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Journal of Clinical Oncology, Vol 25, No 17 (June 10), 2007: pp. 2473-2479 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.08.9821
New Waves of Discovery: Modeling Cancer in Zebrafish
From the Stem Cell Program and Division of Hematology/Oncology, Children's Hospital, and Dana-Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell Institute, Howard Hughes Medical Institute; and the GI Unit, Massachusetts General Hospital, Boston, MA Address reprint requests to Leonard I. Zon, MD, Stem Cell Program, Hematology/Oncology, Children's Hospital, Karp Family Research Building, 7th flr, 1 Blackfan Cir, Boston, MA 02115; e-mail: zon{at}zon.tchlab.org
Cancer is one of the most common causes of death worldwide, affecting almost 1.4 million people annually in the United States alone and leading to more than 560,000 deaths.1 All vertebrate species have the potential to develop cancer. By using other vertebrates as model organisms, we have greatly enhanced our understanding of the mechanisms of human carcinogenesis and cancer progression. Furthermore, studies in invertebrate organisms such as flies and worms have been used to decipher the genetic mechanisms relevant to cancer, such as those regulating tissue specification and growth, cell migration, and apoptosis. Zebrafish, a vertebrate model system, combines the advantages of invertebrate organisms with those of mammalian models; large clutch sizes and relative transparency aid in the identification of molecular genetic pathways involved in organ development and homeostasis, whereas the histology of normal tissue and cancers is highly similar to that of mouse and human samples. Recently, zebrafish have emerged as a model to study cancer susceptibility and carcinogenesis. This review highlights the current use of zebrafish in the field of cancer research, the applicability of the zebrafish to human cancer biology, and the future prospects of using zebrafish to discover novel cancer targets and treatments. Zebrafish belong to the genus of teleosts or bony fish. The split between fish and mammals during evolution dates back 300 million years, yet the genetic programs between these organisms are largely conserved. Zebrafish were originally identified as inhabitants of the river Ganges in India by Hamilton in 1822,2 and to date have served primarily as a fixture in the standard household aquarium. However, during the last two decades they have emerged as an important tool to study embryogenesis. Zebrafish embryos develop ex vivo and are transparent, allowing easy examination of organogenesis in vivo in real time. The adult fish can produce up to 200 offspring per week, aiding in the identification of genetic mutations. Teleosts develop spontaneous tumors in the wild, and, as in mammals, tumorigenesis is a phenomenon of advancing age. Swordtails (Xiphophorus) develop melanomas as a result of a mutation in the receptor tyrosin kinase Xmrk3,4 Medaka fish (Oryzias latipes) develop hepatocellular carcinoma, melanomas, and lymphosarcomas,5 whereas liver tumors were observed in lung fish.6 In addition, fish have been used in toxicology studies for many years; in trout, the development of liver tumors in response to aflatoxin B exposure was investigated widely in the 1970s7,8; more recently, zebrafish have been used to identify embryotoxic drugs.9 Given the propensity for tumor formation in zebrafish, many laboratories have begun to develop techniques to enhance tumorigenesis and thus use the fish to model disease states. These methodologies include chemical carcinogenesis of wild-type (wt) fish, the identification of mutant zebrafish with enhanced susceptibility for tumor development, and the development of transgenic models of specific cancers.
On exposure to water-borne carcinogens, fish develop a wide array of benign and malignant tumors in many different organs.10,11 The effects of dimethyl benzanthracene and N-methyl-N-nitro-N-nitrosoguanidine exposure have been studied extensively and induce a large variety of tumors in zebrafish, most commonly hepatic neoplasms, but also chondromas, hemangiomas, hemangiosarcomas, leiomyosarcoma, rhabdomyosarcomas, and seminomas. Other chemicals, especially nitrosocompounds such as dimethylnitrosamine, diethylnitrosamine, and nitrosomorpholine, provoke predominantly hepatocellular and cholangiolar neoplasms.12-14 The histologic appearance of these tumors is similar to that of human tumors, including increased frequency of mitosis and atypical nuclei (Fig 1). A comparison of the human genome with the nearly completed zebrafish genome reveals the conservation of cancer-relevant oncogenes, tumor suppressor genes, and cell cycle genes.15 The histologic similarity and genetic conservation suggests that the genetic mechanisms underlying carcinogenesis in zebrafish may be highly similar to those in humans. A recent study compared the gene expression signatures in chemically induced liver tumors in zebrafish to human hepatocellular carcinoma.16 Here, a ranked gene list specific for zebrafish liver tumors was constructed by comparing the RNA expression levels in tumor tissue versus normal liver by microarray analysis. It revealed dysregulation of many genes involved in cell cycle, apoptosis, DNA repair, and metastasis; in particular, alterations in the wnt and p53 pathways were noted, which are well-characterized aberrancies in human liver cancer. Gene set enrichment analysis was used to compare the most significantly altered set of pathways in zebrafish versus human liver tumors, and determined that the genetic changes in both species were significantly correlated. In contrast, comparison to other human tumors such as gastric tumors did not demonstrate a high degree of similarity. These results highlight the similarity and applicability of zebrafish biology to human disease.
Forward genetic approaches to gene disruption are well established in zebrafish.17 Random genetic mutations are induced by either chemical mutagenesis, most commonly performed by exposure to ethylnitrosourea (ENU),18 or irradiation.19 Mutated F0 zebrafish are used to create haploid F1 progeny or homozygous diploid embryos for analysis, or the F1 fish can be used to create a F2 diploid generation to reveal developmental changes associated with the induced genetic mutations. Forward genetic screens provide an unbiased approach to identifying genetic pathways that influence various aspects of vertebrate development, including organogenesis. The first large-scale genetic screens, performed in Tuebingen and Boston in the 1990s, were ENU based and revealed hundreds of mutants with a wide array of developmental phenotypes.20,21 Similarly, viral insertion has been used as an efficient means to disrupt genes at random, and has the added benefit of facilitating gene identification by polymerase chain reaction from the viral mutagen into the host genomic sequence.22,23
On the basis of the observation that cancers regularly exhibit cell cycle defects, a screen was undertaken in our laboratory to identify mutants with cell cycling defects based on staining with phosphorylated histone 3 (pH3), a marker of mitosis. In brief, wt male zebrafish were exposed to ENU to produce many germline mutations. Treated males were then bred to untreated wt female fish to produce F1 offspring carrying mutations in one genome. Eggs were expressed from the F1 females and fertilized with UV-inactivated sperm to produce haploid embryos. At 36 hours postfertilization (hpf), the embryos were subjected to immunohistochemistry to assess pH3 expression (Fig 2). Nineteen mutants were identified in the screen. One of these mutants, crash&burn (crb), exhibited a four-fold increase in mitotic cells and carried a mutation in the transcription factor b-myb.25 Although the mutant embryos were embryonic lethal, the heterozygous fish grew to adulthood and after N-methyl-N-nitro-N-nitrosoguanidine exposure developed tumors at two-fold the frequency of treated wt siblings. Other mutants emerging from this screen currently are being analyzed; several genes known to participate in carcinogenesis were also identified by the screen, highlighting the validity of this screening approach.
In a parallel endeavor to identify mutations that predispose organisms to genomic instability and resulting loss of heterozygosity, Moore et al26,27 modified the classical mosaic-eye mutation frequency assay performed on zebrafish carrying the golden mutation.28 Homozygous golden fish display a light-colored eye phenotype, whereas heterozygotes have the wt dark eye; heterozygote fish show mosaic reversion to light eye pigmentation and can be used to estimate the spontaneous frequency of somatic mutation as a measure of genomic stability. Fish heterozygous for the golden mutation were treated with ENU, and in the subsequent generation 12 mutants with increased genomic instability were identified. One mutant, gin-10, demonstrated a 10-fold increased rate of spontaneous tumor formation compared with wt fish by 30 to 34 months. The tumors were found in a wide variety of tissues, which supports the hypothesis that the mutated gene gin10 increases general tumor susceptibility by inducing genome instability. This also supports the notion that the Knudson's two-hit model of carcinogenesis would be applicable in zebrafish.29 These screening approaches document the link of abnormal developmental phenotypes with increased cancer incidence in adult fish. Amsterdam et al23 conducted a tumorigenesis screen of zebrafish lines produced initially for an embryonic development screen created by disrupting the genome with viral inserts. It was noted that 12 zebrafish lines generated in the original screen exhibited increased tumor incidence compared with wt fish, which led to a doubling of the mortality rate during a 2-year period.30 Most of these tumors were found to be malignant peripheral-nerve sheath tumors; other tumors identified in these lines included lymphomas, sarcomas, and gut, pancreatic, and renal tumors. Eleven mutants carried mutations in ribosomal proteins, whereas one was found to have a mutation in the neurofibromatosis type 2 gene, a well characterized tumor suppressor gene. The involvement of ribosomal proteins in carcinogenesis has not been described extensively in mammals. However, it seems that mutation of RPS19 in patients with Diamond-Blackfan anemia leads to misregulation of oncogenes and tumor suppressor genes, and might be associated with leukemogenesis in these patients.31,32 These findings suggest that the genes encoding ribosomal proteins that were identified in zebrafish might also produce human disease phenotypes when mutated.
Although targeted knockout of specific genes is not yet available in zebrafish, recent advances in high-throughput sequencing technology enable the direct identification of gene mutations after ENU mutagenesis. The methodology has been termed targeted induced local lesions in genomes.33,34 In brief, random chemical mutagenesis is achieved by ENU exposure of adult zebrafish, followed by screening for mutations in a specific target gene by high-throughput genome sequencing analysis. With this approach, it is possible to create an allelic series of mutations in a gene of interest to characterize functionally important domains as well as facilitate analysis of gene function in situations in which the null allele is lethal to the embryo. Among the first genes targeted in this fashion were classical tumor suppressor genes.
P53 is a well characterized tumor suppressor gene and the most frequently mutated gene in all human cancers.35,36 Germline mutations in P53 lead to Li-Fraumeni syndrome with an increased risk for the development of sarcomas, leukemia, and breast cancer. Zebrafish embryos that are homozygous for an inactivating mutation, p53M214K, exhibited a suppressed apoptotic response after The adenomatous polyposis coli (APC) gene encodes for a central regulator of the wnt signaling pathway and is the causative genetic mutation in familial adenomatous polyposis (FAP) coli syndrome. Patients with APC mutations develop thousands of colonic polyps and eventually colon cancer.38 In addition, some patients also develop desmoid tumors or congenital hypertrophy of retinal pigment epithelium,39,40 and children with APC mutations may develop hepatoblastomas.41,42 A murine model of FAP, the APCmin mouse, develops multiple small bowel adenomas,43 thereby not completely recapitulating the human tumor spectrum. Zebrafish carrying a mutation in the conserved mutation cluster region (where most of the APC mutations in patients have been found) were identified recently. The homozygous mutant embryos die as a result of cardiac edema arising from cardiac valve defects.44 Subsequently, defects in the intestinal development of these fish were also reported.45-47 We have observed effects on liver specification and growth in the heterozygous and homozygous mutant embryos (Goessling et al, unpublished results). The heterozygous mutant fish developed spontaneous hepatic (18%) and intestinal (12%) adenomas at 14 months.48 The intestinal tumors resembled adenomatous polyps in their cellular architecture, whereas the hepatic lesions demonstrated increased glycogen accumulation, condensed chromatin, nucleolar prominence, and increased apoptotic bodies—features that are found in hepatoblastomas. Therefore, the spontaneously occurring lesions in APC mutant zebrafish more closely resembled the spectrum of tumors found in FAP patients than in the APCmin mouse. When APC-heterozygous fish were exposed to dimethyl benzanthracene, the tumor incidence compared with wt siblings increased dramatically, had an earlier onset of tumor development, and involved a larger organ spectrum: at 6 months, 44% of heterozygous mutant APC fish developed liver tumors, compared with 2% in wt fish; 35% had intestinal tumors (10% in wt), and 35% and 15% of fish develop pancreatic (2% in wt) and biliary tumors (4% in wt), respectively. Both the APC and p53 tumor suppressor mutants exhibited early embryonic phenotypes that make them amenable to use in chemical and genetic screens. The p53-mutant embryos showed an abnormal response to DNA damage, whereas the APC mutants had abnormal cardiac, intestinal, and liver development. Several laboratories are now undertaking forward genetic screens using the p53-mutant zebrafish, and the impaired response to DNA damage to identify mutants that may have potentially additional increased or decreased tumor susceptibility. In addition, a chemical genetic screen using the APC-mutant embryos to identify modifiers of normal and aberrant liver development is currently underway in the Zon laboratory.
Zebrafish can also be used to model human malignancies by targeting expression of a known oncogene to specific organs of interest. This technique has been used extensively in mice, where recent technological advances have allowed the induction of oncogenes at various time points through the use of cre/lox technology as well as switch-on–switch-off strategies controlled by tetracycline or estrogen exposure. The first transgenic tumor model in the zebrafish was the creation of a myc-induced T-cell leukemia.49 Here, the rag2 promoter was used to target m-myc to lymphoid cells. Zebrafish embryos were injected with the oncogene at the one-cell stage, and 6% of fish developed T-cell leukemias, some as early as 30 days postinjection. The tumors arose in the thymus, spread locally to gills and the retro-orbital area, and then disseminated into abdominal organs and muscle (Fig 3). The cells could also be transplanted into irradiated recipient fish and caused new tumors to develop. The leukemia led to early death of the fish before they could mate and propagate, making it challenging to maintain the fish line for subsequent studies. Conditional expression of the oncogene using cre/lox technology enabled the creation of stable lines where 100% of fish developed T-cell leukemia, leading to a more detailed molecular analysis of the leukemia.50 The zebrafish T-cell acute lymphoblastic leukemia expressed scl and lmo2; such expression is also found in a subset of patients with the disease51 and is indicative of an immature or blast-like lymphoid leukemia.52 In another study, using the same oncogene under the control of the myoD promoter, myc overexpression resulted in pancreatic neuroendocrine tumors between 4 and 6 months of age.53 Recently, a zebrafish model for melanoma was developed in the Zon laboratory. Here, the activated BRAFV600E oncogene, encoding a mutated tyrosine kinase, was targeted to melanocytes using the melanocyte-specific mitf promoter.54 When injected into wt fish, this transgene led to an increased formation of nevi. However, when injected into p53-homozygous mutants, 7% of fish developed melanoma by 4 months of age, indicating cooperation between BRAF and p53 pathways in the pathogenesis of these neoplasms (Fig 4). The melanoma was characterized by dense cellularity, high mitotic index, and local invasion. In addition, the melanoma cells could be transplanted into irradiated recipients and caused tumor formation at a high rate. Although BRAF had been identified previously as a melanoma-related oncogene,55,56 the observed cooperation between BRAF and p53 in the acceleration of melanoma formation was newly described in the fish. As evident from the last zebrafish development and genetics meeting in 2006, many more transgenic cancer models are being developed, including those for other leukemias, pancreatic cancer, and rhabdomyosarcoma.
Many vital cancer biology tools are currently under development and will enhance the utility of the zebrafish as a model for carcinogenesis. These include technologies to detect genomic instability and mutations, tumor transplantation, novel cancer imaging approaches, and the use of chemical genetic screens to modify relevant cancer phenotypes. For example, Freeman and Lee57 are developing a validated zebrafish array comparative genomic hybridization platform that already has been used successfully to characterize several tumor models (Fig 5). This technology will provide rapid identification of genomic imbalances in specific zebrafish tumors, and lead to the appreciation of candidate genes involved in tumor growth, progression, and metastasis.
Tumor transplantation has been a tool in cancer research for more than 100 years.58 Several of the zebrafish cancer studies mentioned employed tumor transplantation assays using irradiated fish. More recently, Mizgireuv and Revskoy59 developed clonal zebrafish lines, induced carcinomas by exposure to nitrosodiethylamine, and demonstrated the feasibility of tumor transplants in nonirradiated syngeneic fish. Lee et al60 transplanted human melanoma cells into zebrafish embryos to evaluate cancer cell migration and mitosis. This type of xenograft study allows the assessment of the effects of environmental cues on cancer cell behavior as well as assessment of cell autonomy. Tumor detection of small lesions has been challenging in adult zebrafish. Most studies to date have used unbiased histologic analysis of large cohorts of potential cancer-bound fish to determine tumor incidence. The introduction of organ-specific fluorescent reporter zebrafish lines may help in the gross assessment of enlarging organ size. In addition, several groups are working on the identification of zebrafish mutants that lack pigmentation, thereby allowing direct visualization of internal organs and tumorigenesis in the adult. We have recently adapted high-frequency ultrasound to detect abdominal tumors in adult zebrafish with high accuracy.60a Using an ultrasound head with a frequency of 55 MHz, penetration was adequate to image the entire thickness of the fish, whereas resolution of approximately 30 nm enabled the assessment of internal organs and detection of tumors 2 mm or larger in size. This methodology will allow for in vivo assessment of tumor progression and ultimately cancer therapy. Although the initial transgenic models of cancer in zebrafish have established the feasibility of modeling disease, we still await the application of the unique strengths of this system to perform forward genetic screens. For this, modifier screens can be performed in the cancer-prone fish to identify genetic interactions that can prevent or alter the development of a particular cancer phenotype.61 Mutant male zebrafish would be subjected to ENU mutagenesis and bred to females also carrying the original mutant gene of interest; the progeny would then be examined for the presence of a modified phenotype. If the secondary mutation is a suppressor, cancer would be delayed in onset, decreased in incidence, or completely absent. If the mutation were an enhancer, the phenotypic defect would have an earlier onset or greater severity. Modifying mutations might help to identify new genetic pathways that act in concert with the original gene on which the screen was based, thereby demonstrating a new potential target for chemical intervention. These screens are especially feasible when the tumor phenotype occurs early. The zebrafish also is uniquely suited to be used in chemical genetic screens because a high number of embryos can be generated, arrayed, and exposed to small volumes of a large number of chemical compounds in a relatively short timeframe.62 The observed connection between abnormal embryonic phenotypes and increased cancer susceptibility in adulthood allows one to perform a chemical screen during embryogenesis to identify potential compounds and target pathways that could influence carcinogenesis. The DIVERSet E library of 16,320 compounds was screened in the Zon laboratory to identify chemicals that could bypass the cell-cycle defect and reverse the phenotype of the b-myb mutant crash&burn.63 One chemical, persynthamide, was identified that inhibited the increased phospho H3 staining indicative of high mitotic activity. Although the effect of persynthamide on the increased cancer incidence in the b-myb mutants awaits additional evaluation, the feasibility of performing chemical screens to reverse embryonic phenotypes was demonstrated clearly. Embryonic chemical genetic screens can also help to identify pathways that regulate major functions in cell growth and carcinogenesis. We recently identified the prostaglandin pathway as a regulator of vertebrate hematopoietic stem-cell growth through this methodology. These findings may have an impact not only on our understanding of leukemogenesis but also on recovery after bone marrow transplantation.64 Chan et al65 used chemical inhibition to investigate potential genetic interactions involved in the regulation of angiogenesis. In this study, a chemical inhibitor to vascular endothelial growth factor was used to ablate vessel growth in zebrafish embryos. Injection of Akt mRNA rescued the phenotype, documenting the central importance of this pathway in angiogenesis. This approach could be used to identify novel genes and pathways that are involved in the formation of new blood vessels, a central process during tumor progression. One can envision the potential utility of compounds identified in embryonic chemical screens of fish that are cancer prone either due to mutation or transgene expression: these chemicals might then be tested further to determine whether they can lower tumor incidence or lead to tumor regression in adults. Berghmans et al66 recently outlined plans for such an approach using transgenic myc fish that are susceptible to leukemia development. The zebrafish is a valuable resource for such studies because cancer histology is correlated well to human disease, yet large-scale unbiased chemical screens, even in adults, are still feasible. The varied approaches documented here illustrate the enormous potential for the zebrafish to add significant resources to the inventory of cell lines, and invertebrate and mammalian models in cancer research. Whether with chemical carcinogenesis, forward genetic screens, or targeted deletions in tumor suppressor genes, or by creating transgenic animals, zebrafish can develop tumors that highly resemble human disease. At present, zebrafish cancer biology is on the verge of having a major and uniquely innovative impact on our understanding of carcinogenesis and the development of methodologies to inhibit disease progression. Much effort should be made to push forward on the production of in vivo targeted screens in the zebrafish that are not feasible in the mouse because of early in utero embryonic lethality or high cost. For example, performing genetic screens to identify enhancers or suppressors of known cancer phenotypes should reveal novel genetic pathways for tumor regulation. Furthermore, the feasibility for large-scale chemical screens in the zebrafish may accelerate the discovery of relevant pathways, molecular targets, and effective chemicals for cancer treatment. All of these approaches are promising to bridge the gap from fish tank to bedside more quickly.
The author(s) indicated no potential conflicts of interest.
Conception/design: Wolfram Goessling, Trista E. North, Leonard I. Zon Manuscript writing: Wolfram Goessling, Trista North, Leonard I. Zon Final approval: Wolfram Goessling, Trista E. North, Leonard I. Zon
Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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TOP
INTRODUCTION
CHEMICAL CARCINOGENESIS
-irradiation.
, bottom). Reprinted with permission.
