• melanoma
• zebrafish
• melanoma model
• pigmentation
• edem1
• golden
• MiniCoopR vectors

Melanoma originates from the malignant transformation of melanocytes, neural crest-derived cells that are located between the keratinocytes at the level of the basal layer of the epidermis. A surgical resection is the most valid approach when melanoma is in stage 0 (in situ), however metastatic melanoma (MM) is characterized by an extremely poor prognosis with a median survival of less than 10 months.
One of the most frequent and important mutations in melanoma affects BRAF protein kinase and involves the substitution of the glutamic acid with a valine at the amino acid residue 600 (BRAFV600E). This mutation causes BRAF to be constitutively active as a monomer, hence able to act independently from the upstream activation mediated by RAS. This leads to the deregulation of MAPK cascade, which is responsible for cell growth and proliferation. In animal models, it was shown that transgenic expression of human BRAFV600E together with the loss of tumour suppressor genes (such as PTEN or p53) is responsible for the formation of highly invasive melanomas.
In recent years, selective inhibitors have been developed for BRAFV600E (BRAFi), which are capable of inhibiting only the activity of the mutated kinase but not that of the WT one. However, in spite of the fact that they are effective in 50% of the patients and increase their life expectancy, BRAFi tend to have various types of limitations, such as heavy side effects and a short duration over time, since insurgence of acquired resistance is invariably observed within few months of treatment.
Studying BRAFV600E human melanoma cell lines treated with the BRAFi vemurafenib, in the lab where I have been working a difference was detected between those cells that become more pigmented (“pigmentable”) and those that don’t (“non-pigmentable”). Compared to the non-pigmentable ones, pigmentable cells show an increase in the number of melanosomes (intracellular organelles that produce melanin) and lower sensitivity to vemurafenib. Both in vitro and in vivo experiments confirmed that pigmentation acts as an adaptive cellular mechanism to reduce vemurafenib efficacy, a process largely mediated by miR-211. In this regard, the use of animal models is fundamental to try to increase knowledge about the negative impact that pigmentation has on sensitivity to vemurafenib and its implication on melanoma, in order to later identify efficient therapeutic strategies. In recent years, zebrafish is increasingly establishing itself as a validated cancer model. In particular, transgenic fish lines have been created that express BRAFV600E and develop melanomas, similar in characteristics to human ones.
The aim of my thesis project is to analyse in vivo the effect of the expression of two genes, edem1 and golden, which are negative and positive modulators of pigmentation respectively, using a melanoma model in zebrafish. For the tissue-specific expression of the genes of interest, I used the miniCoopR system. Specifically, I cloned edem1 and golden inside the miniCoopR Vector, an Expression Vector developed by Ceol and colleagues. In addition, I cloned them in the miniCoopR-I, an inducible variant of the miniCoopR Vector that is based on Tet-On technology and was developed in the lab where I have been working. miniCoopR-I vectors will be crucial to better understand how edem1 and golden affect pigmentation, restricting their expression in the adult zebrafish stage.
The practical activities of my research work have envisaged the amplification of the genes of interest, using traditional techniques, in order to integrate them into the appropriate plasmids, taking advantage of the Multisite Gateway cloning, a system that allows to assemble multiple DNA fragments in a single vector. I will microinject the miniCoopR and miniCoopR-I vectors expressing edem1 and golden in zebrafish embryo of the Tg (mitfa:BRAFV600E);p53−/−;mitfa−/− line at the single-cell stage. Finally, I will measure the increase in edem1 and golden gene expression and evaluate the effects on embryonic pigmentation.