Poster Presentation Advances in Neuroblastoma Research Congress 2016

Identification of CASZ1 nuclear export signal (NES) reveals potential mechanism for loss of CASZ1 tumor suppressor activity in neuroblastoma (NB) (#346)

Zhihui Liu 1 , Norris Lam 2 , Evelyn Wang 2 , Ryan A. Virden 2 , Bruce Pawel 3 , Edward F. Attiyeh 3 , John M. Maris 3 , Carol J. Thiele 2
  1. National Cancer Institute, Bethesda, MARYLAND, United States
  2. National Cancer Institute, Bethesda, MD, USA
  3. Children's Hospital of Philadelphia , University of Pennsylvania, Philadelphia, PA, USA

Although a number of chromosome 1p36 loss of heterozygosity (LOH) tumor suppressor genes have been identified in NB, the classic mechanism of mutational inactivation is rarely found to play a role in the loss of function of their remaining allele. How these are inactivated is important for understanding NB pathogenesis. Here we have identified cytosolic sequestration as a novel mechanism for loss of function for the 1p36 NB tumor suppressor CASZ1b in a subset of poor prognosis NB tumors. We performed an analysis of CASZ1b protein expression in a tissue array of 112 primary NB tumors. The results show that low nuclear CASZ1 expression and cytoplasmic-restricted CASZ1 expression is associated with MYCN amplification, unfavorable Shimada histology and high risk (all p<0.05). Remarkably, for the samples with CASZ1 restricted to the cytoplasm, 90% showed unfavorable Shimada histology. To understand potential mechanisms that contribute to CASZ1 cytosolic expression, we performed a structure and function study by generating a series of CASZ1b mutant constructs and assessing their localization in 293T cells or SK-N-AS NB cells. We identified CASZ1b N-terminus AA23-40 as a nuclear localization signal that mediates both nuclear localization and nucleosome remodeling and histone deacetylase (NuRD) complex interaction. Mutations of CASZ1b that result in cytosolic localization or lose its ability to bind NuRD complex abrogate transcriptional activity compared to wild-type CASZ1b (p<0.01). In silico bioinformatic analysis of CASZ1b identified a nuclear export signal (NES) at AA176-192. Structure and function mutational analyses showed that the predicted NES contributes to CASZ1 nuclear-cytoplasmic shuttling in an exportin 1 (XPO1/CRM1)-dependent manner. The finding that treatment of different types of cancer with exportin inhibitors such as KPT-330 results in nuclear accumulation of tumor suppressor genes and growth inhibition, suggests a therapeutic mechanism to restore CASZ1 tumor suppressor properties to a subset of poor prognosis NB tumors that have high cytosolic localization of CASZ1.