Scientists Watch as Healthy Cells Turn Into Melanoma

Alexander M. Castellino, PhD

February 04, 2016

Harvard researchers have engineered zebrafish that can be used to visually track melanoma as it begins — from its initiation from neural crest progenitor (NCP)–like cells to full-blown   melanoma. The study was published online January 29 in Science.

The researchers believe this work could have significant implications for cancer therapeutics, in that it provides clues for stopping cancer before it even begins.

"We can follow a single cell — when it looks like embryonic neural crest cells — and see it become a melanoma," senior investigator Leonard I. Zon, MD, told Medscape Medical News. "Our   study shows that reactivation of the NCP program is responsible for cells proliferating into melanomas," he added.

Dr Zon is Grousbeck Professor of Pediatric Medicine at Harvard Medical School, investigator at Howard Hughes Medical Institute, and director of the Stem Cell Program, Boston Children's   Hospital. His laboratory created the first animal model of a BRAF-driven melanoma.

Dr Zon explains the science and its implications in a short video, Seeing Cancer at its Very Beginning, in which he states, "From a diagnostic standpoint, the turning on of this set of   genes that represent the neural crest could be a new way to diagnose a mole to be malignant. In this case, we would do surgery." He explained that it is possible to develop a therapy that would   turn off the set of genes, possibly leading to a cure.

In an accompanying commentary, Soufiane Boumahdi, MD, PhD, and Cédric Blanpain, MD, PhD, from the Université Libre de Bruxelles,   in Belgium, write: "[The researchers] report the development of an elegant transgenic reporter system that allows early steps of tumor initiation to be tracked in situ. They find that the   oncogene-expressing melanocytes are reprogrammed into neural crest-like progenitors before progressing into invasive tumors."

"This work from Dr Zon's lab provides information on events responsible for cancer initiation from a zebrafish model that have not been obtained in mice and humans," Meenhard Herlyn, DVM, DSc,   Caspar Wistar Professor in Melanoma Research, Wistar Institute, Philadelphia, Pennsylvania, told Medscape Medical News. Dr Herlyn was not involved in the study and was approached for his   expertise. His laboratory has been studying the biology of melanoma and has made significant contributions to understanding stem cells as they relate to cancer.

Dr Herlyn explained that with its reproductive speed, there is no shortage of progeny in zebrafish compared with mice and humans. "For sheer numbers and sample size, this model is a dream," he   said.

He added a cautionary note from past research ― some work in the 1980s on another fish, Xiphophorus, a genus that includes platyfish and swordtails, yielded results that could not be   replicated in humans.

The Zebrafish Melanoma Story

To visualize how melanoma starts, the Harvard researchers used zebrafish with the oncogenes BRAF V600E and p53 -/- . These fish initially develop nevi or benign   moles, then, after several months, invasive melanomas. These are the same oncogenes that are associated with human melanomas, Dr Zon explained.

There was a question as to why melanoma developed after several months and only in some of the cells that had the mutations.

Clues from earlier work suggested that the sporadic melanomas developed in the zebrafish when the crestin gene is reactivated in adult zebrafish harboring the melanoma.

The crestin gene is only found in the genome of zebrafish. It is turned on only during neural crest development during embrogenesis; 3 days after fertilization, it is undetectable.

The crestin gene is, however, turned on and re-expressed in adult zebrafish with melanoma. This provided a way for the Harvard researchers to take their transgenic zebrafish to another level of   visualization — to engineer the double transgenic zebrafish into triple transgenic zebrafish.

The researchers amplified a region of the DNA (4.5 kb) upstream of the crestin gene and cloned this element upstream of an enhanced green fluorescent protein reporter gene (EGFP). The   upstream DNA elements contain promoters/enhancers and are critical for the transcription of DNA to mRNA. When expressed in cells, the EGFP reporter lights up green — like a beacon — under   fluorescent light.

                                                               

A zebrafish with the triple transgene. Dark melanocytes are seen throughout the field of view. The bright patch shows GFP-expressing cells of an early melanoma. (Courtesy of   Dr Charles Kaufman)

                       

Postdoctoral fellow Charles K. Kaufman, MD, PhD, from Dr Zon's laboratory, explained to Medscape Medical News how zebrafish with stable transgenes are obtained. In this instance, the   researchers were looking for zebrafish with the three stable transgenes, BRAF V600E, p53 -/- , and EGFP.

In an institution press release, Dr Zon said: "What's cool about this group of genes is that they also get turned on in human melanoma. It's a change in cell fate, back to neural crest   status."

Embryos from normal fish were injected with the cloned DNA containing the EGFP gene under a dissecting microscope. When the cloned DNA was stably integrated into the zebrafish genome,   the researchers looked for embryos that had fluorescent neural crest cells during early development. These fish were then bred with BRAF V600E, p53 -/- double   transgenic fish, and these developed into adults that displayed green spots under fluorescence light. These green spots invariably developed into tumors.

Dr Kaufman visualized the growing embryos under the dissecting microscope or in a fish tank wearing fluorescent goggles. Videos were taken on an iPhone placed behind the goggles. The iPhone   recorded the results while fluorescent light was flashed on the fish — exciting, but time consuming.

Using this method, the researchers could track single, isolated EGFP+ cells in the triple transgenic zebrafish and could follow their persistence and enlargement into melanomas.

Notably, the researchers confirmed that crestin-EGFP is undetected from days 3 to >21 days after fertilization. In embryos carrying the triple transgene, the crestin-EGFP is   re-expressed in adult zebrafish that develop overt melanomas.

In their commentary, Dr Boumahdi and Dr Blanpain write: "Crestin-[E]GFP is invariably expressed, prior to the malignant transition, in all lesions that will eventually progress into invasive   tumors; this suggests that crestin-[E]GFP marks the point of no return during tumorigenesis and represents one of the earliest molecular states associated with tumor initiation."

The researchers also looked at enhancer and promoter elements to understand why a cell in a "cancerized field" may turn into a melanoma. They identified elements that serve as binding sites for   Sox10, Pax3, Mitf, and Tfap2 — all of which regulate NCP specification and differentiation.

Explaining Cancer Initiation

Genetic events associated with cancer have been characterized across many malignancies. However, still poorly understood is the molecular basis for the reprogramming of normal cells into   tumors. In a "cancerized field" of cells, why do only sporadic cells with the same genetic alterations complete the conversion to a malignant state?

"This work shows that melanoma precursor cells reinitiate an embryonic neural crest signature and activate a melanoma gene program," the Harvard researchers write in their discussion.

"Our data support a model in which the stem/progenitor cell gene programs are an integral part of cancer initiation and not reacquired later," they add.

Dr Kaufman told Medscape Medical News that this may not be peculiar only to melanomas but may be relevant across many other cancers. An analogous situation, for example, exists for the   initiation of basal cell carcinoma and may be true for bladder cancer and lung cancer, he speculated.

"As the initiation stages of more cancers are analyzed, stem cell/progenitor phenotype reacquisition may be a generally observed phenomenon in most cancers," the researchers write.

Richard M. White, MD, PhD, a former postdoctoral fellow from Dr Zon's laboratory who now heads his own laboratory at the Memorial Sloan Kettering Cancer Center, told Medscape Medical   News: "At the molecular level, this study shines. It uses transgenic and imaging to demonstrate the principle that the embryonic program is required for the initiation of at least some   cancers."

Although the notion that cancer cells revert to an embryonic stage has been around for decades, it has been difficult to get conclusive evidence for its support, Dr White explained.

"Crestin is unique to zebrafish, raising the question of whether there is an equivalent maker of NCP and early melanoma-initiating cells in mice and humans," Dr Bouhahdi and Dr Blanpain write   in their commentary.

"Finding an equivalent gene in humans that is as silent in normal adults as crestin could be difficult but likely worth the effort," Dr White said.

Dr Herlyn indicated that his own laboratory has shown that the Notch gene confers a transformed phenotype to human skin cells, resulting from a dedifferentiation and a reprograming into "stem   cell–like" cells. "Notch activation induces a phenotype closely similar to neural crest progenitor cells," he told Medscape Medical News.

A couple of genes in humans come close to crestin, Dr Zon told Medscape Medical News. For example, DLX2 is on in the embryonic neural crest, is off in normal adults, but is on in   melanoma, he explained. He suggested that a combination of genes might satisfy the criteria.

The study was supported by the National Institutes of Health, the National Institute of Arthritis and Musculoskeletal and Skin Diseases, the Ellison Foundation, the Melanoma Research   Alliance, the V Foundation, and the Howard Hughes Medical Institute. Dr Zon is a founder and stockholder of Fate, Inc, and Scholar Rock.

Science. Published online January 29, 2016. Abstract, Commentary

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