Identification and clinical evaluation of senescence-associated markers to distinguish melanocytic nevi from melanomas

Sammanfattning: Melanoma is a form of cancer that develops in melanocytes. While it represents only 5% of skin malignancies, it is the most aggressive and lethal. Benign proliferation of these cells form the melanocytic nevi. The definitive diagnosis of melanocytic nevi or melanoma lesions is histopathologic. However, it is estimated that a correct diagnosis is established by means of standard skin biopsy in only 83% of the melanocytic lesions; of the remaining cases 8% and 9% are overinterpreted (false positives) and under-interpreted (false negatives), respectively. This underscores the importance of additional diagnostic tests. Since cellular senescence is considered to be a tumor suppressive mechanism, immuno-histochemistry using senescence markers has been suggested for the evaluation of difficult melanocytic lesions; however, the routinely used senescence markers lack the ability to distinguish nevi from melanoma. The general aim of this thesis is therefore to identify novel senescence markers that may aid in melanoma diagnosis.In study I, we established a cellular model with nevus-mimicking characteristics consisting in primary melanocytes that become senescent. Transcriptomic analysis allowed expanding the set of senescence-associated markers that could distinguish nevi from melanoma and identifying tubulin β-3 as a potential diagnostic marker. Depletion of tubulin β-3 and pretreatment with tubulin destabilizing drugs in melanocytes and melanoma cells induced a senescence-like phenotype in vitro. In particular, reduced migration capacity and induction of cell cycle arrest in G2/M phase of the cell cycle was demonstrated.In study II, a potential inter-cellular signaling pathway between melanoma cells and stromal fibroblasts, that might facilitate melanoma invasion, was investigated. Ultraviolet (UV) radiation was shown, both in melanoma cells and fibroblasts, to promote the release and activation of TGF-β1 and subsequent increase in expression of the serine protease FAP-α, a protein that plays role in extracellular matrix degradation and therefore facilitates the invasion of melanoma cells. Such mechanism was not functional in senescent melanocytes.In study III, it was shown that tubulin β-3 immunostaining aids in the diagnosis of nevi and melanomas. The diagnostic criterium was the tubulin β-3 gradient within the melanocytic nevi that was no longer apparent in melanoma. Different patterns of tubulin β-3 immunostaining in melanoma were described, dermoscopy-immunohistochemistry associations were found, specific dermoscopic features highlighted, and the prognostic value of this tubulin β-3 marker was examined. The progression rate in patients whose melanomas had areas with loss of tubulin β-3 was 4 times higher than in patients without this feature, although statistical significance could not be reached (p=0.06).In conclusion, transcriptomic analysis expanded the set of senescence-associated markers that could distinguish nevi from melanoma and identified tubulin β-3 as novel immunohistochemistry marker shown to have diagnostic and probably prognostic value. From a mechanistical point of view, ultraviolet radiation was shown to promote not only the formation of melanoma but also its progression by increasing a cathepsin-TGF-β1-FAP-α pathway resulting in extracellular matrix degradation.