Abstract 2867: A novel WT1-derived peptide induces cellular senescence and inhibits tumor growth in a human melanoma cell line and xenograft model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Malignant melanoma is the most aggressive and deadly type of skin cancer. The Wilms tumor protein 1 (WT1) is a transcription factor that has been associated with melanoma oncogenesis and proliferation, hence could be a target of tumor control procedures. A lysine-arginine rich peptide (WT1 pTj) based on WT1 C-terminal zinc finger domain was synthesized and tested for anti-proliferative effects on human and murine melanoma cell lines (A2058 and B16F10-Nex2, respectively), including inhibition of cell growth and colony formation, cell cycle arrest and induction of cell senescence. Internalization of WT1 pTj into A2058 cells was established by confocal microscopy, using biotinylated peptide and staining by streptoavidin-FITC. An effect on p53 activity was measured by a luciferase reporter assay. Competitive ELISA assay was performed to verify whether the peptide interferes with p53-WT1 interaction. In vivo, athymic nude mice and C57Bl/6 were inoculated with A2058 and B16F10-Nex2 cells, respectively, and treated for 5 consecutive days with C-amidated WT1 pTj. Here, we show that WT1 pTj decreased, in a dose and time dependent manner, the number of melanoma viable cells. The anti-proliferative activity of WT1 pTj was linked to an irreversible G1 cell cycle arrest and induction of senescence as suggested by positive staining for beta-galactosidase. Treatment with WT1 pTj reduced the total number of soft-agar colonies by A2058 melanoma cells. The peptide entered the cell within 15 minutes and was diffusely distributed throughout the cell, including the nucleus. Moreover, WT1 pTj enhanced p53 activity and competed with WT1 protein for binding to p53. In vivo, WT1 pTj reduced the number of lung nodules in the B16F10-Nex2 syngeneic model of metastatic melanoma and prolonged survival of mice in the A2058 subcutaneous xenograft model. In conclusion, WT1 pTj peptide entered cancer cells and promoted antitumor activity in vitro and in vivo. The data support the hypothesis that WT1 pTj may prevent important interactions between WT1 protein and its partners via a zinc finger motif, inhibiting melanoma development. Therefore, WT1 pTj is a promising agent against WT1-expressing malignancies (e.g. MCF-7 but not SK-BR-3 breast cancer or normal human fibroblasts; and also HL60 acute myeloid leukemia). WT1 pTj may act also as a trojan carrier peptide for potential drug delivery. Supported by FAPESP grant 2010/51423-0 and the Brazilian National Research Council (CNPq) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2867. doi:1538-7445.AM2012-2867