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MYCN and HDAC2 cooperate to repress miR-183 signaling in neuroblastoma.

ABSTRACT: MYCN is a master regulator controlling many processes necessary for tumor cell survival. Here, we unravel a microRNA network that causes tumor suppressive effects in MYCN-amplified neuroblastoma cells. In profiling studies, histone deacetylase (HDAC) inhibitor treatment most strongly induced miR-183. Enforced miR-183 expression triggered apoptosis, and inhibited anchorage-independent colony formation in vitro and xenograft growth in mice. Furthermore, the mechanism of miR-183 induction was found to contribute to the cell death phenotype induced by HDAC inhibitors. Experiments to identify the HDAC(s) involved in miR-183 transcriptional regulation showed that HDAC2 depletion induced miR-183. HDAC2 overexpression reduced miR-183 levels and counteracted the induction caused by HDAC2 depletion or HDAC inhibitor treatment. MYCN was found to recruit HDAC2 in the same complexes to the miR-183 promoter, and HDAC2 depletion enhanced promoter-associated histone H4 pan-acetylation, suggesting epigenetic changes preceded transcriptional activation. These data reveal miR-183 tumor suppressive properties in neuroblastoma that are jointly repressed by MYCN and HDAC2, and suggest a novel way to bypass MYCN function.

SUBMITTER: Lodrini M

PROVIDER: S-EPMC3695529 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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MYCN and HDAC2 cooperate to repress miR-183 signaling in neuroblastoma.

Lodrini Marco M Oehme Ina I Schroeder Christina C Milde Till T Schier Marie C MC Kopp-Schneider Annette A Schulte Johannes H JH Fischer Matthias M De Preter Katleen K Pattyn Filip F Castoldi Mirco M Muckenthaler Martina U MU Kulozik Andreas E AE Westermann Frank F Witt Olaf O Deubzer Hedwig E HE

Nucleic acids research 20130426 12

MYCN is a master regulator controlling many processes necessary for tumor cell survival. Here, we unravel a microRNA network that causes tumor suppressive effects in MYCN-amplified neuroblastoma cells. In profiling studies, histone deacetylase (HDAC) inhibitor treatment most strongly induced miR-183. Enforced miR-183 expression triggered apoptosis, and inhibited anchorage-independent colony formation in vitro and xenograft growth in mice. Furthermore, the mechanism of miR-183 induction was found ...[more]

PMID: 23625969

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Project description:BackgroundThe MYCN amplification is a defining hallmark of high-risk neuroblastoma. Due to irregular oncogenes orchestration, tumor cells exhibit distinct fatty acid metabolic features from non-tumor cells. However, the function of MYCN in neuroblastoma fatty acid metabolism reprogramming remains unknown.MethodsGas Chromatography-Mass Spectrometer (GC-MS) was used to find the potential target fatty acid metabolites of MYCN. Real-time PCR (RT-PCR) and clinical bioinformatics analysis was used to find the related target genes. The function of the identified target gene ELOVL2 on cell growth was detected through CCK-8 assay, Soft agar colony formation assay, flow Cytometry assay and mouse xenograft. Chromatin immunoprecipitation (ChIP) and Immunoprecipitation-Mass Spectrometer (IP-MS) further identified the target gene and the co-repressor of MYCN.ResultsThe fatty acid profile of MYCN-depleted neuroblastoma cells identified docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid with anti-tumor activity, significantly increased after MYCN depletion. Compared with MYCN single-copy neuroblastoma cells, DHA level was significantly lower in MYCN-amplified neuroblastoma cells. RT-PCR and clinical bioinformatics analysis discovered that MYCN interfered DHA accumulation via ELOVL fatty acid elongase 2 (ELOVL2) which is a rate-limiting enzyme of cellular DHA synthesis. Enforced ELOVL2 expression in MYCN-amplified neuroblastoma cells led to decreased cell growth and counteracted the growth-promoting effect of MYCN overexpression both in vitro and vivo. ELOVL2 Knockdown showed the opposite effect in MYCN single-copy neuroblastoma cells. In primary neuroblastoma, high ELOVL2 transcription correlated with favorable clinical tumor biology and patient survival. The mechanism of MYCN-mediated ELOVL2 inhibition contributed to epigenetic regulation. MYCN recruited PRC1 (Polycomb repressive complex 1), catalysed H2AK119ub (histone 2A lysine 119 monoubiquitination) and inhibited subsequent ELOVL2 transcription.ConclusionsThe tumor suppressive properties of DHA and ELOVL2 are repressed by the MYCN and PRC1 jointly, which suggests a new epigenetic mechanism of MYCN-mediated fatty acid regulation and indicates PRC1 inhibition as a potential novel strategy to activate ELOVL2 suppressive functions.

Project description:Many of the pro-tumorigenic functions of the oncogene MYCN are attributed to its regulation of global gene expression programs. Alternative splicing is another important regulator of gene expression and has been implicated in neuroblastoma development, however, the molecular mechanisms remain unknown. We found that MYCN up-regulated the expression of the core spliceosomal protein, SNRPD3, in models of neuroblastoma initiation and progression. High mRNA expression of SNRPD3 in human neuroblastoma tissues was a strong, independent prognostic factor for poor patient outcome. Repression of SNRPD3 expression correlated with loss of colony formation in vitro and reduced tumorigenicity in vivo. The effect of SNRPD3 on cell viability was in part dependent on MYCN as an oncogenic co-factor. RNA-sequencing revealed a global increase in the number of genes being differentially spliced when MYCN was overexpressed. Surprisingly, depletion of SNRPD3 in the presence of overexpressed MYCN further increased differential splicing, particularly of cell cycle regulators, such as BIRC5 and CDK10. MYCN directly bound SNRPD3, and the protein arginine methyltransferase, PRMT5, consequently increasing SNRPD3 methylation. Indeed, the PRMT5 inhibitor, JNJ-64619178, reduced cell viability and SNRPD3 methylation in neuroblastoma cells with high SNRPD3 and MYCN expression. Our findings demonstrate a functional relationship between MYCN and SNRPD3, which maintains the fidelity of MYCN-driven alternative splicing in the narrow range required for neuroblastoma cell growth. SNRPD3 methylation and its protein-protein interface with MYCN represent novel therapeutic targets. Hypothetical model for SNRPD3 as a co-factor for MYCN oncogenesis. SNRPD3 and MYCN participate in a regulatory loop to balance splicing fidelity in neuroblastoma cells. First MYCN transactivates SNRPD3 to lead to high-level expression. Second, SNRPD3 and MYCN form a protein complex involving PRMT5. Third, this leads to balanced alterative splicing (AS) activitiy that is favorable to neuroblastoma. Together this forms as a therapeutic vulnerability where SNRPD3 perturbation or PRMT5 inhibitors are selectively toxic to neuroblastoma by conditionally disturbing splicing activity.

Project description:BACKGROUND:The oncogene MYCN is critical for tumorigenesis of several types of cancers including neuroblastoma. We previously reported that miR-506-3p repressed MYCN expression in neuroblastoma cells. However, the mechanism underlying such regulation was undetermined since there is no miR-506-3p target site in MYCN 3'UTR. METHODS:By a systematic investigation combining microarray, informatics and luciferase reporter assay, we identified that the transcriptional factor pleiomorphic adenoma gene-like 2 (PLAGL2) is a direct target of miR-506-3p that mediates its regulation on MYCN expression. Using CHIP-PCR and luciferase reporter assay, we validated the transcriptional regulation of MYCN by PLAGL2 and we further demonstrated the transcriptional regulation of PLAGL2 by MYCN. We examined the function of PLAGL2 in regulating neuroblastoma cell fate by cell viability assay, colony formation and Western blotting of differentiation markers. We examined the effect of retinoic acid, the differentiation agent used in neuroblastoma therapy, on miR-506-3p, PLAGL2 and MYCN expressions by quantitative PCR and Western blots. We investigated the clinical relevance of PLAGL2 expression by examining the correlation of tumor PLAGL2 mRNA levels with MYCN mRNA expression and patient survival using public neuroblastoma patient datasets. RESULTS:We found that miR-506-3p directly down-regulated PLAGL2 expression, and we validated a PLAGL2 binding site in the MYCN promoter region responsible for promoting MYCN transcription, thereby establishing a mechanism through which miR-506-3p regulates MYCN expression. Conversely, we discovered that MYCN regulated PLAGL2 transcription through five N-Myc-binding E-boxes in the PLAGL2 promoter region. We further confirmed the reciprocal regulation between endogenous PLAGL2 and MYCN in multiple neuroblastoma cell lines. Moreover, we found that PLAGL2 knockdown induced neuroblastoma cell differentiation and reduced cell proliferation, and combined knockdown of PLAGL2 and MYCN showed a synergistic effect. More strikingly, we found that high tumor PLAGL2 mRNA levels were significantly correlated with high MYCN mRNA levels and poor patient survival in neuroblastoma patients. Furthermore, we found that retinoic acid increased expression of miR-506-3p and repressed expression of MYCN and PLAGL2. CONCLUSIONS:Our findings altogether suggest that the interplay network formed by PLAGL2, MYCN and miR-506-3p is an important mechanism in regulating neuroblastoma cell fate, determining neuroblastoma prognosis, and mediating the therapeutic function of retinoic acid.

Dataset Information

MYCN and HDAC2 cooperate to repress miR-183 signaling in neuroblastoma.

Publications

MYCN and HDAC2 cooperate to repress miR-183 signaling in neuroblastoma.

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