Abstract 4152: Proteomic analysis identifies RAB1A as a novel marker of invasion in neuroblastoma
Authors: Bray Isabella M. , Dowling Paul , Bryan Kenneth R. , Clynes Martin , Stallings Raymond L. 
Published: August 2012

Neuroblastoma is a paediatric cancer arising from precursor cells of the sympathetic nervous system. Extreme heterogeneity in clinical behavior may be attributed to recurrent genomic abnormalities including chromosomal losses/gains, and amplification of the MYCN oncogene. We previously described a signature of 37 miRNAs significantly differentially expressed in MYCN amplified tumours, and have confirmed one such microRNA, miR-542-5p, acts as a tumour suppressor in neuroblastoma. Our current research focuses on identifying the mechanism of action of miR-542-5p. No enrichment for miR-542-5p 3′UTR target sites was found in a panel of mRNA differentially expressed after ectopic over-expression of miR-542-5p. We therefore hypothesize that miR-542-5p has a more significant impact on protein levels through translational inhibition than mRNA degradation. To test our hypothesis, we carried out proteomic analysis using label-free quantification of high sensitive reversed-phase liquid chromatography coupled nanospray tandem mass spectrometry (LC-MS/MS). In total, 27,000 peptides were examined per sample. Of these, 17 proteins changed significantly in MYCN amplified Kelly cells (6 down-regulated and 11 up-regulated) and 14 proteins changed significantly in MYCN single copy SKNAS cells (9 down-regulated and 5 up-regulated). No overlap between significantly differentially expressed mRNA and protein was observed, and none of the proteins identified contained miR-542-5p predicted target sites. Significance was determined based on a confidence score of greater than 40 (applies to protein identification), number of peptides used to identify and quantitate the proteins as ≤ 2, and a p-value &lt; 0.05. Fold changes are based on difference in average protein abundance between the miR-542-5p mimic and negative control cells across 3 biological repeats. The R2 database (http://r2.amc.nl)) was used to assess the clinical relevance of these proteins. Of the 31 differentially expressed proteins, 26% were significantly associated (p &lt; 0.05) with overall (OS), or event-free survival (EFS) in neuroblastoma. Of particular interest was RAB1A, which increased in abundance in both miR-542-5p over-expressing cell lines. Increased expression of RAB1A was associated with better OS (p=0.015) and EFS (p=0.00016), consistent with results for miR-542-5p. Depletion of cellular RAB1A mRNA by siRNA also produced similar result to that seen with miR-542-5p antagomer. In both SKNAS and Kelly cells, RAB1A knock-down resulted in a significant (p&lt;0.001) increase in the number on invading cells, but no change in the rate of proliferation. We therefore conclude that RAB1A is indirectly regulated by miR-542-5p through a yet unknown mechanism. Reduced expression of RAB1A significantly increases in vitro invasion, consistent with clinical data showing that lower expression is highly associated with poor survival in neuroblastoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4152. doi:1538-7445.AM2012-4152