Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Abstract

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors 1,2,3 . A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model 4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined 5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAF V600E );p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis 6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays. Video LinkThe video component of this article can be found at

A population of stress-like cancer cells in melanoma promotes tumorigenesis and confers drug resistance

“…Fish strains and handling. minicoopr fish were generated as previously described (Ceol et al, 2011;Iyengar et al, 2012) . Fish with the genotype mitfa-BRAF V600E ; p53 -/-; mitfa -/-were incrossed.…”

Zebrafish as a model to investigate the effects of exercise in cancer

“…The overall experimental scheme is shown in Figure 3. The transgenic MiniCoopR system (Iyengar et al, 2012) was used to create a zebrafish melanoma, in which the mitfa promoter drives human BRAF V600E in concert with a p53 loss of function allele, along with a GFP marker gene. From that original tumor, we created the ZMEL1-GFP cell line, which can be transplanted into transparent casper fish (White et al, 2008) to image tumor growth and metastasis (Heilmann et al, 2015) .…”

Genomic Approaches to Zebrafish Cancer

“…Transgenic and CRISPR models could help reveal the underlying mechanisms of these poorly understood events. Whether the fish can be used for more translational, actionable, “filtered” list of genes remains to be determined, but the data thus far indicates that for these more clinically relevant questions, the fish can be used to: (1) model potential lists of candidate DNA mutations/CNVs that arise from human TCGA data, as has been described using the miniCoopR system [12, 43], or (2) identify conserved pathways across species using RNA-seq approaches.…”