Oncogenic NRAS cooperates with p53 loss to generate melanoma in zebrafish

Abstract

NRAS mutations are a common oncogenic event in skin cancer, occurring frequently in congenital nevi and malignant melanoma. To study the role of NRAS in zebrafish, a transgenic approach was applied to generate fish that express human oncogenic NRAS(Q61K) under the control of the melanocyte-restricted mitfa promoter. By screening the progeny of the injected animals, two strains stably expressing the NRAS transgene were identified: Tg(mitfa:EGFP:NRAS(Q61K))(1) and Tg(mitfa:EGFP:NRAS(Q61K))(2). Stable expression of this transgene results in hyperpigmented fish displaying a complete ablation of the normal pigment pattern. Although oncogenic NRAS expression alone was found to be insufficient to promote tumor formation, loss of functional p53 was found to collaborate with NRAS expression in the genesis of melanoma. The tumors derived from these animals are variably pigmented and closely resemble human melanoma. Underscoring the pathological similarities between these tumors and human disease and suggesting that common pathways are similar in these models and human disease, gene set enrichment analysis performed on microarray data found that the upregulated genes from zebrafish melanomas are highly enriched in human tumor samples. This work characterizes two zebrafish melanoma models that will be useful tools for the study of melanoma pathogenesis.

FIG. 1.

NRAS^Q61K causes hyperpigmentation in zebrafish. (A) Schematic representation of the transgene. (B) Animals injected with the NRAS construct develop nevi. (C) Hyperpigmentation in the NRAS1 and NRAS2 transgenic animals disrupts the normal pigment pattern. (D) Transgene copy number of NRAS1 and NRAS2 animals. Primers directed against the mitfa promoter were used. Wild-type fish were also used in this comparison and normalized to two copies per genome. (E) Relative transgene expression as measured by EGFP mRNA in both transgenic lines. (F) Low-grade melanocytic lesion observed in NRAS1 transgenic animals. Scale bar is 10 μm.

FIG. 2.

NRAS^Q61K cooperates with p53 loss in melanoma formation. (A) Tumor-bearing fish were identified by visual inspection and checked for fluorescence. The box around the tumor is shown in (B) under fluorescent light. (C, D) Tumor-free survival curves are shown. Three hundred and forty-two NRAS1 sibling and 167 NRAS2 sibling fish were inspected weekly for these studies. The location of tumors in NRAS1;p53^−/− and NRAS2;p53^−/− fish is shown in (E) and (F), respectively. (G, H) Loss of heterozygosity of the wild-type p53 allele is shown. One hundred percentage of NRAS1;p53^+/− fish and 57% of NRAS2;p53^+/− fish lose the wild-type copy of p53.

FIG. 3.

Histological examination of NRAS-driven tumors in zebrafish. (A, B) Hematoxylin and eosin (H&E) staining of melanomas reveals lesions with sporadic pigmentation and high levels of nuclear pleomorphism. (C, D) Standard immunohistochemistry against GFP revealed high levels of EGFP protein in these tumors, suggesting high and ubiquitous NRAS expression. (E, F) In situ hybridization to detect mitfa transcript indicates high levels of expression in these tumors. (G) Limiting dilution analysis of p53-null tumors from both transgenic lines transplanted intramuscularly into sublethally irradiated recipients. Scale bar = 10 μm. (Color figure is available at liebertonline.com.)

FIG. 4.

Gene set enrichment analysis identifies upregulated gene lists that are conserved between zebrafish and human melanoma. The zebrafish gene set enrichment analysis gene list was used to parse human skin and melanoma microarrays. The parsed list is shown as a heat map (A). The enrichment plot for the NRAS1;p53^−/− upregulated gene list (B). The enrichment plot for upregulated NRAS2;p53^−/− is also shown and is significant (C). Downregulated RAS1;p53^−/− and NRAS2;p53^−/− gene lists are not significantly associated with human disease. The enrichment plot for downregulated NRAS1;p53^−/− is shown (D). (Color figure is available at liebertonline.com.)