ZIA BC 010788 (ZIA) | |||
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Title | Regulation of Differentiation of Pediatric Embryonal Tumors- Neuroblastoma | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Thiele, Carol | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $605,348 | Project Dates | 01/01/2006 - 00/00/0000 |
Fiscal Year | 2015 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) Childhood Cancers (100.0%) |
Central Nervous System - Not Including Brain (20.0%) Melanoma (2.0%) Nervous System (80.0%) Neuroblastoma (60.0%) Sarcoma (15.0%) |
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Research Type | |||
Normal Functioning Cancer Initiation: Oncogenes & Tumor Suppressor Genes |
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Abstract | |||
Specific Aim 1. To identify key differentiation genes and characterize their molecular mechanisms of action Part A. Identification, Cloning and Characterization of CASZ1, the human homolog of the drosophila neural fate determination gene castor (dCas). 1. The study of CASZ1 is warranted because it; 1) maps to chromosome 1p36.22 a region lost in almost 98% of NB tumors with 1p36 LOH ; 2) was a highly evolutionarily conserved neural fate determination gene; 3) has not been functionally studied in mammalian model systems; 4) is regulated during NB cell differentiation and 5) is expressed at high levels in primary tumors of NB patients with good overall survival (p=0.0009). 2. EZH2 medicated Epigenetic suppression contributes to CASZ1 loss of function in Neuroblastoma. The classic involvement of a suppressor gene in tumorigenesis requires biallelic gene inactivation. For CHD5 the NB SRD 1p36 tumor suppressor, 1 allele is lost via 1pLOH while the remaining allele is silenced via DNA methylation. The finding that in both 1pLOH and intact1p NB cells, CASZ1 expression is induced after HDACi treatment suggests chromatin modifications play a role in suppressing CASZ1 expression. A bioinformatic analysis of the CASZ1 locus indicated that in Embryonic stem cells (ES) the CASZ1 transcriptional start site (TSS) contains a bivalent chromatin mark- an activation mark characterized by acetylation of histone 3 at lysine 4 (H3K4me3) as well a suppressive mark characterized by methylation of histone 3 at lysine 27(H3K27me3). Bivalent marks in ES cells are typically found at genes poised to be dynamically regulated, such as during develoment. Since the enzymatically active EZH2 component of the polycomb repressor complex (PRC2) mediates the H3K27me3 silencing mark, we performed a series of experiments that directly demonstrated that CASZ1 is a target of PRC2 complex mediated suppression. ChIP-PCR analyses indicated that PRC2 complex proteins and an H3K27me3 suppression mark were enriched over the CASZ1 TSS in NB cells under steady-state conditions. After treatment with an HDACi (Depsipeptide), binding of these proteins to CASZ1 TSS decreased, the CASZ1 TSS lost the chromatin silencing H3K27me3 mark and was enriched in a chromatin activation mark H3K4me3. Pharmacologic and genetic inhibition of EZH2 in NB tumor cells increased CASZ1 expression. Moreover, now we have found that the PRC2 complex proteins suppress not only CASZ1, but repress CLU(clusterin), NGFR, p73, Runx3 and TrkA(NTRK1) expression. This identifies PRC2 as mediating suppression of a number of genes with tumor suppressor activity in NB. A perplexing aspect of the biology of NB tumors has been their genetic heterogeneity and their biologic plasticity. This study identifies EZH2 as a potential regulatory mechanism that may link these two aspects and is therapeutically tractable. Part B. Mechanism of CASZ1 Function 1. Structure Function Study identifies critical domains regulating CASZ1 transcriptional activity. Despite prior studies implicating CASZ1 in neural (Drosophila) and heart (Xenopus) development, the key structural domains mediating its transcriptional programs have not been elucidated in any species. Since the CASZ1b isoform is the most evolutionarily conserved isoform, we utilized site-directed mutagenesis and deletion mapping to identify key domains in CASZ1b required to 1) activate transcription of a tyrosine hydroxylase promoter-luciferase (TH-Luc) reporter construct and 2) regulate endogenous transcription of several CASZ1 neural (TH, TrkA, NGFR) or muscle differentiation-associated (MYO7A) target genes. Mutation of the C2H2 motif-defining cysteine to an alanine in any one of ZF1, 2, 3, or 4 caused a 60-80% loss in CASZ1b transactivation activity. Mutation of ZF5 did not. Next, a series of N-terminal truncations revealed a critical activation domain at AA31-185, which despite retaining nuclear localization completely lost transactivation function. Deletion of other predi |