Abstract 3817: The potential oncogenic significance of Wip1 in neuroblastoma.

Abstract

Abstract Background: In neuroblastoma (NB) 17q+ is the most powerful genetic predictor of adverse clinical outcome. We found aberrations of chromosome 17 in 85% in a national study of NB with 17q+ in 46% and whole 17 gain in 39%. Gain of 17q correlated with poor survival in our population-based material. In NB gain of wild-type p53-induced phosphatase 1 (Wip1) at 17q23 is frequently detected. Wip1 is a serine/threonine phosphatase and was described as a gatekeeper in the Mdm2-p53 regulatory loop by promoting Mdm2-mediated proteolysis of p53. Methods: 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. H2AX phosphorylation and clonogenicity of Wip1 knockdown were examined using flow cytometry and clonogenic assay, respectively. In vivo, Wip1 knocked- and non-silenced cells were injected in nude NMRI mice. Tumor formation was followed by daily evaluation by digital caliper. Results: Detailed CGH analysis revealed that Wip1 is present in at least one extra genomic copy in all 63 tumor samples containing 17q gain. Results from our wide range of NB cell lines demonstrate 17q aberrations in all samples. These findings have been confirmed by analyzing the expression pattern of Wip1 using different immunostaining techniques. Moreover, mRNA levels of Wip1 correlate to Wip1 protein expression. Transfection experiments with shRNA against Wip1 showed decreased cell viability, proliferation and colony formation in NB cells. In addition, substantial increase of gamma-H2AX expression after Wip1 knockdown compared to the non-silencing transfection was observed. Tumor xenograft development was significantly delayed showing median tumor development (0.10 mL) to be more than doubled (15 days median, vs. 33 days) after Wip1 downregulation compared to animals injected with cells transfected with the scrambled construct. Conclusions: Wip1 downregulation inhibits tumor development. Our results showed that Wip1 is present in at least one extra genomic copy in the majority of primary NB. We identified NB cell lines expressing high, moderate and low levels of Wip1 where Wip1 knocked cells exhibited decreased cell growth and clonogenic capacity compared to non-silenced cells. Moreover, knockdown of Wip1 induced phosphorylation of histone H2AX, indicating a significant role of Wip1 in the DNA damage response of these tumors. Our data suggest that Wip1 expression have significant oncogenic function in neuroblastoma development. Citation Format: Jelena Milosevic, Malin Wickström, Lotta Elfman, Ninib Baryawno, Baldur Sveinbjörnsson, Tommy Martinsson, John Inge Johnsen, Per Kogner. The potential oncogenic significance of Wip1 in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3817. doi:10.1158/1538-7445.AM2013-3817