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Fish Model for Melanoma Fingers Culprit Mutations

Zebrafish have become one of the most versatile models for studying vertebrate biology. Their size, transparency, rapid growth, and numerous offspring make them ideal for investigating diseases of the cardiovascular, nervous, and immune systems. Now skin cancer joins this list. In the Feb. 8 Current Biology, Leonard Zon and his colleagues report that they have developed a zebrafish model for melanoma. “This model, coupled with the genetics of the zebrafish, will enable us to discover mutations that can cause melanomas in humans,” said Zon, a Howard Hughes investigator at Children’s Hospital Boston, who recently was named the Grousbeck professor of pediatrics.

Leonard Zon and colleagues (clockwise from back row, Zon, Caitlin Bourque, Michael Dovey, Ryan Murphey, Richard White, and Craig Ceol) have devised a zebrafish model that will accelerate screening for genetic mutations that cause melanomas. (Photo by Graham Ramsay)

The development of the model comes at a time when the incidence of melanoma is on the increase. Last year in the U.S., an estimated 50,000 people were diagnosed with the skin cancer, and in about 7,000 of those cases, the melanoma proved fatal. These numbers are expected to rise to almost 60,000 and 7,700, respectively, in 2005, according to the American Cancer Society. Currently, however, there are very few known mutations that cause human melanoma.

Turning Up the Risk
Zon’s model is based on a mutation in a gene called BRAF (pronounced bee-raf), which codes for a serine/threonine kinase that activates the MAP kinase pathway. This cascade stimulates cell proliferation. Most, but not all, melanomas are found to have a mutation in BRAF, making the kinase ten times more active. Such activating mutations are also found in moles, or nevi, which epidemiologically are associated with increased risk of melanoma. These correlations suggest that mutations in BRAF may facilitate, or even initiate, melanomas, but this hypothesis had never been tested in an animal model.

To address it, research fellow Elizabeth Patton introduced human BRAF into the zebrafish. In cooperation with David Fisher, HMS professor of pediatrics and director of the melanoma program at the Dana–Farber Cancer Institute, the human gene was placed under the control of a promoter for the gene Mitfa, a melanocyte-specific gene discovered in the Fisher lab. This construction ensured that the human BRAF was only expressed in zebrafish melanocytes. Patton injected embryos with either the normal BRAF gene or one with an activating mutation—a glutamic acid for a valine at position 600 of the protein. Once the fish developed, Patton saw that the mutation resulted in fish that had a lot of large black spots. “When we looked at these under the microscope, we found that they looked like normal melanocytes that had proliferated,” said Zon. In other words, they looked a lot like a human mole, but they were not melanomas (see image).

The data indicated that the BRAF mutation was sufficient to create moles, but not to transform mel-anocytes into tumor cells. “At that point, we thought perhaps we needed another hit, another mutation for the melanoma to develop,” said Zon.

The p53 Connection
In collaboration with Thomas Look, HMS professor of pediatrics at Dana–Farber, Zon had just recently developed zebrafish harboring a germline missense mutation in the p53 gene. Because this mutation, equivalent to a methionine to lysine switch in human p53, appears in a wide variety of human cancers, and because lesions in the p53 pathway have been implicated in melanomas, Patton crossed fish containing germline BRAF mutations with the p53 mutants to see if it might have some impact on fish melanocytes.

BRAF mutation causes moles. Leopard zebrafish embryos injected with the activating BRAF mutation develop into adult fish with numerous large skin moles (asterisks). These fish can be used to screen for additional mutations that cause melanomas. (Image courtesy of Leonard Zon)

Zon, a physician, recalls that he was soon “called to the tankside” to see a fish that had developed a black spot on its tail. That spot first turned white, then over the next ten days or so, developed into a large black lesion. Because fish tails can regenerate, Patton was able to remove the lesion and test it in the pathology lab. She found that it was, indeed, malignant with histology that closely resembled human melanoma—a video of the swimming fish can be seen at the lab website.

In fact, when the researchers transplanted part of the tumor into immunosuppressed fish, the cells rapidly metastasized, spreading to the liver, heart, pancreas, and kidney marrow within three weeks of injection. “This is very typical for human melanomas once they begin to metastasize,” said Zon. In addition, Patton found that the genomes of the fish melanomas were unstable, a fate that also befalls human melanoma cells. Only 11 percent of the cells in zebrafish melanomas appeared to be normal diploid cells, seven percent were polyploid, while 82 percent were aneuploid.

“This model, coupled with the genetics of the zebrafish, will enable us to discover mutations that can cause melanomas in humans.”

The findings show that the loss of p53 activity, coupled with activating mutations in BRAF, turns normal fish melanocytes into melanomas that have properties very similar to those in humans. This may seem unusual, given that p53 mutations are found in fewer than five percent of human melanomas. But on the other hand, the most common melanoma mutations are those that ablate the tumor suppressor p19ARF, which, it turns out, acts in the p53 pathway. This connection seems to validate the BRAF mutant background as a valuable tool to screen for mutations that can transform melanocytes.

To date, family history explains only eight to 12 percent of human melanomas, and these can almost all be attributed to mutations in three genes, p19ARF, p16INK4a, and CDK4. The mutations that cause the vast majority of melanomas are unknown. Zon and colleagues are now starting to use the genetic power of the zebrafish to screen for mutations that might intersect with the BRAF pathway to create melanomas. “This is a fantastic opportunity. In principle, we could do similar screens in mammalian models, such as mice, but it would be a much slower process,” said Fisher.

Zebrafish are only a few centimeters in length, requiring little space. Zon, who has about 150,000 of them at Children’s, also plans to chemically mutate the BRAF/p53 double mutants to look for other mutations that might accelerate or prevent melanomas.

—Tom Fagan

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