TGF-β is a cytokine thought to function as a tumor promoter in advanced malignancies.,In this setting, TGF-β increases cancer cell proliferation, survival, and migration, and orchestrates complex, pro-tumorigenic changes in the tumor microenvironment.,Here, we find that in melanoma, integrin β1-mediated TGF-β activation may also produce tumor suppression via an altered host response.,In the A375 human melanoma cell nu/nu xenograft model, we demonstrate that cell surface integrin β1-activation increases TGF-β activity, resulting in stromal activation, neo-angiogenesis and, unexpectedly for this nude mouse model, increase in the number of intra-tumoral CD8+ T lymphocytes within the tumor microenvironment.,This is associated with attenuation of tumor growth and long-term survival benefit.,Correspondingly, in human melanomas, TGF-β1 correlates with integrin β1/TGF-β1 activation and the expression of markers for vasculature and stromal activation.,Surprisingly, this integrin β1/TGF-β1 transcriptional footprint also correlates with the expression of markers for tumor-infiltrating lymphocytes, multiple immune checkpoints and regulatory pathways, and, importantly, better long-term survival of patients.,These correlations are unique to melanoma, in that we do not observe similar associations between β1 integrin/TGF-β1 activation and better long-term survival in other human tumor types.,These results suggest that activation of TGF-β1 in melanoma may be associated with the generation of an anti-tumor host response that warrants further study.
Aurora kinase A (AurkA) is over-expressed in melanoma and its inhibition has been observed to limit tumor growth, suggesting a potential role in melanoma treatment.,A human melanoma cell line with the B-RAF (V600E) mutation (A375mel) was exposed to B-RAF inhibitor (GSK2118436), MEK inhibitor (GSK1120212) and AurkA inhibitor (MLN8054) as single agents or in various combinations (BRAF plus AurkA inhibitor, MEK plus AurkA inhibitor or triple combination BRAF plus MEK plus AurkA inhibitor).,Cell proliferation was assessed using xCELLigence technology.,Total protein extracts were examined for p53 and c-Myc protein expression by Western blot analysis.,Drug anti-tumor effects were further assessed using a 3D-human melanoma skin reconstruction model, in which tissues were incubated with serum-free medium containing control, B-RAF plus MEK inhibitor, MEK plus AurkA inhibitor or the triple combination.,AurkA inhibitor plus B-RAF inhibitor, AurkA inhibitor plus MEK inhibitor or triple combination had a markedly greater anti-proliferative effect on A375 (BRAFV600E) melanoma cells than single agents.,In the 3D human skin model, the triple combination had a greater anti-tumor effect at the epidermal/dermal junction than control or either double combination.,However, S-100 and Ki-67 positively stained spindle-shaped cells were detected in the dermal stratum, suggesting the presence of alive and proliferating melanoma cells.,These findings provide new prospects for melanoma research, including combined B-RAF/AurkA inhibition for B-RAF mutated melanomas and MEK/AurkA inhibitor combination for patients without B-RAF mutations.,Moreover, for the first time, we have shown that a B-RAF, MEK and AurkA inhibitor triple drug combination offers increased efficacy against melanoma cell growth and might be considered as a potential treatment strategy for enhancing clinical response in melanoma.,However, although this triple drug combination was more effective at the epidermal/dermal junction, the suggested presence of alive and proliferating melanoma cells in the dermal stratum could result in drug resistance and disease recurrence.,Molecular characterization of these dermal cells may be critical for the development of novel therapeutic strategies.