Abstract 4734: A LIN28B/RAN/AURKA signaling network promotes neuroblastoma tumorigenesis
Authors: Schnepp Robert W. , Khurana Priya , Attiyeh Edward F. , Chodosh Sara , Raman Pichai , Oldridge Derek A. , Gagliardi Maria E. , Conkrite Karina , Asgharzadeh Shahab , Seeger Robert C. , Madison Blair , Rustgi Anil , Maris John M. , Diskin Sharon J. 
Published: August 2015

Abstract
Background: Neuroblastoma, a childhood cancer of the sympathetic nervous system, accounts for approximately 10-15% of pediatric oncology deaths. The genetic basis of neuroblastoma has grown clearer, with genome-wide association studies performed by our laboratory uncovering CASC15, BARD1, NBPF23, LMO1, HACE1, TP53 and LIN28B as susceptibility genes, with many (if not all) playing a major role in tumorigenesis. Here we focus on LIN28B, which binds mRNAs directly and is a master regulator of the let-7 family of tumor suppressor microRNAs, as we previously showed that high LIN28B expression is associated with advanced stage disease and worse patient outcome.
Methods: To discover LIN28B-associated pathways in neuroblastoma, we performed gene set enrichment analysis (GSEA) on mRNA expression datasets and analyzed SNP-array based DNA copy number datasets. We used siRNAs, shRNAs, and microRNA mimetics to perturb transcripts of interest in neuroblastoma cells and measured effects on downstream signaling, protein-protein interactions, and proliferation.
Results: We applied GSEA to mRNA expression profiles from 250 neuroblastoma tumors and found LIN28B expression to be robustly correlated with several biologically relevant gene sets, including “RAN signaling.” We focused on RAN signaling as RAN is a member of the Ras family of GTPases implicated in the pathogenesis of several malignancies and we demonstrated a strong positive correlation between LIN28B and RAN expression, most strikingly in the MYCN-amplified context (p = 2.2×10−10). We next analyzed 374 high-risk neuroblastoma tumors and found that 28% of them displayed recurrent somatic copy number gain of chromosome 12q24, the genomic location of RAN, which was associated with increased RAN expression (p = 0.0004) and was inversely related to MYCN amplification (p = 0.0021). Increased RAN expression was associated with stage 4 disease (p = 0.0047) and decreased overall survival (p = 0.0002). To further dissect the LIN28B-RAN relationship, we depleted LIN28B using shRNAs, showing that it reduced RAN RNA and protein levels. LIN28B directly bound RAN mRNA, likely enhancing its translation. As RAN promotes the phosphorylation and activation of Aurora kinase A (AURKA), we then demonstrated that LIN28B leads to AURKA activation via RAN. Moreover, we demonstrated that AURKA is a direct let-7 target, defining a separate mechanism by which LIN28B/let-7 influences AURKA expression. Finally, we showed that RAN depletion resulted in decreased neuroblastoma proliferation, phenocopying LIN28B depletion.
Conclusions: These results demonstrate that enhanced LIN28B expression and chromosome 12q24 gain each independently promote RAN expression and that LIN28B and RAN signaling further converge on AURKA. Collectively, our studies support LIN28B as a master regulator of multiple oncogenes implicated in neuroblastoma pathogenesis, nominating it as a candidate for therapeutic targeting.
Citation Format: Robert W. Schnepp, Priya Khurana, Edward F. Attiyeh, Sara Chodosh, Pichai Raman, Derek A. Oldridge, Maria E. Gagliardi, Karina Conkrite, Shahab Asgharzadeh, Robert C. Seeger, Blair Madison, Anil Rustgi, John M. Maris, Sharon J. Diskin. A LIN28B/RAN/AURKA signaling network promotes neuroblastoma tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4734. doi:10.1158/1538-7445.AM2015-4734