From Zebrafish Transgenic Models of Cancer to Evidence from Mouse and Man

“…These models all recapitulate human melanoma with respect to their hyperpigmentation, histology and where tested, their transcriptomic gene expression profiles. The expression of human oncogenes under the control of tissue specific promoters has been employed to create representative cancer models for various organs, including the skin [23], intestine [25], pancreas [26] and brain [27] (See Table 1). Zebrafish models of cancer commonly feature fluorescently tagged oncogenes, which label cancer cells and allow real time monitoring of tumour promotion and progression.…”

“…However, inducible systems for transgene expression have been appropriated from the Drosophila and mouse fields, including the Tet/On system [35], the Lex/PR system [36], the tamoxifen-inducible GAL4/UAS system [37] and the heat-shock-inducible Cre/Lox system [38]. These systems have recently been used to develop inducible cancer models, which now enable temporal precision for the study of tumour initiation in both larval and adult fish (see Table 1) [25,[39][40][41].In addition to the use of zebrafish for the study of cancer biology, the zebrafish has also been widely used for the study of haematopoiesis [42] and the innate immune response [43][44][45]. Both macrophages and neutrophils share comparable developmental origins with their mammalian counterparts [46][47][48][49], and exhibit a high degree of functional conservation, for example, with respect to host-pathogen interactions [50] and wound healing [51,52].…”

“…However, inducible systems for transgene expression have been appropriated from the Drosophila and mouse fields, including the Tet/On system [35], the Lex/PR system [36], the tamoxifen-inducible GAL4/UAS system [37] and the heat-shock-inducible Cre/Lox system [38]. These systems have recently been used to develop inducible cancer models, which now enable temporal precision for the study of tumour initiation in both larval and adult fish (see Table 1) [25,[39][40][41].…”

Zebrafish Models of Cancer—New Insights on Modeling Human Cancer in a Non-Mammalian Vertebrate

“…Many recent studies are looking at the pathogenesis of KRAS -driven cancer. A zebrafish model with inducible expression of a mutated version of KRAS V12 in the intestine (under the ifabp promoter) developed tubular adenoma of the intestine until adulthood [59]. The effects of cancer cachexia, a syndrome affecting cancer patients, which can result in weight loss, muscle wasting, and is predictive of low survival, was studied in an inducible KRAS G12V -driven HCC zebrafish model [60].…”

Genetic Engineering of Zebrafish in Cancer Research

“…However, the LexPR system has been used in transgenic zebrafish to generate hepatocellular carcinoma models through the expression of LexPR under the control of the liver-specific promoter fabp10a and LexOp driving the expression of kras G12V or tgfb1a [ 215 , 216 , 217 ]. Similarly, when the LexOp Nras Q61K /Kras G12V target oncogene is activated by LexPR under the control of the melanocyte-specific mitfa promoter or under the control of the intestine-specific ifabp ( fabp2 ) promoter, double-transgenic zebrafish lines develop, in presence of mifepristone, melanoma or intestinal tumors, respectively [ 218 , 219 ]. In addition, the reversibility of the LexPR system allows the investigation of oncogene addiction of tumors in the transgenics.…”