Abstract 2291: Oncogenic hsa-miR-1323 down-regulates MYCN/ALK and reveals PAG1 as a novel tumor suppressor for neuroblastoma

Abstract

Abstract MicroRNAs (miRNAs) are 22-24 nucleotides non-coding RNAs that can simultaneously influence the levels of multiple target genes and therefore have the potential to strongly silence cancer gene networks. MYCN and ALK are well established oncogenes implicated in the pathogenesis and progression of neuroblastoma. Using multiple web based algorithms we have identified potential tumor suppressor miRNAs that can control the pathogenesis and/or progression of neuroblastoma by down-regulating both MYCN and ALK. We found hsa-miR-1323 as a candidate tumor suppressor for neuroblastoma. Lipofectamine based mimic transfection and lentiviral vector based over-expression of miR-1323 in cell lines led to a decrease in MYCN and ALK transcripts as well as signals from luciferase reporter constructs carrying the 3′-UTR of MYCN or ALK validating MYCN and ALK as direct targets of miR-1323. However, unexpectedly for this predicted tumor suppressor microRNA, gain of function studies for miR-1323 in neuroblastoma cell lines demonstrated increased proliferation, colony formation and tumor growth of orthotopic xenograft tumors. As SiRNA against MYCN strongly limits neuroblastoma proliferation, our data suggests that miR-1323 targets an alternative tumor suppressor pathway which by-passes the growth suppressive effects of MYCN inhibition and drives proliferation. Further bio-informatics analysis of putative miR-1323 targets pointed to PAG1 (or Cbp) as a possible tumor suppressor target of miR-1323. The transmembrane adaptor protein PAG1 inhibits proliferation driven by the Src family of kinases (SFKs) through multiple mechanisms and is a known tumor suppressor in leukemia and non-small cell lung cancers. Importantly, analysis of PAG1 in clinical cohorts of neuroblastoma reveals that low expression of PAG1 strongly correlates with poor overall survival compared to patients with high levels of PAG1 expression (p <0.001). We have confirmed that miR-1323 targets PAG1 transcript via qRT-PCR assays, and this is associated with increased p-ERK levels in neuroblastoma cell lines. We therefore propose that miR-1323 mediated repression of PAG1 activates the Src-dependent oncogenic pathways, overriding its effect on MYCN or ALK, and contributing to the aggressive clinical behavior of high-risk neuroblastoma. These studies further demonstrate the complexity of microRNA functions in normal and cancer cells and mandate careful evaluation of putative ‘tumor suppressive’ microRNAs as therapeutic tools. 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 2291. doi:1538-7445.AM2012-2291