Background: Melanoma is one of the most common skin cancers with poor prognosis and survival rate. Oncolytic viruses, with the ability of causing replicative oncolysis and expessing toxic or immunostimulatory genes, are an appealing addition to the melanoma therapy. Tanapoxvirus (TPV) is potentially a great candidate for melanoma therapy, as it is a benign virus that has exhibited tumor regression in human colon cancer xenografted in nude mice. TPV-66R gene encodes thymidine kinase (TK) that is more abundant in cancerous cells, and deletion of viral TK gene has widely been used for increasing the virus tumor-selectivity. Previous studies have shown that the TPV-15L protein functionally mimics human neuregulin (NRG) and that NRG promotes the proliferation of melanoma. We hypothesized that TPV-15L protein would enhance melanoma proliferation, and aimed to generate TPV recombinants with either the 15L gene deleted (TPVΔ15L) or with both the 15L and the 66R gene ablated (TPVΔ15LΔ66R). We sought to determine the oncolytic effectiveness of TPVΔ15L and TPVΔ15LΔ66R in human melanoma.
Materials and Methods: Owl monkey kidney (OMK) cells, human lung fibroblasts (WI38) and human melanoma cell line SK-MEL-3 were used. Construction of the recombinant viruses was done by transfecting the virus-infected cells with the plasmids targeted for gene deletion from the viral genome. Western blot was conducted for determining the protein expression. Melanoma tumors were induced by injecting SK-MEL-3 cells subcutaneously in the nude mice. Treatments consisted of intratumoral injection of viruses when the tumor volumes reached 45 ± 4.5 mm3.
Results: The TPV-15L protein exhibits similar effectiveness as NRG in promoting melanoma growth in vitro. The replication kinetics of TPVΔ15L was similar to that of wtTPV. However, TPVΔ15LΔ66R replicated less efficiently than TPVΔ15L and the parental virus in vitro. TPVΔ15L exhibited more robust tumor reduction in the human melanoma-bearing nude mice than other recombinant TPVs in vivo. Our results indicate that the deletion of the 66R gene, but not 15L gene, adversely affected virus replication, and that deletion of 15L (which elevates melanoma proliferation) enhanced virus oncolytic efficacy. Interestingly, an anti-viral activity, which was identified as Interferon-λ1 (IFN-λ1) was secreted in a significantly higher quantity by the cells infected with TPVΔ15L. We show that IFN-λ exhibits a more pronounced antiproliferative effect in melanoma than IFNα and IFNβ in vitro. Further, we demonstrate that anti-IFN-λ1 exerts an inhibitory effect on melanoma cell apoptosis caused by TPVΔ15L infection, which indicates that TPVΔ15L regresses melanoma growth partially through inducing IFN-λ1 release.
Conclusion: Our results indicate that deletion of TPV-15L gene product which facilitates the growth of human melanoma cells can be an effective strategy to enhance the oncolytic potential of TPV for the treatment of melanoma. In addition, we demonstrate the immuno-modulatory activities associated with TPVΔ15L and suggest further exploration of TPVΔ15L as a melanoma virotherapy.