Chan-k Woo

Brain-Derived Neurotrophic Factor Activation of TrkB Induces Vascular Endothelial Growth Factor Expression via Hypoxia-Inducible Factor-1α in Neuroblastoma Cells

The extent of angiogenesis and/or vascular endothelial growth factor (VEGF) expression in neuroblastoma tumors correlates with metastases, N-myc amplification, and poor clinical outcome. Recently, we have shown that insulin-like growth factor-I and serum-derived growth factors stimulate VEGF expression in neuroblastoma cells via induction of hypoxia-inducible factor-1alpha (HIF-1alpha). Because another marker of poor prognosis in neuroblastoma tumors is high expression of brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor, TrkB, we sought to evaluate the involvement of BDNF and TrkB in the regulation of VEGF expression. VEGF mRNA levels in neuroblastoma cells cultured in serum-free media increased after 8 to 16 hours in BDNF. BDNF induced increases in VEGF and HIF-1alpha protein, whereas HIF-1beta levels were unaffected. BDNF induced a 2- to 4-fold increase in VEGF promoter activity, which could be abrogated if the hypoxia response element in the VEGF promoter was mutated. Transfection of HIF-1alpha small interfering RNA blocked BDNF-stimulated increases in VEGF promoter activity and VEGF protein expression. The BDNF-stimulated increases in HIF-1alpha and VEGF expression required TrkB tyrosine kinase activity and were completely blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) pathways. These data indicate that BDNF plays a role in regulating VEGF levels in neuroblastoma cells and that targeted therapies to BDNF/TrkB, PI3K, mTOR signal transduction pathways, and/or HIF-1alpha have the potential to inhibit VEGF expression and limit neuroblastoma tumor growth.

EZH2 mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3 and NGFR

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with an undifferentiated status and generally poor prognosis, but the basis for these characteristics remains unknown. In this study, we show that upregulation of the Polycomb protein histone methyltransferase EZH2, which limits differentiation in many tissues, is critical to maintain the undifferentiated state and poor prognostic status of NB by epigenetic repression of multiple tumor suppressor genes. We identified this role for EZH2 by examining the regulation of CASZ1, a recently identified NB tumor suppressor gene whose ectopic restoration inhibits NB cell growth and induces differentiation. Reducing EZH2 expression by RNA interference-mediated knockdown or pharmacologic inhibiton with 3-deazaneplanocin A increased CASZ1 expression, inhibited NB cell growth, and induced neurite extension. Similarly, EZH2(-/-) mouse embryonic fibroblasts (MEF) displayed 3-fold higher levels of CASZ1 mRNA compared with EZH2(+/+) MEFs. In cells with increased expression of CASZ1, treatment with histone deacetylase (HDAC) inhibitor decreased expression of EZH2 and the Polycomb Repressor complex component SUZ12. Under steady-state conditions, H3K27me3 and PRC2 components bound to the CASZ1 gene were enriched, but this enrichment was decreased after HDAC inhibitor treatment. We determined that the tumor suppressors CLU, NGFR, and RUNX3 were also directly repressed by EZH2 like CASZ1 in NB cells. Together, our findings establish that aberrant upregulation of EZH2 in NB cells silences several tumor suppressors, which contribute to the genesis and maintenance of the undifferentiated phenotype of NB tumors.

Oncogene MYCN regulates localization of NKT cells to the site of disease in neuroblastoma

Valpha24-invariant natural killer T (NKT) cells are potentially important for antitumor immunity. We and others have previously demonstrated positive associations between NKT cell presence in primary tumors and long-term survival in distinct human cancers. However, the mechanism by which aggressive tumors avoid infiltration with NKT and other T cells remains poorly understood. Here, we report that the v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN), the hallmark of aggressive neuroblastoma, repressed expression of monocyte chemoattractant protein-1/CC chemokine ligand 2 (MCP-1/CCL2), a chemokine required for NKT cell chemoattraction. MYCN knockdown in MYCN-amplified neuroblastoma cell lines restored CCL2 production and NKT cell chemoattraction. Unlike other oncogenes, MYCN repressed chemokine expression in a STAT3-independent manner, requiring an E-box element in the CCL2 promoter to mediate transcriptional repression. MYCN overexpression in neuroblastoma xenografts in NOD/SCID mice severely inhibited their ability to attract human NKT cells, T cells, and monocytes. Patients with MYCN-amplified neuroblastoma metastatic to bone marrow had 4-fold fewer NKT cells in their bone marrow than did their nonamplified counterparts, indicating that the MYCN-mediated immune escape mechanism, which we believe to be novel, is operative in metastatic cancer and should be considered in tumor immunobiology and for the development of new therapeutic strategies.

Use of RNA interference to elucidate the effect of MYCN on cell cycle in neuroblastoma

MYCN amplification marks poor prognosis in neuroblastoma (NB) tumors. In evaluating the mechanisms by which retinoic acid (RA) or nerve growth factor (NGF) decrease cell number in MYCN amplified NB cells, we have identified a number of proteins whose expression either decreases (E2F, CDC2, CDK6, cyclin dependent kinase activity) or increases (p27) in association with a decrease in MYCN expression. However, it was still unclear which were MYCN dependent effects or not.

NGF activation of TrkA decreases N-myc expression via MAPK path leading to a decrease in neuroblastoma cell number

In neuroblastoma (NB), expression of the TrkA receptor is correlated with good prognosis while N-myc amplification is correlated with poor prognosis. Decreased N-myc levels are key to controlling growth and inducing differentiation in NB cells. In this report, we detail mechanisms by which nerve growth factor (NGF) decreases N-myc levels in TrkA-transfected NB cells and its effect on NB cell proliferation. NGF induced a decrease in N-myc mRNA within 1 h of treatment that occurred in the presence of cycloheximide. The stability of N-myc mRNA was not affected by NGF, indicating a transcriptional control of N-myc mRNA by NGF. NGF but not brain-derived neurotrophic factor (BDNF) decreased N-myc levels demonstrating that p75 alone was not involved. The NGF-induced decrease in N-myc expression was blocked by the Trk tyrosine kinase (TK) antagonist K252a indicating that signals transduced by Trk TK downstream targets were involved. Pharmacologic inhibitors implicated the mitogen-activated protein kinase (MAPK) path. This was supported by the finding that expression of a constitutively activated component of the MAPK path, MAPK kinase (MEK), decreased N-myc levels. Alterations in the level of N-myc are known to alter NB cell cycle progression by affecting the levels of E2Fs and p27kip1. Consistent with these findings, NGF decreased NB cell number and decreased cyclin E-dependent kinase activity via an increase in p27kip1. Thus, our results indicate that the MAP kinase is selectively involved in the NGF-induced N-myc downregulation through a transcriptional mechanism. Furthermore, NGF affects the time required for 15N TrkA cells to complete a replication cycle by decreasing N-myc, E2Fs, cyclin E kinase activity and increasing p27kip1 binding to cyclin E kinase.

Abstract 4880: CASZ1, a neuroblastoma tumor suppressor gene, is epigenetically silenced by EZH2

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Casz1 is a C2H2 Zn-finger transcriptional factor that localizes to a region of LOH on Chr1p36.22 in tumors of Neuroblastoma patients who have a poor prognosis. Moreover, high CASZ1 expression is found in tumors of patients with a good prognosis while tumors from patients with a poor prognosis express low levels of CASZ1 (58 patients, p= 2.07×10-6). Studies indicate that CASZ1 functions as a tumor suppressor gene. Our finding that treatment of NB cells with HDAC inhibitors results in a 10-fold increase in CASZ1 expression suggests that epigenetic silencing contributes to the functional loss of CASZ1 expression from the remaining Chr1p36.22 allele. In studying the mechanism of CASZ1 epigenetic silencing, we found a bivalent chromatin modification region around the CASZ1 Transcription Start Site (TSS) that contains both a H3K27 methylation mark as well as an H3K4 methylation mark. The H3K27 methylation is thought to be a gene repressive marker, which is catalyzed by Polycomb repressor complex 2 (PRC2) proteins. PRC2 contains three core components, EZH2, EED and SUZ12, in which EZH2 (enhancer of zeste 2) is a methyltransferase catalyzing H3K27 methylation. Using Chromatin Immunoprecipitation (ChIP) we identified a 5-fold decrease in binding of EZH2, EED and SUZ12 to the bivalent region after HDAC inhibitor treatment. Consistent with decreased PRC2 binding is the finding of decreased tri-methylation of H3K27 and an increase in H3K4me3, a histone mark associated with transcriptionally active genes. The changes in histone modifications are associated with the activation of the CASZ1 gene. RNA silencing of EZH2 also leads to increases in CASZ1 expression. Consistent with this, CASZ1 expression is also up-regulated (3- fold) in EZH2 knockout MEFs compared to EZH2 wild type MEFs. This indicates that CASZ1 expression on the remaining allele is silenced by epigenetic modifications mediated by EZH2. High expression of EZH2 and EED are also found in tumor samples of neuroblastoma patients with a poor prognosis (58 patients, p=2.6×10-3 for EED and p=5.87×10-4 for EZH2). This is consistent with a model in which aberrant epigenetic gene silencing of the tumor suppressor gene CASZ1 contributes to neuroblastoma tumorigenesis. 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 4880.