RESUMO
Activating mutations in the neuroblastoma rat sarcoma viral oncogene homolog (NRAS) gene are common genetic events in malignant melanoma being found in 15-25% of cases. NRAS is thought to activate both mitogen activated protein kinase (MAPK) and PI3K signaling in melanoma cells. We studied the influence of different components on the MAP/extracellular signal-regulated (ERK) kinase (MEK) and PI3K/mammalian target of rapamycin (mTOR)-signaling cascade in NRAS mutant melanoma cells. In general, these cells were more sensitive to MEK inhibition compared with inhibition in the PI3K/mTOR cascade. Combined targeting of MEK and PI3K was superior to MEK and mTOR1,2 inhibition in all NRAS mutant melanoma cell lines tested, suggesting that PI3K signaling is more important for cell survival in NRAS mutant melanoma when MEK is inhibited. However, targeting of PI3K/mTOR1,2 in combination with MEK inhibitors is necessary to effectively abolish growth of NRAS mutant melanoma cells in vitro and regress xenografted NRAS mutant melanoma. Furthermore, we showed that MEK and PI3K/mTOR1,2 inhibition is synergistic. Expression analysis confirms that combined MEK and PI3K/mTOR1,2 inhibition predominantly influences genes in the rat sarcoma (RAS) pathway and growth factor receptor pathways, which signal through MEK/ERK and PI3K/mTOR, respectively. Our results suggest that combined targeting of the MEK/ERK and PI3K/mTOR pathways has antitumor activity and might serve as a therapeutic option in the treatment of NRAS mutant melanoma, for which there are currently no effective therapies.
Assuntos
GTP Fosfo-Hidrolases/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Proteínas de Membrana/genética , Inibidores de Fosfoinositídeo-3 Quinase , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Nus , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mutations in the receptor tyrosine kinase c-KIT (KIT) are frequent oncogenic alterations in melanoma and are predominantly detected in tumors of acral, mucosal, and chronically sun-damaged skin. Research indicates that melanocytes with aberrant KIT signaling can be found in the distant periphery of the primary tumor; However, it is hitherto unknown whether KIT might confer a migratory advantage, thereby enabling genetically abnormal cells to populate a distal area. In this study, we investigated the role of mutant KIT in melanocyte- and melanoma cell migration using KIT mutant lines as well as genetically manipulated murine and primary human melanocytes. Our results revealed that melanocytes, stably transduced with mutant KIT closed a gap inflicted on cell monolayers faster than wild-type controls. Similarly, KIT mutant human melanoma lines were able to populate a larger area in a 3D in vitro skin model compared to KIT wild type and BRAF mutant lines. Genomic profiling revealed that genes associated with increased cell-dispersal of KIT mutant variants were linked to a statistically significant up-regulation of 60 migratory genes (z-score 1.334; p=0.0001). In addition, in vivo experiments harnessing a mouse xenograft model of early melanoma development demonstrated rapid lateral migration of KIT mutant cells compared to respective controls. The specific kinase inhibitors imatinib and nilotinib, could abrogate this migratory advantage in vitro and in vivo. Our work suggests that KIT inhibition might help to target migratory active, KIT mutant melanoma cells, thus representing a potential strategy to reduce spread and local recurrence.
Assuntos
Movimento Celular/genética , Melanócitos/patologia , Melanoma/patologia , Proteínas Proto-Oncogênicas c-kit/genética , Neoplasias Cutâneas/patologia , Animais , Movimento Celular/efeitos dos fármacos , Humanos , Mesilato de Imatinib/farmacologia , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirimidinas/farmacologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Toxic epidermal necrolysis is a rare disease observed as a consequence of adverse reactions to drugs. It results in the widespread apoptosis of epidermal cells and has a high mortality rate. The mechanisms leading to this apoptosis are not yet elucidated. We investigated whether the cytokines present in the blister fluid, which accumulates under necrotic epidermis, originated from T lymphocytes and may play a role in the propagation of keratinocyte apoptosis. Interferon gamma (IFN-gamma), soluble tumor necrosis factor alpha (TNF-alpha), soluble Fas ligand (sFas-L) were present in much higher concentration in the blister fluids of 13 toxic epidermal necrolysis (TEN) patients than in control fluids from burns. The results of RT-PCR studies, however, indicated that only IFN-gamma and to a lesser extent interleukin (IL)-18 were produced by mononuclear cells present in the fluid. That suggests that the other cytokines also present (TNF-alpha, sFas-L, IL-10) rather originated from activated keratinocytes. Fas-L was indeed overexpressed on the membranes of keratinocytes in lesional skin in situ. The Th1 profile of T lymphocyte activation found in the blister fluid of patients with TEN is consistent with a key role for drug-specific cytotoxic T lymphocytes (CTL) as previously reported, the activation of keratinocytes by IFN-gamma making them sensitive to cell-mediated cytolysis. We propose the hypothesis that the production of Fas-L, TNF-alpha, and IL-10 by keratinocytes could be a defense mechanism against CTL rather than a way of propagating apoptosis among epidermal cells.
Assuntos
Citocinas/imunologia , Síndrome de Stevens-Johnson/imunologia , Apoptose , Vesícula/imunologia , Vesícula/metabolismo , Líquidos Corporais/imunologia , Líquidos Corporais/metabolismo , Queimaduras/imunologia , Queimaduras/metabolismo , Citocinas/metabolismo , Proteína Ligante Fas , Humanos , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-18/imunologia , Interleucina-18/metabolismo , Queratinócitos/imunologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Síndrome de Stevens-Johnson/metabolismo , Síndrome de Stevens-Johnson/patologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Receptor fas/imunologia , Receptor fas/metabolismoRESUMO
Psoriasis is a chronic skin disorder that affects 1% to 3% of the general population worldwide. Streptococcal infection, especially streptococcal pharyngitis, has been shown to be a significant trigger of psoriasis in some patients, possibly by sensitizing T cells to keratin epitopes in the skin. Due to the role of the palatine tonsils as an immunological organ that may generate autoreactive T cells, tonsillectomy has been investigated as a treatment for psoriasis. Tonsillectomy originally gained acceptance in Japan as a treatment for palmoplantar pustulosis, a condition that shares features with pustular psoriasis. Subsequently, tonsillectomy has been used for the treatment of plaque psoriasis and guttate psoriasis. Recently, the first randomized, controlled clinical trial of tonsillectomy was performed. Here, we review the available evidence for the benefit of tonsillectomy as a treatment for palmoplantar pustulosis and psoriasis. We also discuss molecular studies aimed at understanding the role of tonsils in skin disease.
Assuntos
Tonsila Palatina/imunologia , Tonsila Palatina/cirurgia , Psoríase/imunologia , Psoríase/cirurgia , Tonsilectomia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tonsila Palatina/microbiologia , Infecções Estreptocócicas/imunologia , Linfócitos T/imunologia , Tonsilite/imunologia , Adulto JovemRESUMO
Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC(-/-) background. XPC(-/-) cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.
Assuntos
Carcinoma de Células Escamosas/genética , Reparo do DNA/genética , Genoma Humano/genética , Heterocromatina/genética , Taxa de Mutação , Neoplasias Cutâneas/genética , Transcrição Gênica , Empacotamento do DNA/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Células Germinativas/metabolismo , Humanos , Proteínas Proto-Oncogênicas/genéticaAssuntos
Dermoscopia , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Dermatopatias Vasculares/congênito , Adulto , Diagnóstico Diferencial , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Masculino , Pele/patologia , Dermatopatias Vasculares/diagnóstico , Dermatopatias Vasculares/patologiaRESUMO
BACKGROUND: Toxic epidermal necrolysis (TEN) is a very rare but extremely severe drug reaction characterized by widespread apoptosis of epidermis with extensive blisters. We previously found drug-specific cytotoxic CD8 T lymphocytes in the blisters of a single patient. OBJECTIVE: To confirm the role of drug specific cytotoxic lymphocytes in a larger series, to test the cytotoxicity on keratinocytes, and to look for cross-reactivity between chemically related drugs. METHODS: The phenotype of lymphocytes present in the blister fluids of 6 patients with TEN was analyzed by flow cytometry. Cytotoxic functions were tested by chromium release assay on a variety of target cells (autologous or MHC class I-matched EBV-transformed lymphocytes, autologous keratinocytes) after nonspecific (CD3 monoclonal antibody) or specific (suspected and potentially cross-reactive drugs) activation. RESULTS: Blister lymphocytes were CD8 + HLA-DR + CLA + CD56 + . In all 6 cases, they were cytotoxic after nonspecific activation. A drug-specific cytotoxicity was observed in 4 cases (3 related to cotrimoxazole and 1 to carbamazepine) toward lymphocytes. Blister cells also killed IFN-gamma-activated autologous keratinocytes in the presence of drug in the 2 patients tested. Blister cells showed a strong immunoreactivity for granzyme B, and cytotoxicity was abolished by EGTA, but not by anti-Fas/CD95, suggesting perforin/granzyme-mediated killing. By using several sulfonamides for testing the specificity of the drug T-cell receptor interaction, we observed cross-reactivity only between 4 structurally closely related medications. CONCLUSION: These results strongly suggest that drug-specific, MHC class I-restricted, perforin/granzyme-mediated cytotoxicity probably has a primary role in TEN.