The Zon Lab @ Children's Hospital, Boston

Zebrafish Cancer

A screen for small molecule suppressors of the cancer-susceptible crash&burn cell cycle mutant

1, 2*Howard M. Stern, 2*Ryan D. Murphey, 2Jennifer L. Shepard, 2James F. Amatruda, 3Randall W. King, and 2Leonard I. Zon

*These two authors contributed equally.1Brigham and Women's Hospital, 2Children's Hospital, 3Institute of Chemistry and Cell Biology, Boston, MA.

The use of small molecules to perturb specific cancer pathways is a powerful tool, both to study neoplasia and to develop chemotherapeutic or chemopreventative agents. The zebrafish system is well suited to organism-based small molecule screens because large numbers of embryos can be arrayed to multi-well plates with water and test compounds. Chemical screens using embryos select for compounds active in a multi-cellular organism, an advantage over traditional screens using transformed cell lines or in vitro protein binding assays. We performed a screen for small molecules that suppress the embryonic phenotype of crash&burn (crb), a cell cycle mutant that exhibits an accumulation of cells in G2/M, illustrated by increased anti-phospho-histone H3 (pH3) antibody staining in homozygous mutants. Crb homozygotes also develop brain necrosis and polyploidy, and adult heterozygotes exhibit an increased incidence of cancer. 50 crb heterozygote pairs were mated to generate ~3,000 offspring weekly. 20 embryos at 4 hpf were added to each well of a 48-well plate, and test chemicals were robotically transferred at a concentration of ~20 mM. Chemical treatments resulting in the absence of mutants as determined by pH3 staining at 24 hrs were considered candidates for crb suppression. In addition, because each well contained a mixture of wild-type, heterozygous and mutant embryos, this screen enabled the identification of ten novel compounds that increase or decrease pH3 staining in wild-type embryos. In 5 weeks we screened 5,000 compounds and, strikingly, found one novel compound that suppressed the crb phenotype, including the accumulation of cells in G2/M and brain necrosis. DNA content analysis of chemical-treated wild-type embryos by FACS reveals an accumulation of cells in G1/S, suggesting that this effect could be the mechanism of crb rescue. Future experiments will explore the compound's effect on cancer predisposition in heterozygous crb adults. This example illustrates that the zebrafish can be used to identify small molecule suppressors of a disease-relevant phenotype.

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A Genetic Screen for Mutations Affecting Cell Proliferation and Cancer Susceptibility in the Zebrafish

1Jennifer L. Shepard*, 1James F. Amatruda*, 1James Ziai, 1, 2Howard M. Stern, 1David Finkelstein,
1Kathleen Lindahl, 1Candace Hersey, 2Jon Aster, 2Jeffrey Kutok , 2Jonathan Glickman, 1Matthew Freedman,
3Jan Spitsbergen, 1Yi Zhou, and 1Leonard I. Zon. (*these authors contributed equally)

1Children's Hospital, 2Brigham & Women's Hospital, Boston, MA 3Oregon State University, Corvallis, OR

Zebrafish is an ideal system for cancer gene discovery owing to its combination of forward genetics and long use as a carcinogenesis model. Zebrafish exposed to carcinogens develop a wide variety of tumors, including sarcomas, schwannomas, germ-cell tumors, and carcinomas in virtually all organs with a histology closely resembling that of human tumors. To construct a zebrafish cancer model, we screened F2 embryos from mutagenized stocks, using whole-mount anti-phosphohistone H3 antibody staining to detect mutations causing alterations in cell proliferation. 750 F1 females were screened and eight homozygous lethal mutants with distinct cell cycle abnormalities were identified. We analyzed these mutants for DNA content, mitotic spindle abnormalities, BrdU incorporation, apoptosis, and mRNA expression of cell cycle genes. crash&burn (crb) and cease&desist (cds) have aberrant spindle formation and demonstrate polyploidy. standstill (sds) has an increased proportion of cells in G2/M and decreased pH3 staining while shortstop (srp) has an increased G1 fraction. Other mutants also showed changes in the number of cells present in S phase. Positional cloning projects are ongoing for several of the mutants; crb has been mapped within 0.1 cM and cds within 0.06 cM (n=3000 meioses for each). Because the mutations are homozygous lethal, we have studied cancer incidence in adult heterozygotes after exposure to the carcinogens MNNG or DMBA. Compared with wild-type siblings, crb heterozygotes have significantly increased cancer incidence. After DMBA exposure, 43/183 (23%) crb heterozygotes developed cancer, vs 18/182 (10%) of wild-type (p=0.0007). After MNNG, 11/77 (14%) crb heterozygotes developed cancer, vs. 3/74 (4%) of wild-type (p=0.05). While data continue to be collected, two other mutants, cds and srp, also demonstrate a trend toward increased tumor incidence. The zebrafish has great promise as a system for discovering cancer genes and developing novel cancer therapies.

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