The “cancerized field” concept posits that cells in a given tissue sharing an oncogenic mutation are cancer-prone, yet only discreet clones within the field initiate tumors. Studying the process of cancer initiation has remained challenging because of (i) the rarity of these events, (ii) the difficulty of visiualizing initiating clones in living organisms, and (iii) the transient nature of a newly transformed clone emerging before it expands to form an early tumor. A more complete understanding of the molecular processes that regulate cancer initiation could provide important prognostic information about which precancerous lesions are most prone to becoming cancer and also implicate druggable molecular pathways that, when inhibited, may prevent the cancer from ever starting.

The majority of benign nevi carry oncogenic BRAF^V600E mutations and can be considered a cancerized field of melanocytes, but they only rarely convert to melanoma. In an effort to define events that initiate cancer, we used a melanoma model in the zebrafish in which the human BRAF^V600E oncogene is driven by the melanocyte-specific mitfa promoter. When bred into a p53 mutant background, these fish develop melanoma tumors over the course of many months. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and is specifically reexpressed only in melanoma tumors, making it an ideal candidate for tracking melanoma from initiation onward.

We developed a crestin:EGFP reporter that recapitulates the embryonic neural crest expression pattern of crestin and its expression in melanoma tumors. We show through live imaging of transgenic zebrafish crestin reporters that within a cancerized field (BRAF^V600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, and this establishes that a fate change occurs at melanoma initiation in this model. Early crestin⁺ patches of cells expand and are transplantable in a manner consistent with their possessing tumorigenic activity, and they exhibit a gene expression pattern consistent with the NCP identity readout by the crestin reporter. The crestin element is regulated by NCP transcription factors, including sox10. Forced sox10 overexpression in melanocytes accelerated melanoma formation, whereas CRISPR/Cas9 targeting of sox10 delayed melanoma onset. We show activation of super-enhancers at NCP genes in both zebrafish and human melanomas, identifying an epigenetic mechanism for control of this NCP signature leading to melanoma.

This work using our zebrafish melanoma model and in vivo reporter of NCP identity allows us to see cancer from its birth as a single cell and shows the importance of NCP-state reemergence as a key event in melanoma initiation from a field of cancer-prone melanocytes. Thus, in addition to the typical fixed genetic alterations in oncogenes and tumor supressors that are required for cancer development, the reemergence of progenitor identity may be an additional rate-limiting step in the formation of melanoma. Preventing NCP reemergence in a field of cancer-prone melanocytes may thus prove therapeutically useful, and the association of NCP genes with super-enhancer regulatory elements implicates the associated druggable epigenetic machinery in this process.

The crestin:EGFP transgene is specifically expressed in melanoma in BRAF^V600E/p53 mutant melanoma-prone zebrafish. (Top) A single cell expressing crestin:EGFP expands into a small patch of cells over the course of 2 weeks, capturing the initiation of melanoma formation …