Neuroblastoma (NB) is an enigmatic pediatric cancer for which highly successful therapies have been developed for tumors displaying favorable characteristics, while poor prognoses persist for unfavorable tumors. Chromosome 1p36 is frequently lost in the tumors of many patients with unfavorable prognoses and harbors the locus of the zinc finger (ZF) transcription factor CASZ1. Low CASZ1 expression is significantly associated with lower overall patient survival in NB. Functionally, CASZ1 suppresses NB tumor cell growth and regulates a number of key genes important in neural development (e.g. TrkA, p75 and tyrosine hydoxylase [TH]). It has been shown that high expression of TrkA and p75 are both independently associated with good prognoses in NB patients. Despite the important role of CASZ1 in neural development and NB tumor suppression, the key domains that mediate the activation role of CASZ1 have never been elucidated. In order to address this question, we performed a detailed analysis of CASZ1 protein isoform A (CASZ1a), which contains five ZF's. Abrogation of CASZ1a function was determined by loss of the ability to induce endogenous TrkA, p75 and TH mRNA transcription as well as by loss of the ability to activate a TH promoter-luciferase reporter construct. Using site-directed mutagenesis, we individually mutated each of the five ZF's and discovered that loss of any one of ZF1-4 resulted in a 72-98% loss of function compared to wild-type CASZ1a (p < 0.001), without altering protein stability or nuclear localization. Also, deletion of the C-terminal sequence AA728-1166 (a 38% truncation of the protein) did not appreciably alter function. However, a series of N-terminal truncations revealed a novel nuclear localization signal (NLS) at AA23-29, and it was found that CASZ1a depends on this NLS to localize to the nucleus. In addition, a critical activation domain was mapped to AA31-185 of the N-terminus, which also coincides with a newly identified CASZ1a homodimerization domain. Loss of this activation domain resulted in a complete loss of CASZ1a-mediated induction of the target genes TrkA, p75 and TH (p < 0.001), without affecting protein stability or nuclear localization. Therefore, the discovery and analysis of critical domains in this study has linked CASZ1a loss of function to the loss of induction of the positive NB prognostic factors p75 and TrkA. This demonstrates how the loss of CASZ1a may contribute to the pathogenesis and abysmal survivability of high stage neuroblastoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5014.