Rodents are known as common research models, but zebrafish have been gaining ground as an attractive alternative for many reasons. It is easy to generate large numbers of zebrafish quickly, and new genetic tools have enabled scientists to manipulate the zebrafish genome easily. Now, researchers have engineered zebrafish so that certain genes will emit light when they turn on, indicating when cancer begins to develop. That model allows for many different chemicals to be tested and assessed as potential suppressors or promoters of tumor growth. Scientists have also shown that we can learn more about the molecular pathways behind that growth and how to treat tumors from the zebrafish model. New research reported in Science used the model to reveal a novel cancer gene.
Researcher Leonard Zon, MD, and his colleagues at Boston Children's have been developing zebrafish as a cancer research model for many years and wanted to use their tools to study mucosal melanomas. These rare tumors grow in the linings of the mouth, respiratory, GI and genitourinary tracts, and are usually only discovered after they are quite advanced. Patient survival is only about 33 percent, and because these tumors don’t usually carry the genetic mutations linked to skin melanoma, they are poorly understood and difficult to treat.
"Zebrafish offer the ability to test genetic hypotheses quite rapidly," said postdoctoral researcher Julien Ablain, Ph.D. "Genetic sequencing gives you a lot of information, but not a functional understanding of what the genetic alterations do. Our melanoma model allows us to ask these questions in large numbers of fish, and make statistically significant observations."
At UCSF, the lab of Iwei Yeh, MD, Ph.D. was hard at work studying melanoma tumors. They have genetically sequenced many tumor samples, including 43 patients with mucosal melanomas.
A survey of mutations revealed the SPRED1 gene, which has not been implicated in cancer before. The team found that it was not active in 37 percent of mucosal melanomas (and only five percent of skin melanomas). Another gene was also found to be mutated more frequently in mucosal tumors, a known oncogene called KIT.
With this information, the Zon lab could study what these genes were up to in zebrafish. They altered the SPRED1 gene in cells that produced melanin, modeling SPRED1 loss in human epithelium. Because cancer often begins when different genes change - for example, a gene that promotes tumors becomes activated while one suppressing tumors is lost. The researchers developed a way to model the effect in zebrafish, mining and matching mutations in different genes.
The researchers tested all the combinations, introducing human melanoma genes: KIT, BRAF, and NRAS and deleting genes that prevent tumors: tp53, pten, and cdkn2a. When tumors developed in the fish due to those genetic manipulations, they were similar to human mucosal melanomas and grew in places like internal organs that were not skin.
Zebrafish that carried deletions in the SPRED1 gene had more severe melanomas, which were even worse when they also carried KIT mutations. The team followed up on the results in human cell lines. Their work determined that SPRED1 is a tumor suppressor.
SPRED1 inactivation drove melanoma growth, and the opposite was true too. "If you overexpress SPRED1 in a KIT-driven zebrafish melanoma model, you delay melanomas," explained Ablain. "That confirms that SPRED1 acts as a tumor suppressor."
The loss of SPRED1 ramped up a signaling pathway called MAPK, which is known to be linked to skin melanomas. "Before, it was unclear whether the mucosal subtype of melanomas was driven by MAPK activity," said Ablain. "Our data suggest that they are, even though the genetic alterations are different."
The researchers also determined that SPRED1 loss makes tumors resistant to drugs that interfere with KIT. Patients, therefore, may not benefit from KIT inhibitors if they carry mutations in SPRED1. They may be helped by drugs that work downstream of KIT in the MAPK pathway, however. The researchers will be able to follow up on that prediction by assessing past clinical trials. The Zon lab also wants to see if SPRED1 is implicated in the growth of other cancers.
"For targeted therapies, you really need to understand the molecular mechanism. That is the first thing this paper is going to change. This paper establishes the zebrafish as an extremely quick model to study the many genes mutated and regulated in human cancer," added Zon. "In the future, patients could have their specific mutations modeled in zebrafish to guide decisions about their treatment."
Zon is the Director of the Stem Cell Research Program at Boston Children's Hospital and a senior physician with the Dana-Farber/Boston Children's Cancer and Blood Disorders Center. He is featured discussing his research in the video.