MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells

Oncogene (2007) 26, 5017–5022 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc SHORT COMMUNICATION MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells C Welch, Y Chen and RL Stallings Children’s Cancer Research Institute and Department of Pediatrics, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Neuroblastoma (NB) is one of the most common forms of cancer in children, accounting for 15% of pediatric cancer deaths. The clinical course of these tumors is highly variable and is dependent on such factors as age at presentation, stage, ploidy and genomic abnormalities. Hemizygous deletion of chromosome 1p occurs in approximately 30% of advanced stage tumors, is associated with a poor prognosis, and likely leads to the loss of one or more tumor suppressor genes. We show here that microRNA (miRNA)-34a (1p36.23) is generally expressed at lower levels in unfavorable primary NB tumors and cell lines relative to normal adrenal tissue and that reintroduction of this miRNA into three different NB cell lines causes a dramatic reduction in cell proliferation through the induction of a caspase-dependent apoptotic pathway. As a potential mechanistic explanation for this observation, we demonstrate that miR-34a directly targets the messenger ribonucleic acid (mRNA) encoding E2F3 and significantly reduces the levels of E2F3 protein, a potent transcriptional inducer of cell-cycle progression. Furthermore, miR-34a expression increases during retinoic acidinduced differentiation of the SK-N-BE cell line, whereas E2F3 protein levels decrease. Thus, adding to the increasing role of miRNAs in cancer, miR-34a may act as a suppressor of NB tumorgenesis. Oncogene (2007) 26, 5017–5022; doi:10.1038/sj.onc.1210293; published online 12 February 2007 Keywords: apoptosis; CGH; E2F3; microRNA; neuroblastoma; tumour suppressors Neuroblastoma (NB) is a pediatric tumor originating from precursor cells of the sympathetic nervous system. These tumors account for 15% of childhood cancer deaths and are particularly noted for a wide range in clinical behavior, ranging from spontaneous regression to rapid progression and death owing to disease (Brodeur, 2003). Loss of chromosome 1p material, Correspondence: Professor RL Stallings, Children’s Cancer Research Institute and Department of Pediatrics, University of Texas Health Science Center at San Antonio, 8403 Floyd Curl Drive, MC 7784, San Antonio, TX, USA. E-mail: Received 6 October 2006; revised 1 December 2006; accepted 13 December 2006; published online 12 February 2007 occurring predominately through an unbalanced t(1;17) that also results in gain of 17q (Van Roy et al., 1994), is a common chromosomal imbalance found in NB and occurs preferentially in tumors with MYCN amplification (MNA) (Fong et al., 1989). All three of these nonrandom genetic abnormalities, loss of 1p, gain of 17q and MNA, are independently associated with a poor clinical outcome (Brodeur et al., 1984; Spitz et al., 2003; Attiyeh et al., 2005; Vandesompele et al., 2005). Although the importance of 1p loss in NB pathogenesis has been known for some time, the genes and genetic pathways affected by this loss are not completely understood. The region of 1p loss is quite large, containing many potential candidate genes. Furthermore, smaller homozygous deletions that might further pinpoint the genes of interest are exceedingly rare (Mosse et al., 2005; Stallings et al., 2006) and do not define a single shortest region of overlap (Schleiermacher et al., 1994). Expression microarray studies of primary tumors have also led some investigators to suggest that multiple genes on chromosome 1p contribute to NB pathogenesis (Janoueix-Lerosey et al., 2004; Wang et al., 2006), a concept supported by the fact that in vitro functional studies have shown that more than one gene from chromosome 1p can have anti-tumorigenic effects when ectopically expressed in NB cell lines (Ejeskar et al., 2005; Valentijn et al., 2005). Here, we explore the possibility that some of the chromosome 1p sequences with tumor suppressor effects in NB are not conventional protein coding gene sequences, but rather microRNAs (miRNAs), which regulate gene expression at a post-transcriptional level by either inhibiting mRNAs from being translated or causing them to be degraded. MiRNAs play major roles in the differentiation of neural cells (Miska, 2005), and the dysregulation of these sequences can have tumor suppressor or oncogenic activity in different forms of cancer (Esquela-Kerscher and Slack, 2006). For example, two miRNAs (miR-15 and miR-16) mapping to chromosomal region 13q14 are frequently deleted and downregulated in chronic lymphocytic leukemia and thus act as tumor suppressor genes (Calin et al., 2002). MiRNAs acting in a dominant oncogenic manner are illustrated by miR-21 on chromosome 17q, which, when overexpressed in some forms of cancer have anti MicroRNA-34a as a tumor suppressor in neuroblastoma C Welch et al 5018 Figure 1 Analysis of 1p loss in 13 primary tumors and the SK-N-AS cell line using oligonucleotide array CGH, as described previously (Stallings et al., 2006). The vertical axis of each plot represents the fluorescence ratio of tumor to normal DNA on a log2 scale, whereas the horizontal axis represents genomic position on chromosome 1p in base pairs. The array CGH data was generated by NimbleGen Systems Inc (Iceland). Positions of miR-34a and miR-30e are noted at the top of the figure. Chromosome 1p breakpoints are denoted with arrows. Oncogene MicroRNA-34a as a tumor suppressor in neuroblastoma C Welch et al 5019 Figure 2 Reverse transcriptase QPCR expression analysis of miR-34a in low stage hyperdiploid tumors with favorable histopathology (grey), high stage 11q- tumors with unfavorable histopathology (clear), high stage MYCN amplified tumors with unfavorable histopathology (black), NB cell lines Kelly, SK-N-AS, NGP, SK-N-BE and adrenal gland (dark grey). Reverse transcription with stemloop primers was carried out as described by Chen et al. (2005) (Supplementary methods). Samples designated with an * have 1p loss. apoptotic effects (Chan et al., 2005; Cimmino et al., 2005). An in silico search for miRNAs encoded within the 1p region identified miR-34a (1p36.23) and miR-30e (1p34.2) as potential candidates. Using array comparative genomic hybridization (CGH) data on a published set of tumors, we determined that the region encoding miR-34a is located in the minimal region of 1p loss (Figure 1) whereas the region encoding miR-30e is lost in 79% of tumors (Stallings et al., 2006). Using realtime PCR analysis, the expression of miR-34a was generally reduced in NB primary tumors as compared to normal adrenal gland (Figure 2), and tumors with 1p loss have a 30% reduction in miR-34a expression relative to tumors with an intact 1p locus. Similarly, miR-34a expression is reduced in (...truncated)