A giant step backward into discovery.
by Zhenya Senyak
Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field …a single melanocyte reactivates the NCP [neural crest progenitor] state, revealing a fate change at melanoma initiation….
We’re all too familiar with the effects of cancer but we have little understanding of its beginnings.
What is the triggering event at the molecular level that prods the genetic alteration of a cell to convert to a malignant state?
We may have moved a giant step closer to understanding this process through the work done by Dr. Leonard Zon’s lab and his colleagues at Boston Children’s Hospital and Harvard University. By moving backwards from observation of cancer lesions to discovery of genetic mutations and events within the cancer field, they realized somewhere within this niche was the precipitating event. What was it?
Immersed in the deep study of melanoma, Dr. Len Zon’s lab – along with those of his colleagues – knew that a couple of oncogenes – cancer genes – were necessary to produce melanoma, skin cancer. Every melanoma cell showed the presence of those genes. Problem was, the same is true of most skin moles, almost all of which are usually benign Why?
There is some urgency in figuring out what’s going on.
Skin cancer, already the most common cancer, is rapidly increasing in human populations. Each year there are more new cases of skin cancer than the combined incidence of cancers of the breast, prostate, lung and colon. Melanoma, the least common of these skin cancers, is also the most deadly.
Melanoma is a cancer of transformed melanocytes, the pigment-producing cells. Melanoma accounts for only 2% of total skin cancers but the vast majority of skin cancer deaths. This year, it’s estimated over 75,000 people in the United States will be diagnosed with melanoma and 10,000 will die.
Dr. Zon’s lab was hunting a killer whose secret was deeply hidden. His quarry, the genes of his nearly transparent zebrafish swimming in his lab’s tanks.
The zebrafish, a Himalayan minnow that’s a favorite in aquarium fishtanks, has the unenviable job of participating in scientific discoveries. It was the very first verterbrate whose genome was fully sequenced and the resulting similarities to its human counterpart – along with its ease of upkeep and rapid breeding cycle — sealed its fate.
This is the story of research into melanoma. And the ability of this lowly minnow to help provide answers to the origins of skin cancer with implications for other cancers including myeloproliferative neoplasms.
Moles on human skin are common. And almost always insignificant. Every now and then one turns into an aggressive and malignant melanoma. Skin cancer. Why?
More than 10 years ago, Dr. Leonard Zon looked at moles and saw most contained the oncogene BRAF. So do melanomas. Using zebrafish animal models he concluded BRAF mutations, in the presence of mutated or disabled p53 tumor-preventing protein, are sufficient to promote formation of moles and cooperate in the genesis of melanoma.
After intensive investigation and near endless genomic sequencing, finally a breakthrough. Last month, Zon’s group published the results of its four year study of the origins of melanoma in zebrafish, in the lead research article in SCIENCE and reported in the New York Times.
Those cells in his zebrafish that did develop melanoma were found to contain a gene, crestin, that no one expected to find. The crestin gene is expressed in the earliest embryonic stages of life. It’s part of what’s called the neural crest, a stem cell population that plays a role in development of the skin, bones, blood, nerve sheath. Once embryonic cells are differentiated and on their way to their specific destinies, the crestin gene becomes undetectable, usually about 72 hours after fertilization of the zebrafish embryo.
And yet here it was, reexpressed in melanoma tumors in adult zebrafish. In the most studied of stem cell populations, those involved in hematopoiesis, such reversion is unknown. Cells on their way to become red blood cells, for example, never return to precursor states. And yet here was a cell reverted to its neural crest progenitor status at the birth of a melanoma.
To unpack the cycle of a melanocyte reverting to its original progenitor – or near stem cell state- could lead to deeper understanding of the molecular processes that drive development of melanomas from pre-cancerous lesions.
To get there, Dr. Zon’s lab combined newly developed molecular biology tools with old-fashioned handmade trial and error techniques. They constructed a sequence of DNA to link regulatory elements of the gene crestin plus an enhanced green fluorescent protein (EGFP). This construct, injected into a single cell embryo destined to become a transgenic zebrafish expressing the human oncogene, BRAF, and the mutated tumor suppressor protein, p53. The constructed sequence would ultimately permit visual notification of crestin expression in advance of the melanoma tumor’s appearance. Three days after fertilization, just as in wild type crestin expression, the gene was no longer expressed in this transgenic animal.
Succeeding generations bred from these transgenic zebrafish could now signal the onset of melanoma with a green fluorescent light on a fish scale long before the melanoma lesion appeared. As crestin is expressed in tumors arising in these triple transgenic adult zebrafish but nowhere else in the zebrafish, the researchers concluded crestin expression is specific to its melanoma.
“Once we traced it back to it those scales,” said Zon,” we wanted to see if the very beginning lesion truly had the activity to become cancerous. What we did initially was remove the green scale to see if the fish got cancer, Once we removed the first scale or two, it never got cancer. That meant we were removing a tumor causing element.
“To establish that pretumor patches of crestin: EGFP+ cells are tumorigenic … we performed scale auto-transplants on p53/BRAF/ crestin:EGFP zebrafish. We put the scale in a different location to see if the tumor arises in that new location .” The lab removed a single green scale from a transgenic zebrafish under anesthesia and transferred it to a new site on the animal in which a scale had been removed.
This stage of the operation was clearly low-tech. “It was a mix of 1970’s immunology with 2016, molecular biology, “ recalls Zon. The donor scale from the transgenic fish resting anesthetized on a wet sponge, was physically inserted into recipient site and held in place by surrounding scales. The procedure proved successful about 80% of the time. (A few scales got dislodged.) But it did prove the point. Just as in the case of embryonic molecular introduction of the genetic insert, simply incorporating the crestin gene produced melanoma cells in the subject.
“We were able to detect single isolated melanoma cells in p53/BRAF/crestin/:EGFP [Enhanced Green Fluorescent Protein] zebrafish. Beyond that, we could track their persistence and enlargement.”
“We tracked individual small patches of crestin EGFPt tagged cells over time as they progressed into fully formed raised melanoma lesions and found that all melanomas traced in this manner initiated from crestin:EGFP patches of cells (30 of 30). Thus, if a patch is seen in the p53/BRAF background it will become an overt melanoma.” (Kaufman et al,,“A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation,” )
Discovering how the re-introduction of progenitor cells might turbo-charge proliferation in a tumor could open the door to new understanding of disease process and drug targeting in other cancers that share this rapid expansion of cellular mass.
What this means for myeloproliferative research
Consider: This giant step backwards into the etiology of a cancer cell revealed something unsuspected in biology – the reversion of an adult melanocyte to its stem-cell state lighting up a dormant crestin gene.
As reported in The Harvard Gazette, “Zon and [Charles] Kaufman posit a new model for cancer formation, going back to a decades-old concept of “field cancerization.” They propose that normal tissue becomes primed for cancer when oncogenes are activated and tumor suppressor genes are silenced or lost, but that cancer develops only when a cell in the tissue reverts to a more primitive, embryonic state and starts dividing. They believe this model may apply to most if not all cancers, not just melanoma.”
The implications for myeloproliferative neoplasms are enormous. Despite clear identification of genetic mutations capable on their own of generating MPNs, humans negative for all those mutations are still able to become acutely proliferative and develop full blown advanced MPN phenotypes. Thus, there must be in the thick soup of the blood-bone niche a similar, undiscovered process that reawakens the fast-growing immortalized stem or progenitor cell under stresses and signals we do not yet know. The work of Leonard Zon’s group should spur research into the epigenetic effects – inflammation, stress, biochemical signals – that turn a mutated cell into a stable and deadly clone.
And with this piece of the puzzle solved, the fundamental question framed by the zebrafish work emerges: What is the event that causes reversion of the melanocyte and the resultant re-emergence of the crestin gene?
It’s the next giant step backward to root causes, a discovery that could rock the cancer research world.
“We are close,” says Zon. “We are doing screens to find this initiating event and need to do a lot of experiments.”
- BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma. Patton, E.E., et al. Curr. Biol. (2005) [PubMed]
- Ectopic expression of MITF, a gene for Waardenburg syndrome type 2, converts fibroblasts to cells with melanocyte characteristics. Tachibana, M., et al. Nat. Genet. (1996) [Pubmed]
- A zebrafish melanoma model reveals emergence of neural crest identify during melanoma initiation, (Kaufman et al. Science vol.351 Issue 6272)
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