• melanoma
• neural crest cells
• Crestin
• drug screening
• BRAFV600E

Melanoma is one of the most common skin cancers and is the deadliest, due to its ability to form metastases. In our lab, we study melanoma driven by BRAF substitution V600E. This proto-oncogene encodes for a constitutively active kinase that promotes cell growth and division through MEK/ERK signaling pathway.
In the Tg(mitfa:BRAFV600E);p53-/- transgenic line, the most widely used melanoma model in zebrafish, tumors form in adult fish due to the absence of p53 and to the melanocyte lineage-restricted expression of BRAFV600E. Interestingly, in this genetic context, melanoma cells are characterized by a transcriptional reprogramming that leads to the re-activation of many genes in common with neural crest cells (NCCs). These genes, such as crestin, are physiologically expressed in embryonic multipotent NCCs and are downregulated after their differentiation, but they get aberrantly re-expressed in melanoma cells in the adult fish.
Our aim is to use embryos of the Tg(mitfa:BRAFV600E);p53-/- line and to take a decrease in crestin levels as read-out of efficacy of anti-melanoma drugs. In fact, we have preliminarily confirmed that, once injected with crestin:mCherry or crestin:luciferase constructs at 1-cell stage, 30 hours post fertilization (hpf) embryos show lower levels of fluorescent signal or luciferase activity, upon 24h of treatment with drugs (e.g. BRAF inhibitors (BRAFi) and/or pigmentation inhibitors (PIGMi)).
During my thesis, I have contributed to generate transgenic zebrafish lines expressing reporter constructs that allow to monitor the decrease in crestin levels qualitatively and/or quantitatively. Specifically, crestin:mCherry and crestin:luciferase single-reporter constructs, as well as crestin:luciferase-P2A-mCherry dual-reporter construct, express mCherry (as qualitative marker) and/or Luciferase (as quantitative marker), under the control of crestin promoter.
Whereas the crestin:mCherry single-reporter construct was already available, I generated crestin:luciferase;γ-cryst:VenusGFP single-reporter construct, and crestin:luciferase-P2A-mCherry;cryst:VenusGFP dual-reporter construct, taking advantage of the Gateway® Cloning System. In this technology, the recombination among DNA fragments is mediated by specific sequences (Gateway® att sequences) and LR clonase enzyme. In my case, the fragments were: destination vector (γ-cryst:VenusGFP), 5’E plasmid (crestin promoter), middle plasmid (luciferase or luciferase-P2A-mCherry) and 3’E plasmid (polyA).
Once obtained, I injected the above listed constructs in 1-cell stage embryos of the Tg(mitfa:BRAFV600E);p53-/- zebrafish line. The integration of the constructs in the zebrafish genome is mediated by Tol2 transposase system. At 24hpf, I screened embryos of the crestin:mCherry single-reporter line and of the crestin:luciferase-P2A-mCherry dual-reporter line for mCherry fluorescence. Analogously, at 48hpf, I screened embryos of the crestin:luciferase single-reporter line and crestin:luciferase-P2A-mCherry dual-reporter line for GFP expression in the crystallin.
Currently, I am selecting fish at filial generation 3/4 for a well-defined and homogeneous crestin pattern, in order to finally obtain stable reporter lines.
As future perspective, embryos of crestin reporter lines will be used for drug screenings in melanoma. In order to assess the decrease in crestin expression after drug treatments, in the crestin:mCherry line the decrease in mCherry fluorescence can be first evaluated qualitatively by fluorescence microscopy. Then, for a quantitative result, the percentage of mCherry-positive cells can be measured using flow cytometry. In the luciferase lines (single- and dual-reporter crestin lines), luciferase signal can be quantified using a luminometer.
In summary, the crestin lines that I have contributed to generate in zebrafish can be used as an effective read-out in drug screening. Our plan is to select and further use the line that allows to assess, in the most accurate and reproducible way, the efficacy of single pharmacological treatments, as well as of their combinations.