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Educational Session: Zebrafish as an Animal Model in Cancer Research |
Children's Hospital/Dana-Farber Cancer Institute, Boston, MA
Abstract
The zebrafish is emerging as a powerful model system for cancer research (1). Several key aspects of the fish system make it particularly well suited to cancer gene discovery and to the genetic study of tumor biology in vivo. First, zebrafish exhibit true vertebrate cancer biology. They are susceptible to spontaneous and carcinogen-induced tumors, and the cancers they develop—including carcinomas, sarcomas, hematopoietic neoplasms and germ-cell tumors—closely resemble their human counterparts (2). Most of the known oncogenes, tumor-suppressor genes and apoptosis genes are present in the zebrafish genome and are highly conserved compared with mouse and human homologs (3) . Second, the fish is a genetically tractable system. Fish can be cheaply maintained in large numbers, adults mate year-round and can produce clutches of several hundred embryos weekly. Thus, large-scale forward genetic approaches are feasible (4). Because the embryos are transparent and develop outside the mother, it is possible to investigate key processes such as cell proliferation and migration, apoptosis and angiogenesis in a living organism. Finally, an array of tools for the analysis and manipulation of gene expression now exists. An international effort has produced high-resolution physical and genetic maps, and a full genome sequence is nearing completion (5–8). Through transgenic approaches, oncogenes can be expressed in a tissue-specific or inducible manner (9); morpholino oligonucleotides can be used to transiently knock-down expression of specific genes (10); and high-throughput genomics techniques have been successfully used to generate animals inactivated for specific genes (11).
Our main interest has been in using the genetics of the zebrafish system to identify novel genes associated with susceptibility to cancer. To create a zebrafish cancer model, we screened the progeny of mutagenized zebrafish for mutations causing defects in embryonic cell proliferation. Screening fish at the embryonic level is rapid and efficient, and there is good reason to believe that mutations affecting cell proliferation in embryos will be relevant to cancer in adults (for example, inactivation of most tumor-suppressor genes in mice causes an embryonic phenotype). A screen using antiphosphohistone H3 immunostaining as a proliferation marker yielded eight mutants, and we showed that the mutants have distinct and specific cell-cycle phenotypes. These phenotypes include arrest or delay at specific stages of the cell cycle, aneuploidy, mitotic abnormalities and activation of DNA-damage checkpoints. All of the mutations are homozygous lethal, and mutant embryos exhibit extensive apoptosis. A major question is whether adult fish with cell-proliferation mutations will have increased cancers. Because homozygotes have embryonic lethality, we developed a carcinogenesis assay to test for excess cancer susceptibility in adult heterozygotes. There is a wide spectrum of tumors that develop after carcinogen exposure. These tumors closely resemble human tumors morphologically, and some lesions appear to progress through different stages of tumorigenesis. One of the mutations, called crash&burn;, causes a 2-fold increase in cancers in heterozygous carriers compared to their wild-type siblings. We cloned the mutation in crash&burn; and showed that it lies in the B-myb gene (Shepard JL, Amatruda JF and Zon LI, submitted). Currently we are assessing tumors from B-myb +/– animals for loss of heterozygosity at the B-myb locus, and assaying human cancer cell lines for mutations in B-myb. This work represents the first direct evidence that mutations in B-myb are associated with a cancer phenotype. Data are still being collected, and at least one other cell proliferation mutant shows a similar cancer predisposition phenotype.
In general, the cell-cycle mutants isolated via the genetic screen do not develop excess spontaneous tumors. However, in parallel to the cell-cycle screen we have identified a family of zebrafish that develops spontaneous tumors at high penetrance, with dominant inheritance, and as early as three months of age. Interestingly, more than 90% of these tumors are testicular germ cell tumors (seminomas). Little is known about the molecular pathways causing seminomas in humans. To gain insight into this disease, we will clone the mutant gene. Through interval haplotype analysis we have mapped the germ cell tumor mutant to a 20cM interval on zebrafish chromosome 10, and we continue to collect more affected animals. The early success of the zebrafish system in generating transgenic cancer models, inactivating key genes and identifying cancer-related genes is encouraging. The next step is likely to come from combinations of these approaches. For example, expressing oncogenes in the context of an underlying cell proliferation mutation may provide new insight into cancer-related pathways. A particularly exciting prospect is the further use of large-scale screens. For example, given a phenotype due to the loss of a tumor-suppressor gene, a second site mutation that suppresses the phenotype could identify molecules interacting with the tumor-suppressors. These molecules might be excellent therapeutic targets. This intersection of classical genetics with vertebrate cancer biology may allow the fish system to make major contributions to our understanding of cancer biology.
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