The Zebrafish as a Tool to Cancer Drug Discovery

Review Article

Austin J Pharmacol Ther. 2015; 3(2).1069.

The Zebrafish as a Tool to Cancer Drug Discovery

Huiting LN¹, Laroche FJF¹ and Feng H¹*

¹Departments of Pharmacology and Medicine, Cancer Research Center, Section of Hematology and Medical Oncology, Boston University School of Medicine, Boston, MA, USA

*Corresponding author: : Feng H, Departments of Pharmacology and Medicine, Cancer Research Center, Section of Hematology and Medical Oncology, Boston University School of Medicine, Boston, MA, USA

Received: March 23, 2015; Accepted: April 23, 2015; Published: May 04, 2015


The ability of zebrafish to faithfully recapitulate a variety of human cancers provides a unique in vivo system for drug identification and validation. Zebrafish models of human cancer generated through methodologies such as transgenesis, gene inactivation, transplantation, and carcinogenic induction have proven similar to their human counterparts both molecularly and pathologically. Suppression of cancer-relevant phenotypes provides opportunities to both identify and evaluate efficacious compounds using embryonic and adult zebrafish. After relevant compounds are selected, preclinical evaluation in mammalian models can occur, delivering lead compounds to human trials swiftly and rapidly. The advantages of in vivo imaging, large progeny, and rapid development that the zebrafish provides make it an attractive model to promote novel cancer drug discovery and reduce the hurdles and cost of clinical trials. This review explores the current methodologies to model human cancers in zebrafish, and how these cancer models have aided in formation of novel therapeutic hypotheses.

Keywords: Zebrafish; Cancer; Drug discovery; Small molecule screens; Efficacy; Toxicity


MPNST: Malignant Plural Nerve Sheath Tumor; T-ALL: T-cell acute lymphoblastic leukemia; hpf: Hours Post-Fertilization; DHODH: Dihydroorotate Dehydrogenase; B-ALL: B-cell Acute Lymphoblastic Leukemia; AML: Acute Myeloid Leukemia; MPN: Myeloproliferative Neoplasm

Current Challenges in Drug Discovery

The pharmaceutical industry is experiencing lapses in drug development productivity. The predominant drug discovery methodologies for the past 50 years have been target centric. The drug development process in its entirety, from compound identification through preclinical animal models, takes approximately 10-15 years [1]. A high number of compounds are often filtered out during the preclinical animal testing stage, due to failure to meet standards of Absorption, Distribution, Metabolism, and Excretion (ADME). This is because multiple iterations of in vivo studies are performed on preclinical animal models in later stages of drug development (i.e., prior to compounds being relinquished for human trials). Specifically, more than 70% of compounds in oncology fail in phase II clinical trials, while 59% of the remaining compounds are discarded in phase III due to intolerable toxicities [2]. To increase success rates, it is extremely important to test compounds using inexpensive, whole organism vertebrate models during early stages of drug development. Whole organism testing not only provides information on tissue specificity and toxicity, but also determines compound bioavailability that may not be accurately accounted for in a small number of murine models. The zebrafish has emerged as an ideal complementary model system for drug discovery, as it is capable of high throughput screening for discoveryof novel therapeutic compounds or testing of candidate cancer modulators. Research in the past few years has proven the potential of zebrafish to significantly improve the capacity of predicting clinical efficacy and reduce the time and money lost in pushing ineffective drugs to market [3].

The Advantage of the Zebrafish System in Drug Discovery

Zebrafish have emerged as powerful models for drug discovery and biosafety studies because they develop most of the organs found in mammals including those of the nervous, digestive, reproductive, immune, excretory, and cardiovascular systems [4,5]. Zebrafish have a number of unique advantages positioning them for rapid drug discovery and toxicity testing: (i) zebrafish generate large numbers of progeny, offering high confidence in statistical analysis; (ii) zebrafish can absorb compounds solubilized in water, making drug administration simple and feasible; (iii) zebrafish develop rapidly, allowing for assays of drug toxicities on organ development; (iv) the maintenance cost for zebrafish is less expensive than for mammals, decreasing the cost associated with animal husbandry [6]; (v) zebrafish and human share high molecular and genetic homologies, especially for enzymes and cell surface receptors [7]; (vi) zebrafish embryos are as accessible and proliferative as cell culture systems and thus lend themselves to being as applicable as in vitro systems; and (vii) multiple cancer models have been generated in zebrafish and proven similar to their human counterparts molecularly and pathologically, providing excellent tools for anti-cancer drug discovery through large-scale screens, candidate drug testing, and target identification [8,9]. Taken together, these features indicate that the zebrafish is a simple, cost-effective, and faithful model for both drug discovery and toxicological studies.

Zebrafish Models of Human Cancer

Zebrafish cancer models induced by chemicals

While maintaining zebrafish in laboratory conditions, researchers observed diseases developing in adult fish, including cancer. Later studies clarified that after exposure of certain mutagens, zebrafish spontaneously developed almost any tumor type known from humans with similar morphology and comparable signaling pathways. The most common locations for this spontaneous neoplasia to arise include gut, thyroid, and liver. Lower levels of spontaneous neoplasia occur in blood vessels, brains, and gills. In light of spontaneous tumor acquisition, detailed chemical approaches to induce cancer have been developed [10]. To chemically induce cancer, zebrafish are soaked in water dissolved with carcinogens for varied periods of time. Advantageously, zebrafish can endure treatments at a variety of chemical concentrations and durations. For instance, smaller doses, from 5 mM or less can be applied for up to 24 hours, while doses greater than 20 mM are to be applied for 8 hours or less [11]. The treatment of zebrafish with the mutagen 7,12-dimethylbenz(a) anthracene induces the broadest range of tumors, from epithelial tumors in intestines to mesenchymal tumors in blood vessels and lymphoid malignancies (Table 1) [12]. Treatment with N-nitrosodiethylamine is reported to induce pancreatic and liver carcinomas, while N-nitrosodimethylamine specifically induces liver tumors (Table 1) [13].

Zebrafish cancer models resulted from tumor suppressor inactivation

Reverse genetics aims to discover the function of a gene by characterizing phenotypic changes upon gene inactivation. Targeting, and subsequent inactivation of specific tumor suppressor genes has led to zebrafish cancer models of Malignant Plural Nerve Sheath Tumor (MPNST), ocular, liver, and intestinal cancer [14,15]. These cancer types are modeled through silencing of the p53, ptenb, apc, or nf1tumor suppressor genes respectively (Table 1) [15-19]. Targeted in activation of tumor suppressor genesis done by taking advantage of: site-directed mutagenesis, recombination mediated by Cre or Flp recombinases, Targeted Induced Local Lesions In Genomes (TILLING), Zinc Finger Nucleases (ZFN), Transcription Activator-Like Effector Nucleases (TALENs), or Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated (CRISPR/ Cas) technologies [12].

In forward genetic screens, mutations are introduced to the adult zebrafish’s genome through chemical, viral, or transposon-based mutagenesis. The progeny of these mutagenized adult zebrafish are screened for abnormal phenotypes. Genes that harbor genetic mutations are then identified through gene mapping, sequence analysis, and phenotype validation. Using zebrafish in forward genetic screens provides a powerful approach to identify cancer susceptible or novel modifier genes in a specific oncogenic cascade based on their ability to accelerate or suppress tumor phenotypes [20].It has been found that mutations in genes encoding ribosomal proteins have led to development of MPNST, as well as mutations affecting genomic instability that have also been associatedwith MPNST in zebrafish [21,22]. Shephard et al. have discovered that the separase and bmybloss-of-function mutants are susceptible to liver and testicular cancers respectively, through forward genetic screens (Table 1) [23,24].

Transgenic zebrafish cancer models

Transgenic zebrafish expressing mammalian oncogenes provide a convenient platform for modeling human cancers through the misexpression of wild-type or constitutively active form of oncogenes under a zebrafish tissue-specific promoter. To generate transgenic zebrafish models, exogenous DNA is microinjected into one-cell-stage zebrafish embryos. Traditionally, linear or circular DNA plasmids, or artificial bacterial chromosomes are injected into fertilized zebrafish eggs. A large number of eggs need to be injected and screened to compensate for low germline transmission of the transgene of interest to the F1 generation. As transgenic lines are passed on through generations, repetitive DNA becomes susceptible to methylation, leading to the silencing of transgenes [4,5]. Modifications of these early transgenic techniques have led to the development of transposon- or I-SceI meganuclease-mediated transgenesis approaches that significantly improved germline transmission rates in zebrafish [25,26].With these improved techniques, modeling human cancers in zebrafish through transgenesis becomes much easier. There are multiple types of cancers in zebrafish developed through the use of transgenesis (Table 1) [27-44]. MYC-induced T-cell Acute Lymphoblastic Leukemia (T-ALL) and melanoma models in particular have not only been used extensively to gain mechanistic insights into disease pathogenesis, but also in small molecule screens to successfully identify candidate therapeutics for human cancers (Table 2) [25,29,30,32-37,39,40,45-48].