• adaptive response
• melanoma
• pigmentation
• zebrafish

Melanoma is one of the most aggressive skin malignancies and, while being one of the least frequent forms of cutaneous cancers, it is by far the deadliest. The most common driving mutation in melanoma is BRAFV600E, consisting of an amino acid substitution that makes the kinase constitutively active and thus upregulates the MAPK pathway, increasing the cell's proliferation rate. The introduction of targeted therapy greatly improved patients’ prognosis, but still its efficacy is limited by the inevitable acquisition of resistance to treatment. Thus, there is a significant interest in finding new approaches to elude the development of resistance to targeted therapy.
In the lab where I performed my thesis, melanin is regarded as an interesting target to impair the tolerance of melanoma cells to BRAF inhibitors (BRAFi). In pigmentable melanoma cells, treatment with BRAF inhibitors has a pro-pigmentation outcome. In turn, the increased melanin production has a protective effect and makes them less sensitive to the drug compared to non-pigmentable melanoma cells.
My thesis focuses on pigmentation as a hallmark of tolerance towards targeted therapy in melanotic melanoma and seeks to exploit its inhibition as a novel approach to boosting BRAF inhibitors' efficacy.
The first aim of my thesis was to investigate the genes involved in the modulation of pigmentation by observing the effect of their over-expression or down-regulation in the in vivo model zebrafish (Danio rerio). Taking advantage of a line devoid of all melanocytes, I microinjected one-cell stage embryos with MiniCoopR plasmids containing the mitfa minigene allowing for the rescue of melanocytes and one gene putatively involved in the modulation of pigmentation. At five days post fertilization, I quantified the embryos showing pigmentation rescue and characterized them using qRT-PCR.
The project's second aim was to develop a pipeline to screen pigmentation inhibitors for their ability to hamper vemurafenib-induced pigmentation in melanotic melanoma cells and enhance BRAF-inhibitors efficacy. To do so, I took advantage of both in vitro cell culture models (melanoma cell lines) and the in vivo model. Compounds able to reduce vemurafenib-induced pigmentation after seventy-two hours of co-treatment were further studied for their capacity to increase reactive oxygen species (ROS) production in the same setting. Those pigmentation inhibitors that successfully increased oxidative stress in cancer cells were brought to the next step and used in a proliferation assay in combination with vemurafenib. Finally, the compounds able to unravel the antiproliferative effect of vemurafenib in vitro were screened in zebrafish embryos for a final assessment.
The microinjection of MiniCoopR plasmids allowed us to obtain important information regarding genes involved in the modulation of pigmentation, which will be useful for drug sensitivity studies on adult zebrafish. Furthermore, thanks to the development of a multistep process to screen pigmentation inhibitors, we evaluated several compounds as candidates for an innovative combination treatment of melanoma, consisting in the simultaneous administration of MAPK inhibitors and pigmentation inhibitors.