The pigment phenotype found in the NRAS1 and NRAS2 transgenic fish is striking yet similar to the observed hyperpigmentation in BRAFQ61K
transgenic fish. Activation of the MAPK pathway in zebrafish melanocytes clearly has clear effects on patterning. The difference in pigmentation and DNA content between the two transgenic lines is intriguing, especially because the less severe fish phenotypically (RAS2) has higher levels of RAS expression. This result agrees with the previous observations that extremely high levels of RAS expression can have different cellular effects than lower levels of RAS expression. Specifically, high-intensity RAS signaling has been shown to activate senescence pathways in addition to MAPK signaling.28
In these studies, it was found that high-intensity, but not low-intensity, RAS signaling leads to senescence by activating MEKK3/6-p38, which leads to an increase in Ink4a
transcription. It is possible that the higher levels of transgene expression in RAS2 fish prevent more severe cellular and pigmentation phenotypes by contributing to premature cell-cycle arrest. Although we were unable to detect apoptosis or senescence in adult scale-associated melanocytes, it is possible that with improved assays we would be able to see this phenotype in 3–4 week juveniles, the age when hyperpigmentation becomes apparent.
Although NRAS1 and NRAS2 fish express oncogenic NRAS, they do not readily develop cancer. This result indicates that in fish, analogous to the mammalian systems, the presence of a single oncogenic stimulus is insufficient for tumorigenesis. Indeed, the observation that p53-deficient transgenic animals do not develop cancer until at least 10 weeks of age further confirms a multihit tumor model and the need to accumulate additional genomic alterations. It is interesting that the rate of tumor onset in p53-matched fish, either RAS1;p53−/− versus RAS2;p53−/− or RAS1;p53+/− versus RAS2;p53+/−, is similar. This suggest that there may be a threshold effect for RAS signaling in zebrafish melanocytes, above which excess NRAS does not impart an additional oncogenic stimulus. Instead, in the context of oncogenic NRAS coupled with p53 loss, the rate of tumor onset is dependent on the accumulation of spontaneous mutations. The multihit nature of this tumor model further underscores its similarity with human disease.
In the p53 heterozygous background, tumor progression requires the loss of heterozygosity of the remaining wild-type p53 allele. The fact that wild-type p53 is selectively lost in these fish confirms the required role in zebrafish for p53 in genome maintenance and cell-cycle regulation. The observation that p53 is lost in 100% of NRAS1;p53+/−
-derived tumors but only 57% of NRAS2;p53+/−
-derived tumors is not a significant difference (p
0.3) and likely reflects technical challenges associated with the difference in tumor locations instead of different mechanisms of tumor progression. NRAS1 tumors were collected from fins and were therefore easy to dissect away from normal tissue. NRAS2 tumors, however, arose laterally, and clean dissections were often not possible.
In addition to the histological similarities, whole-genome expression arrays indicate that NRAS1;p53−/− and NRAS2;p53−/− tumors have high degrees of molecular similarity to human disease. Not only does this indicate that the transcriptional pathways activated in human melanoma are conserved in zebrafish but also it validates the utility of zebrafish melanoma as a model for studying human disease. Although both NRAS models develop tumors highly similar to human melanomas, they are unique from each other. Although the similarities between the two models may provide insights into some of the more common features of human disease, exploring the differences may be equally useful in studying relevant subclasses of human disease.
It is interesting to note that only upregulated genes are conserved between zebrafish and human melanoma. Although there are many possible causes, a major contributor is likely the fact that normal skin was used for the initial comparisons to melanoma. Ideally, purified melanocytes would have been used as the normal tissue control. However, because zebrafish melanocytes cannot be purified, this was not possible. With significantly fewer melanocytes in normal skin than melanoma, even modest increases in expression would appear amplified in melanoma. Additionally, there is effectively no upper limit to overexpression. In an attempt to control for these issues, zebrafish melanoma arrays were compared with the mean observed expression of the chip. The most highly expressed genes on the chip were the same genes identified in our original analysis.
In contrast, detection of downregulated genes may require purified melanocytes as a control. A normally expressed gene in a relatively small number of normal melanocytes may simply resemble, on an array, the expression profile for that gene when downregulated in a melanoma sample. Plus, there is a lower limit on downregulation. If a probe set registers zero when data are collected, it cannot be evaluated and is automatically excluded from analysis. This phenomenon that only upregulated genes are conserved between human and zebrafish cancer has been observed previously in other zebrafish cancer models and may reflect a limitation of the system or the assay.25
In summary, we have described two new zebrafish models of NRAS-driven human melanoma. We found that when expressed in the melanocyte compartment, human oncogenic NRASQ61K causes hyperpigmentation in zebrafish and cooperates with p53 loss to generate melanomas. The phenotypic similarities between zebrafish melanoma and human disease underscore both the importance and the utility of further zebrafish research. Further, the similarities in gene expression between human and zebrafish disease may provide clues as to what aspects of human melanoma will be most amenable to dissection through zebrafish models.