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Cell migration is an essential element in the immune response on the one hand and in cancer metastasis on the other hand. The architecture of the actin network in lamellipodia determines the elasticity of the leading edge and contributes to the regulation of migration. We have implemented a new method for the analysis of actin network morphology in the lamellipodia of B16F1 mouse melanoma cells. This method is based on fitting multilayer geometrical models to electron microscopy images of lamellipodial actin networks. The chosen model and F-actin concentrations are thereby deterministic parameters. Using this approach, we identified distinct structural features of actin networks in lamellipodia. The mesh size which defines the elasticity of the lamellipodium was determined as 34 and 78 nm for a two-layer network

Ultraviolet radiation (UVR) stimulates melanogenesis in melanocytes, primarily via release of alpha-melanocyte stimulating hormone from keratinocytes. UVR also induced an inflammatory response in the skin in which Interferon-gamma (IFNγ) cytokine plays an important orchestrating role. Here we report that recombinant IFNγ induces a temporal increase of melanogenesis in mouse melanoma cells. IFNγ elevates expression of microphthalmia-associated transcription factor (Mitf), which is the master regulator of melanogenesis by initiating transcription of melanogenic enzymes, tyrosinase (Tyr), tyrosinese-related protein 1 (Tyrp1) and dopachrome tautomerase (Dct). Interestingly, tyrosinase protein, but not mRNA expression, accumulated in response to IFNγ treatment and was consistent with tyrosinase activity. In addition, glycosidase digestion showed that IFNγ induced ER-resistant, fully mature tyrosinase via post-transcriptional mechanisms, rather than increased de novo synthesis or early processing in the ER. Most strikingly, IFNγ mediated alkalization of melanosomes by elevating Oca2 expression, which leads to facilitate melanosome maturation and sequential accumulation of mature tyrosianse. Both Jak1/Jak2 inhibitor Ruxolitinib and knockout of Stat1 mediated by CRISPR-CAS9 blocked the IFNγ-induced Mitf, tyrosinase, Oca2 expressions and melanin biosynthesis. Our data reveals that IFNγ-Jak1/2-Stat1 axis regulates melanogenesis by inducing maturation of melanosomes and accumulation of mature tyrosinase via post-translational mechanisms. CTLA4 is a cell surface receptor on T cells that functions as an immune checkpoint molecule to enforce tolerance to cognate antigens. Anti-CTLA4 immunotherapy is highly effective at reactivating T cell responses against melanoma, which is postulated to be due to targeting CTLA4 on T cells. Here we report that CTLA4 is also highly expressed by most human melanoma cell lines, as well as in normal human melanocytes. Interferon-gamma (IFNγ) signaling activated the expression of the human CTLA4 gene in a melanocyte and melanoma cell-specific manner. Mechanistically, IFNγ activated CTLA4 expression through JAK1/2-dependent phosphorylation of STAT1, which bound a specific gamma-activated sequence (GAS) site on the CTLA4 promoter, thereby licensing CBP/p300-mediated histone acetylation and local chromatin opening. In melanoma cell lines, elevated baseline expression relied upon constitutive activation of the MAPK pathway. Notably, RNA-seq analyses of melanoma specimens obtained from patients who had received anti-CTLA4 immunotherapy (ipilimumab) showed upregulation of an IFNγ -response gene expression signature, including CTLA4 itself, which correlated significantly with durable response. We also show that ectopic expression of Ctla4 in mouse melanoma cells promotes tumor growth in immunocompetent mice. Ctla4-enhanced melanomagenesis is blocked in immunodeficient NSG mice. In addition, ligation of CD86 (one of Ctla4 ligands) in T cells inhibits CD8 T cells proliferation in vitro. Expression of Ctla4 in melanoma cells are resistant to CD8 T cell cytoxicity in vitro. Our data demonstrates and highlights the novel and unrecognized functions of CTLA4 in melanoma cells that aids their survival, immunoevasion and tumorigenic capabilities. Taken together, these findings have potential implications for the conventional and prototypical roles of the IFNγ signaling pathway and CTLA4 in tumor immunosurveillance and tumor immunoevasion. More importantly, our results raise the possibility that CTLA4 targeting on melanoma cells may contribute to the clinical immunobiology of anti-CTLA4 responses. / Biomedical Sciences

β-catenin is a multifunctional protein that plays crucial roles in embryonic development, physiological homeostasis, and a wide variety of human cancers. Previously, we showed that in vivo targeted ablation of β-catenin in melanoma-associated fibroblasts after melanoma formation significantly suppressed tumor growth. However, when the expression of β-catenin was ablated in melanoma-associated fibroblasts before tumor initiation, melanoma development was surprisingly accelerated. How stromal β-catenin deficiency leads to opposite biological effects in melanoma progression is not completely understood. Here, we report that β-catenin is indispensable for the activation of primary human stromal fibroblasts and the mediation of fibroblast-melanoma cell interactions. Using coimmunoprecipitation and proximity ligation assays, we identified Yes-associated protein (YAP) as an important β-catenin-interacting partner in stromal fibroblasts. YAP is highly expressed in the nuclei of cancer-associated fibroblasts (CAFs) in both human and murine melanomas. Mechanistic investigation revealed that YAP nuclear translocation is significantly modulated by Wnt/β-catenin activity in fibroblasts. Blocking Wnt/β-catenin signaling in stromal fibroblasts inhibited YAP nuclear translocation. In the absence of YAP, the ability of stromal fibroblasts to remodel the extracellular matrix (ECM) was inhibited, which is consistent with the phenotype observed in cells with β-catenin deficiency. Further studies showed that the expression of ECM proteins and enzymes required for remodeling the ECM was suppressed in stromal fibroblasts after YAP ablation. Collectively, our data provide a new paradigm in which the β-catenin-YAP signaling axis regulates the activation and tumor-promoting function of stromal fibroblasts.

No / Genome-wide microarray technology has facilitated the systematic discovery of diagnostic biomarkers of cancers and other pathologies. However, meta-analyses of published arrays often uncover significant inconsistencies that hinder advances in clinical practice. Here we present an integrated microarray analysis framework, based on a genome-wide relative significance (GWRS) and genome-wide global significance (GWGS) model. When applied to five microarray datasets on melanoma published between 2000 and 2011, this method revealed a new signature of 200 genes. When these were linked to so-called ‘melanoma driver’ genes involved in MAPK, Ca2+, and WNT signaling pathways we were able to produce a new 12-gene diagnostic biomarker signature for melanoma (i.e., EGFR, FGFR2, FGFR3, IL8, PTPRF, TNC, CXCL13, COL11A1, CHP2, SHC4, PPP2R2C, and WNT4). We have begun to experimentally validate a subset of these genes involved in MAPK signaling at the protein level, including CXCL13, COL11A1, PTPRF and SHC4 and found these to be over-expressed in metastatic and primary melanoma cells in vitro and in situ compared to melanocytes cultured from healthy skin epidermis and normal healthy human skin. While SHC4 has been reported previously to be associated to melanoma, this is the first time CXCL13, COL11A1, and PTPRF have been associated with melanoma on experimental validation. Our computational evaluation indicates that this 12-gene biomarker signature achieves excellent diagnostic power in distinguishing metastatic melanoma from normal skin and benign nevus. Further experimental validation of the role of these 12 genes in a new signaling network may provide new insights into the underlying biological mechanisms driving the progression of melanoma.

The skin has a system that can detect light in a fashion similar to the retina. Although its presence was initially reported almost 20 years ago, only in 2011 functional studies started to be reported. The biological clock of the skin has also been reported in the beginning of the century, but its function and relevance still remain unexplored. Thus, this Ph.D. project was designed to explore the functionality of both systems in melanocytes, and whether the disruption of these systems is associated with the development of melanoma cancer. Using in vitro, in vivo, and bioinformatics approaches, we have shown that: 1) the biological clock of malignant melanocytes is more responsive to visible light, UVA radiation, estradiol, and temperature compared to normal cells; 2) UVA radiation is detected by melanopsin (OPN4) and rhodopsin (OPN2), which triggers a cGMP related cascade that leads to immediate pigment darkening (IPD) in normal and malignant melanocytes; 3) in addition to detecting UVA radiation, OPN4 also senses thermal energy, which activates the biological clock of both normal and malignant melanocytes; 4) regarding the biological clock, we have provided several layers of evidence that proves that in melanoma a chronodisruption scenario is established compared to healthy skin and/or normal pigment cells; 5) in vivo tumor samples display a low amplitude circadian rhythm of clock gene expression and an ultradian oscillatory profile in melanin content; 6) a non-metastatic melanoma leads to a systemic chronodisruption, which we suggest that could favor the metastatic process; 7) in human melanoma, we demonstrated the role of BMAL1 as a prognostic marker and a putative marker of immune therapy success. Taken altogether, these results significantly contributed to the literature as it brought to light new and interesting targets and processes, which will be explored in future projects / A pele possui um sistema que pode detectar luz de forma análoga à retina. Embora a presença deste sistema tenha sido inicialmente descrita quase há 20 anos, apenas no ano de 2011 estudos funcionais começaram a ser relatados. Sabe-se que o relógio biológico da pele também foi identificado no início do século, mas sua função e relevância ainda continuam pouco exploradas. Diante deste cenário, este projeto de doutorado foi desenhado para investigar a funcionalidade de ambos os sistemas em melanócitos e se perturbação dos mesmos estaria associada com o desenvolvimento de melanoma. Através do uso de abordagens in vitro, in vivo e de bioinformática, nós demonstramos que: 1) o relógio biológico de melanócitos malignos é mais responsivo à luz visível, radiação UVA, estradiol e temperatura comparado ao de células normais; 2) a radiação UVA é detectada por melanopsina (OPN4) e rodopsina (OPN2), que ativam uma via de sinalização dependente de GMPc, levando ao processo de pigmentação imediata (IPD) em melanócitos normais e malignos; 3) além de detecção de radiação UVA, a OPN4 também detecta energia térmica que, por sua vez, ativa o relógio biológico de melanócitos normais e malignos; 4) relativo ao relógio biológico, provamos por diferentes abordagens que, no melanoma, um cenário de cronoruputura está estabelecido em comparação a pele saudável e/ou melanócitos; 5) tumores in vivo apresentam um ritmo circadiano de baixa amplitude na expressão dos genes de relógio e um ritmo ultradiano oscilatório no conteúdo de melanina; 6) um melanoma não metastático leva a um quadro sistêmico de cronoruptura, o qual sugerimos favorecer o processo de metástase; 7) em melanoma humano, demonstramos o papel do gene BMAL11 como marcador de prognóstico e um possível indicador de sucesso de imunoterapias. Portanto, este projeto contribuiu de forma significante para a literatura científica uma vez que trouxe à luz novos e interessantes alvos terapêuticos e processos, os quais serão explorados em projetos futuros

Dados recentes do nosso laboratório demonstram que somente a co-transdução, não a tradução individual, com vetores adenovirais portadores de Interferon-beta (IFN?) (citocina imuno modulatória) e p19Arf (parceira funcional da proteína supressora de tumor p53) resulta na morte celular massiva do melanoma murino B16F10. A capacidade desse tratamento combinado de induzir uma resposta imune antitumoral ainda não foi avaliada. Dessa maneira, o objetivo do presente trabalho foi investigar se células B16F0 tratadas por essa combinação são capazes de induzir uma resposta imune antitumoral em um modelo de vacinação profilático de melanoma. Para isso, essas células foram co-transduzidas com os vetores AdPGp19 e AdPGIFN? e 48 horas depois, inoculadas como agente vacinal no flanco esquerdo (sítio da vacina) de camundongos C57BL/6 imunocompetentes. Sete dias após a última vacinação, esses animais foram desafiados com células B16F10 naïve no flanco direito (sítio do desafio). A progressão tumoral do desafio foi significativamente reduzida, mesmo quando o desafio tumoral foi feito 73 dias após da vacinação. Porém, como os animais imunizados desenvolveram tumores no sítio da vacina, condições para o uso dessas células tratadas foram avaliadas, revelando que: o número de células e de aplicações usadas durante a vacinação tem influência no aparecimento desse tumores, e que apenas com o tratamento combinado os camundongos permanecem livres de tumor. A influência do sistema imune para este resultado foi revelada após protocolo de imunussupressão. Em seguida, o papel da p19Arf e do IFN? na proteção antitumoral da combinação foi estudado. In vitro, os efeitos antitumorais da combinação parecem ser mais influentes da reposição de p19Arf do que da expressão de IFN?, mas já in vivo, na presença do sistema imune, foram mais dependentes do IFN?. Com a combinação estes efeitos mostraram-se mais pronunciados, induzindo uma proteção antitumoral e maior sobrevida aos animais vacinados. Estes resultados indicam que a combinação p19Arf e IFN? pode ser aplicada como um agente imunoterápico e sugerem que a associação entre morte celular e imuno estimulação pode beneficiar o tratamento contra o câncer / Previously, we have shown in a mouse melanoma model of in situ gene therapy that co-transduction, but not individual application, with adenovirus vectors expressing the Interferon-beta (IFN?) (immune modulatory cytokine) and p19Arf (functional partner of the p53 tumor suppressor) transgenes results in massive cell death and reduced tumor progression. However, the capability of this combined treatment to stimulate an antitumor immune response has not been evaluated. Therefore, the aim of this work was to investigate, trough a prophylactic vaccine model, if B16F10 cells treated by the p19Arf and IFN? combination could induce such immune response. To do so, these cells were co-transduced by the AdPGp19 e AdPGIFN? adenoviral vectors and 48 hours after, inoculated as a vaccine agent in the left flank (vaccine site) of immune competent C57BL/6 mice. Seven days after the last vaccine, a tumor challenge was done with naïve B16F10 cells in the right flank (challenge site). Tumor progression was markedly reduced, even when challenge was done 73 days after the vaccination. However, since these animals developed tumors where the vaccine was applied, more appropriate conditions for the use of these treated cells were pursued, thus revealing that: the number of cells and inoculations can dictate tumor development, and also, that only with the combined treatment was tumor formation abolished. The influence of the immune system for this result was revelead by performing an immune supression protocol. Next, the roles of p19Arf and of IFN? were studied. In vitro, the antitumor effects were stronger upon the introduction of p19Arf than IFN?, but in vivo, in the presence of the immune system, the effects were more IFN? dependent. In fact, these effects were more pronouced with the combined treatment, inducing protection against tumor formation and progression and increasing survival in the vaccinated animals. Taken together, these results demonstrate the application of cells treated by the p19Arf e IFN? combination as an effective vaccine agent and also indicates that the association between cell death and immune stimulation may benefit the treatment of cancer

INTRODUÇÃO: O melanoma cutâneo constitui cerca de 3% de todos os tumores da pele. Atinge indivíduos jovens com média de idade de aparecimento entre 50 e 58 anos. Em torno de 20% dos doentes apresentarão doença avançada e morrerão antes de completar cinco anos de sobrevida. CASUÍSTICA E MÉTODOS: Neste estudo retrospectivo de 364 casos acompanhados de maio de 1993 a janeiro de 2006 descreveram-se as variáveis: sexo, idade, cor, localização da lesão primária, tipo de crescimento, espessura de Breslow, nível de Clark, presença de ulceração, estadiamento e suas correlações. RESULTADOS: Predominou o sexo feminino (58,8%) resultando em uma proporção de 1,4 mulheres para cada homem. A média das idades dos pacientes foi de 58,9 anos e a mediana de 61,0 anos. Pacientes não-brancos constituíram 13,7% da amostra. Para homens e mulheres o melanoma cutâneo localizou-se, predominantemente no tronco (24,3-38,0%) e pés (21,4-23,9%). O melanoma acrolentiginoso representou 22,3% de toda amostra. Os padrões melanoma expansivo superficial e melanoma nodular (p < 0,001) e lesões no tronco (52,8%) predominaram nos indivíduos brancos. O melanoma acrolentiginoso (64%) e a localização nos pés (68,2%)prevaleceram nos pacientes não-brancos. Observou-se minoria de casos com lesão primária in situ (14,6%- EC 0) e alto percentual de melanoma cutâneo espesso (39,7% > 4,0 mm). Presença de ulceração foi observada em 13,4% para tumores finos (= 1,0 mm). Homens apresentaram lesões mais espessas (p = 0,011) e ulceradas (p < 0,001) em relação às mulheres, assim como idosos em relação à não idosos (p = 0,021 para a espessura e p = 0,015 para ulceração). A sobrevida média para os pacientes com doença localizada foi de 97,8 meses e a taxa de sobrevida específica para melanoma cutâneo foi de 85,1% em três anos. CONCLUSÕES: Esta amostra constituiu-se de pacientes com tumores espessos e ulcerados denotando diagnóstico tardio do melanoma cutâneo e pior prognóstico. Caracterizou-se por apresentar predomínio de mulheres, de pacientes não-brancos, de lesões nas extremidades e de melanoma acrolentiginoso. / INTRODUCTION: Cutaneous melanoma represents around 3% of all skin tumors. Affecting young patients, with mean age between 50 and 58 years old. About 20% of the patients will have advanced disease and will die before five years of survival. CASUISTIC AND METHODS: In this retrospective study of 364 cases recorded from May 1993 to January 2006 at the Melanoma Unit of Santa Casa de São Paulo the following variables were described: sex, age, skin color, tumor site, growth pattern, tumor thickness, Clark level, ulceration, staging and their correlations. RESULTS: Female (58,8%) prevailed resulting in 1,4 women for each man. The mean age of the patients was 58,9 years old and the median, 61,0 years old. Non-white patients were 13,7% of the sample. The anatomic site of cutaneous melanoma on men and women prevailed at trunk (24,3 – 38,0%) and feet (21,4 – 23,9%). Acral entiginous melanoma represented 22,3% of the cohort. Superficial expansive melanoma and nodular melanoma patterns (p<0,001) and trunk lesions (52,8%) predominated on white patients. Acral lentiginous melanoma (64%) and feet anatomic site 68,2%) prevailed on non-white patients. In situ primary lesions were observed in few cases (14,6% - EC 0) and there was high percentage of thick cutaneous melanoma (39,7% > 4,0 mm). In thin tumors (=1,0 mm) were found 13,4% of ulceration. Thickener (p = 0,011) and ulcerated lesions (p < 0,001) were found more in male and in elderly patients (p = 0,021 for thickeness and p = 0,015 for ulceration). The mean survival of patients with local disease was 97,8 months and the three-year survival rate for cutaneous melanoma was 85,1%. CONCLUSIONS: The cohort consisted mostly of thick and ulcerated tumors, which meant late diagnosis and bad prognosis. Also distinguished by considerable prevalence of female, non-white patients, limb lesions and acral lentiginous melanoma.