RESUMO
BACKGROUND: The molecular mechanisms underlying melanoma tumor development and progression are still not completely understood. One of the new candidates that emerged from a recent gene expression profiling study is fatty acid-binding protein 7 (FABP7), involved in lipid metabolism, gene regulation, cell growth and differentiation. METHODS: We studied the functional role of FABP7 in human melanoma cell lines and using immunohistochemistry analyzed its expression pattern and clinical role in 11 nevi, 149 primary melanomas and 68 metastases. RESULTS: FABP7 mRNA and protein level is down-regulated following treatment of melanoma cell lines with a PKC activator (PMA) or MEK1 inhibitor (PD98059). Down-regulation of FABP7 using siRNA decreased cell proliferation and invasion but did not affect apoptosis. In clinical specimens, FABP7 was expressed in 91% of nevi, 71% of primary melanomas and 70% of metastases, with a cytoplasmic and/or nuclear localization. FABP7 expression was associated with tumor thickness in superficial spreading melanoma (P = 0.021). In addition, we observed a trend for an association between FABP7 expression and Ki-67 score (P = 0.070) and shorter relapse-free survival (P = 0.069) in this group of patients. CONCLUSION: Our data suggest that FABP7 can be regulated by PKC and the MAPK/ERK1/2 pathway through independent mechanisms in melanoma cell lines. Furthermore, FABP7 is involved in cell proliferation and invasion in vitro, and may be associated with tumor progression in melanoma.
Assuntos
Proteínas de Transporte/fisiologia , Proliferação de Células , Melanoma/patologia , Nevo Pigmentado/patologia , Neoplasias Cutâneas/patologia , Proteínas Supressoras de Tumor/fisiologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinógenos/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Progressão da Doença , Inibidores Enzimáticos/farmacologia , Proteína 7 de Ligação a Ácidos Graxos , Flavonoides/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Melanoma/metabolismo , Melanoma/secundário , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica , Nevo Pigmentado/genética , Nevo Pigmentado/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Taxa de Sobrevida , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in malignant cells while leaving normal cells unharmed, making it a desirable anticancer target. In the present study, metastatic melanoma cell lines were treated with lexatumumab (Human Genome Sciences, Inc.) a high-affinity monoclonal antibody agonistic to TRAIL receptor 2 (DR5). Binding of the antibody to the receptor led to activation of the extrinsic apoptosis pathway in approximately 20% of the treated cells. However, by combining subtoxic concentrations of the protein translation inhibitor anisomycin with lexatumumab, we obtained synergistic effects on cell viability compared with single agent treatment. Even the low doses of anisomycin could inhibit protein synthesis in melanoma cells with up to 30%, which might result in the shift in the levels of the proteins involved in apoptosis. Co-treatment with anisomycin increased activation of caspases and cleavage of the anti-apoptotic protein Livin, leading to formation of truncated p30-Livin α and p28-Livin ß proteins with potential pro-apoptotic functions. Furthermore, ansiomcycin treatment decreased levels of antiapototic XIAP. In summary our results suggest that combinational treatment with anicomycin and lexatumumab represents a novel therapeutic strategy in the treatment of melanoma.
Assuntos
Anisomicina/farmacologia , Apoptose , Melanoma/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
Notoriously resistant malignant melanoma is one of the most increasing forms of cancer worldwide; there is thus a precarious need for new treatment options. The Wee1 kinase is a major regulator of the G(2)/M checkpoint, and halts the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15). Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis. To assess the role of Wee1 in development and progression of malignant melanoma we examined its expression in a panel of paraffin-embedded patient derived tissue of benign nevi and primary- and metastatic melanomas, as well as in agarose-embedded cultured melanocytes. We found that Wee1 expression increased in the direction of malignancy, and showed a strong, positive correlation with known biomarkers involved in cell cycle regulation: Cyclin A (p<0.0001), Ki67 (p<0.0001), Cyclin D3 (p = 0.001), p21(Cip1/WAF1) (p = 0.003), p53 (p = 0.025). Furthermore, high Wee1 expression was associated with thicker primary tumors (p = 0.001), ulceration (p = 0.005) and poor disease-free survival (p = 0.008). Transfections using siWee1 in metastatic melanoma cell lines; WM239(WTp53), WM45.1(MUTp53) and LOX(WTp53), further support our hypothesis of a tumor promoting role of Wee1 in melanomas. Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G(1)/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively. Both latter cell lines displayed DNA damage and induction of apoptosis, in the absence of Wee1, indicating that the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells. Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas, and indicate a potential role for targeted therapy, alone or in combination with other agents.