Abstract 2499: The significance of Wip1 in neuroblastoma and medulloblastoma

Abstract

Abstract Background: The most common cytogenetic lesions in the embryonal neural tumors medulloblastoma (MB) and neuroblastoma (NB) affect chromosome 17, with 17q+ or isochromosome 17q, in approximately one-third of MB with these aberrations being a significant indicator of poor clinical outcome. Similarly, in 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 (PNAS 2010) with 17q+ in 46% and whole 17 gain in 39%. In both MB and NB gain of wild-type p53-induced phosphatase 1 (Wip1) at 17q23 is frequently detected. Wip1 is a serine/threonine phosphatase of the type 2C protein phosphatase family (PP2C). The type 2C phosphatases are linked to the regulation of cellular stress responses. Methods: Comparative genomic hybridization (CGH) with the Affymetrix250k SNP array was used to analyze primary NB and MB and a large set of cell lines. Cell lines from MB, NB and breast cancers were examined for Wip1 expression using western blotting, immunohistochemistry and Real-Time quantitative PCR (Q-PCR). Stable transfections of Wip1 silenced cell lines were generated by shRNA specific for Wip1 and puromycin selection. The phosphorylation of histone H2AX and clonogenicity of Wip1 knockdown were examined in cell lines using flow cytometry and clonogenic assay, respectively. Results: Detailed CGH analysis revealed that Wip1 is present in at least one extra genomic copy in all 54 tumor samples containing 17q gain. Results from our large set of MB and NB cell lines demonstrate 17q aberrations with more complex patterns in MB corroborating previous results in clinical tumors. 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 substantial decrease in cell viability in MEB-MED8A, D283 MED, D324 MED and SK-N-BE(2) cell lines. MED-MED8A and SK-N-BE(2) showed a significant decrease in cell growth and colony formation. The MB cell lines D283 MED and D324 MED but not MEB-MED8A showed a substantial increase of gamma-H2AX expression after Wip1 knockdown compared to the non-silencing transfections. Conclusions: Our study indicates an important oncogenic role of Wip1 in NB and MB. Our results showed that Wip1 is present in at least one extra genomic copy in the majority of primary NB and MB. We identified NB and MB cell lines expressing high, moderate and low levels of Wip1 where Wip1 knocked cell lines exhibited decreased cell growth and clonogenic capacity compared to non-silenced cells. Moreover, knockdown of Wip1 induced phosphorylation of histone H2AX in NB and MB cell lines indicating a significant role of Wip1 in the DNA damage response of these tumors. 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 2499. doi:1538-7445.AM2012-2499