Abstract 3971: PPM1D/WIP1, a potential target in neuroblastoma

Abstract

Abstract Background: Gain of 17q is the most powerful genetic predictor of adverse clinical outcome in neuroblastoma (NB). 17q+ correlated with poor survival in our population-based material where we found aberrations of chromosome 17 in 85% of primary neuroblastoma tumors. Specifically, gain of WIP1 at 17q23 is frequently detected in NB. WIP1 is a serine/threonine phosphatase encoded by the gene PPM1D, member of the PP2C family known to be regulators of DNA repair pathways. Methods: A formazan-based spectrophotometric method was used to measure cell viability in several NB cell lines treated with different WIP1 inhibitors. Comparative genomic hybridization (CGH) was used to analyze primary NB tumors and cell lines. NB cells were examined for WIP1 expression using immunoblotting, immunohistochemistry and qPCR. Stable WIP1 knockdown NB cells were generated by shRNA transfections. Clonogenicity of WIP1 knockdown were examined by clonogenic assay. In vivo, WIP1 knocked- and non-silenced cells were injected in nude NMRI mice followed by daily evaluation of tumor formation. Results: Detailed CGH analysis revealed that genomic gain of WIP1 is evident with at least one extra copy in all tumor samples showing 17q gain. Results from our NB cell lines demonstrate 17q aberrations in all samples. These findings were corroborated by expression pattern of WIP1 mRNA and different immunostaining techniques. In a wide range of NB cell lines we found that all WIP1 inhibitors had good cytotoxic effect at higher concentrations (50µM) while some showed cytotoxic effects at lower concentrations. Transfection experiments with shRNA against WIP1 showed decreased cell viability, proliferation and colony formation in NB cells. Tumor xenograft development after WIP1 downregulation was significantly delayed with time to tumor development (0.10 mL) more than doubled compared to animals injected with cells transfected with the scrambled construct (15 days median, vs. 33 days). Conclusions: Our data suggest that WIP1 expression have significant oncogenic function in neuroblastoma development. Our results showed that WIP1 is present in at least one extra genomic copy in the majority of primary NB. WIP1 knocked cells exhibited decreased cell growth and clonogenic capacity compared to non-silenced cells. In addition, WIP1 inhibition could be a potential target in treating high-risk neuroblastoma but more studies investigating the effects of WIP1 inhibition in neuroblastoma are needed to evaluate its clinical significance. Citation Format: Jelena Milosevic, Malin Wickström, Diana Treis, Hjalmar Ståhlberg Nordegren, Lotta Elfman, Ninib Baryawno, Susanne Fransson, Baldur Sveinbjörnsson, Tommy Martinsson, John Inge Johnsen, Per Kogner. PPM1D/WIP1, a potential target in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3971. doi:10.1158/1538-7445.AM2014-3971