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

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.Malignt melanom är en form av cancer som utvecklas i melanocyter, de celler i huden som bildar pigmentet melanin. Även om malignt melanom endast utgör 5% av alla hudcancrar, är den den mest aggressiva och dödliga. Godartad ansamling av melanocyter bildar en leverfläck eller ett sk nevus. Vid minsta misstanke om melanom, opereras nevuset bort och analyseras i mikroskop. Det uppskattas dock att en korrekt diagnos fastställs endast i 83% av de bortopererade lesionerna. Av de återstående fallen är 8% övertolkade och 9% undertolkade. Detta understryker vikten av att förbättra melanomdiagnostik. Melanocyter i nevi anses i motsats till melanom ha avtagande förmåga att föröka sig efter ett visst antal celldelningar på grund av cellåldrande. Påvisande av cellåldrande-markörer skulle därför kunna förbättra diagnostiken av svårbedömda lesioner. De idag rutinmässigt använda cellåldrande-markörerna saknar dock förmåga att skilja nevi från melanom. Det övergripande syftet med denna avhandling var därför att försöka identifiera nya cellåldrande-markörer för att förbättra melanom-diagnostiken. I studie I etablerade vi en cellmodell med nevus-efterliknande egenskaper bestående av melanocyter som åldras. Med genuttryck-analys studerades cellåldrande-markörer som kunde skilja nevi från melanom. Proteinet tubulin β-3 identifierades som en potentiell diagnostisk markör. Minskning av tubulin β-3 och förbehandling med tubulin-destabiliserande läkemedel i melanocyter visade sig inducera ett tillstånd som liknar cellåldrandet. I studie II undersöktes en signalväg mellan melanomceller och fibroblaster, som kan underlätta melanomcellers förmåga att infiltrera och aktivt förstöra omgivande vävnad. Fibroblaster är en typ av bindvävsceller i läderhuden som tillverkar ämnen som finns mellan celler, s.k. extracellulär matrix. Ultraviolett (UV)-strålning visade sig, både i melanomceller och fibroblaster, främja signalvägen som resulterar i ökat proteinuttryck av FAP-α, ett protein som bidrar till nedbrytning av extracellulär matrix vilket kan underlätta infiltration av melanomceller. En sådan signalväg var inte funktionell i åldrande melanocyter. I studie III visade vi att proteinet tubulin β-3 är en markör som underlättar diagnostik av nevi respektive melanom. Det diagnostiska kriteriet var tubulin β-3 gradienten som kunde ses i nevi men som ej var synligt i melanom. Olika mönster av tubulin β-3 analys i melanom beskrevs, kliniska associationer hittades och värdet av denna markör för att förutsäga sjukdomens förlopp undersöktes. Melanompatienter, vars melanom hade områden med förlust av tubulin β-3, hade fyra gånger högre progressionstakt av sjukdomen jämfört med patienter utan detta mönster. Sammanfattningsvis studerade vi cellåldrande-markörer som kunde skilja nevi från melanom och vi identifierade proteinet tubulin β-3 som en markör med diagnostiskt och förmodligen prognostiskt värde. Vi visade att ultraviolett strålning inte bara främjar melanombildning utan också dess progression genom att öka en signalväg mellan melanomceller och fibroblaster som resulterar i extracellulär matrixnedbrytning

Evaluation of tubulin β-3 as a novel senescence-associated gene in melanocytic malignant transformation.

Malignant melanoma might develop from melanocytic nevi in which the growth-arrested state has been broken. We analyzed the gene expression of young and senescent human melanocytes in culture and compared the gene expression data with a dataset from nevi and melanomas. A concordant altered gene expression was identified in 84 genes when comparing the growth-arrested samples with proliferating samples. TUBB3, which encodes the microtubule protein tubulin β-3, showed a decreased expression in senescent melanocytes and nevi and was selected for further studies. Depletion of tubulin β-3 caused accumulation of cells in the G2/M phase and decreased proliferation and migration. Immunohistochemical assessment of tubulin β-3 in benign lesions revealed strong staining in the superficial part of the intradermal components, which faded with depth. In contrast, primary melanomas exhibited staining without gradient in a disordered pattern and strong staining of the invasive front. Our results describe an approach to find clinically useful diagnostic biomarkers to more precisely identify cutaneous malignant melanoma and present tubulin β-3 as a candidate marker. This article is protected by copyright. All rights reserved.Funding agencies: Swedish Research Council; Welander-Finsen Foundation; Ostgotaregionens Cancer Foundation; Swedish Cancer Society; County Council of Ostergotland; Olle Engkvist Foundation</p

UV radiation promotes melanoma dissemination mediated by the sequential reaction axis of cathepsins-TGF-beta 1-FAP-alpha

Background: Ultraviolet radiation (UVR) is the major risk factor for development of malignant melanoma. Fibroblast activation protein (FAP)-alpha is a serine protease expressed on the surface of activated fibroblasts, promoting tumour invasion through extracellular matrix (ECM) degradation. The signalling mechanism behind the upregulation of FAP-alpha is not yet completely revealed. Methods: Expression of FAP-alpha was analysed after UVR exposure in in vitro co-culture systems, gene expression arrays and artificial skin constructs. Cell migration and invasion was studied in relation to cathepsin activity and secretion of transforming growth factor (TGF)-beta 1. Results: Fibroblast activation protein-a expression was induced by UVR in melanocytes of human skin. The FAP-alpha expression was regulated by UVR-induced release of TGF-beta 1 and cathepsin inhibitors prevented such secretion. In melanoma cell culture models and in a xenograft tumour model of zebrafish embryos, FAP-alpha mediated ECM degradation and facilitated tumour cell dissemination. Conclusions: Our results provide evidence for a sequential reaction axis from UVR via cathepsins, TGF-beta 1 and FAP-alpha expression, promoting cancer cell dissemination and melanoma metastatic spread.Funding Agencies|Swedish Research Council; Welander-Finsen Foundation; Ostgotaregionens Cancer Foundation; Swedish Cancer Society; County Council of Ostergotland; Olle Engkvist Foundation</p