Abstract
BackgroundMYCN is amplified in small cell lung cancers and several pediatric tumors, including alveolar rhabdomyosarcomas and neuroblastomas. MYCN protein is known to play a key oncogenic role in both alveolar rhabdomyosarcomas and neuroblastomas. MYCN opposite strand (MYCNOS) is a gene located on the antisense strand to MYCN that encodes alternatively spliced transcripts, two of which (MYCNOS-01 and MYCNOS-02) are known to be expressed in neuroblastoma and small cell lung cancer with reciprocal regulation between MYCNOS-02 and MYCN reported for neuroblastomas. We sought to determine a functional role for MYCNOS-01 in alveolar rhabdomyosarcoma and neuroblastoma cells and identify any associated regulatory effects between MYCN and MYCNOS-01.MethodsMYCNOS-01, MYCNOS-02 and MYCN expression levels were assessed in alveolar rhabdomyosarcoma and neuroblastoma cell lines and tumor samples from patients using Affymetrix microarray data and quantitative RT-PCR. Following MYCNOS-01 or MYCN siRNA knockdown and MYCNOS-01 overexpression, transcript levels were assayed by quantitative RT-PCR and MYCN protein expression assessed by Western blot and immunofluorescence. Additionally, effects on cell growth, apoptosis and cell cycle profiles were determined by a metabolic assay, caspase activity and flow cytometry, respectively.ResultsMYCNOS-01 transcript levels were generally higher in NB and RMS tumor samples and cell lines with MYCN genomic amplification. RNA interference of MYCNOS-01 expression did not alter MYCN transcript levels but decreased MYCN protein levels. Conversely, MYCN reduction increased MYCNOS-01 transcript levels, creating a negative feedback loop on MYCN protein levels. Reduction of MYCNOS-01 or MYCN expression decreased cell growth in MYCN-amplified alveolar rhabdomyosarcoma and neuroblastoma cell lines. This is consistent with MYCNOS-01-mediated regulation of MYCN contributing to the phenotype observed.ConclusionsAn alternative transcript of MYCNOS, MYCNOS-01, post-transcriptionally regulates MYCN levels and affects growth in MYCN-amplified rhabdomyosarcoma and neuroblastoma cells.
Highlights
MYCN is amplified in small cell lung cancers and several pediatric tumors, including alveolar rhabdomyosarcomas and neuroblastomas
MYCN opposite strand (MYCNOS)-01 is predicted to be non-protein coding and correlations between MYCNOS and MYCN transcript levels in RMS and NB patients and cell lines Previous data showed protein encoding potential for MYCNOS-02 transcripts [15]
There was a significant positive correlation between both MYCNOS transcripts and MYCN transcript expression in NB patient samples (Fig. 1e and f ) and cell lines (Fig. 1g and h) that is consistent with the literature for MYCNOS-02 [17, 19]
Summary
MYCN is amplified in small cell lung cancers and several pediatric tumors, including alveolar rhabdomyosarcomas and neuroblastomas. MYCN protein is known to play a key oncogenic role in both alveolar rhabdomyosarcomas and neuroblastomas. MYCN opposite strand (MYCNOS) is a gene located on the antisense strand to MYCN that encodes alternatively spliced transcripts, two of which (MYCNOS-01 and MYCNOS-02) are known to be expressed in neuroblastoma and small cell lung cancer with reciprocal regulation between MYCNOS02 and MYCN reported for neuroblastomas. Several pediatric cancers feature amplification at the chromosomal region 2p24 including alveolar rhabdomyosarcoma (ARMS), neuroblastoma (NB), medulloblastoma, Wilms’ tumor, and retinoblastoma [1,2,3]. In ARMS, MYCN transcription is driven by PAX3-FOXO1, the protein product of a fusion between the PAX3 and FOXO1 genes that has prognostic significance in these tumors [9, 13]. RMS and NB are a major cause of cancer related death in children with a five-year survival rate of around 50% for high-risk NB cases, including those with MYCN amplification, and 40% for PAX3-FOXO1 positive RMS cases [13, 14]
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