The oncoproteins gelsolin, periostin and thrombospondin are enriched in Merkel cell carcinoma exosomes, and their promoter activity is stimulated by Large T-antigen of Merkel cell polyomavirus

Abstract

Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine form of skin cancer with high mortality rate and increasing incidence. Around 80 % of MCC tumours are positive for Merkel cell polyomavirus (MCPyV), the only polyomavirus known to induce cancer in humans. Continuous expression of the viral oncogenes LT-ag and st-ag is necessary for MCPyV positive MCC tumour cell survival, suggesting that it could be a causative agent in MCC. Exosomes are 30-150 nm vesicles of endocytic origin that play a crucial role in cell-cell communication both under normal and pathophysiological conditions like cancer, through transfer of various nucleic acid species, proteins, lipids or viral particles. The role of exosomes in MCC remains elusive. Previous proteomic studies from our group have demonstrated that MCC-derived exosomes contain the proteins gelsolin, periostin and thrombospondin, all of which have tumorigenic properties. It is not known whether the MCPyV proteins LT-ag or/and st-ag affect the expression of these proteins and their exosomal concentrations. By using western blot and RT-PCR we confirmed that gelsolin, periostin and thrombospondin were present and possibly enriched in exosomes derived from four MCC cell lines. The effect of LT-ag and st-ag on gelsolin, periostin and thrombospondin promoter activity was also studied using transient transfection studies with luciferase reporter constructs, and it was found that LT-ag but not st-ag induces promoter activity of the genes encoding these proteins. Furthermore, the effect of the three proteins on cell proliferation was assessed using MTT assay. However, the addition of increasing concentrations of recombinant proteins to MCC cell cultures did not give conclusive results. In conclusion, the results suggest that MCPyV LT-ag may contribute to MCC by enhancing the expression of the oncoproteins gelsolin, periostin and thrombospondin. Furthermore, the proteins seem to be enriched in MCC exosomes and therefore may play a role in exosome-mediated processes in carcinogenesis.