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

SHORT COMMUNICATION MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells C Welch, Y Chen and RL Stallings (2007) 26, 5017 5022 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc 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, accountingfor 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 duringretinoic acidinduced differentiation of the SK-N-BE cell line, whereas E2F3 protein levels decrease. Thus, addingto the increasingrole of mirnas in cancer, mir-34a may act as a suppressor of NB tumorgenesis. (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: STALLINGS@uthscsa.edu 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

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 log 2 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.

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 the majority of NB cell lines tested. Initially, we determined if ectopic expression of either mir-30e or mir-34a had a biological effect in three different NB cell lines, two with MNA (Kelly, NGP) and one lacking MNA (SK-N-AS), using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay. Each cell line transfected with pre-mir-34a had a significant reduction in metabolically active cells compared with the scrambled negative control over a 5-day interval (Figure 3a). A reduction in cell viability was detected in each cell line by day 2 following transfection, and dramatic loss of cell viability was apparent by day 5. In contrast, transfection of cell lines with mir-30e had no effect on cell viability even though its expression was at the same level as mir-34a, as evaluated by real-time quantitative polymerase chain reaction (data not shown). To determine the possible mechanism of the antiproliferative activity of mir-34a, activation of the caspase 3/7 apoptotic pathway was assessed following transfection of pre-mir-34a into each cell line. Caspase- 3 and -7 enzymatic activities (key executioners of apoptosis) significantly increased by 72 h post-transfection in Kelly cells treated with pre-mir-34a relative to the scrambled RNA transfected control cells Figure 3 (a) MTT cell viability assay, as described in the Supplementary methods, was performed on days 1 to 5 after Lipofectamine transfection of Kelly, NGP and SK-N-AS cells with either a pre-mir-34a molecule (10 nm) or a negative control scrambled oligonucleotide that does not encode for any known mirna (Ambion). Cell populations transfected with the negative control oligo had a significantly greater number of metabolically active cells than cells transfected with the pre-mir-34a. (b) Caspase 3/7 assay, as described in the Supplementary methods, was carried out on Kelly, NGP and SK-N-AS cells transfected with either the negative control oligo (dark grey) or the pre-mir-34a (diagonal stripe). Kelly showed a maximal effect at day 3, whereas the other two cell lines showed the greatest effect on day 5. Statistical significance for caspase-3/7 activity was assessed using a Student s paired t-test.

5020 Figure 4 (a) Validation of mir-34a mrna targets. Luciferase constructs were made by ligating oligonucleotides containing the putative target site into the XbaI site of the pgl3-control vector (promega) as described previously (Guo et al., 2006) (see Supplementary methods). A significant reduction in luciferase activity is detected when an oligonucleotide containing the exact complementary sequence of mir-34a is cloned into the construct (pgl3_mir-34a). Neither the construct containing the putative MYCN target sequence (pgl3_mycn oligo) or a 625 bp fragment of the 3-UTR (pgl3_mycn UTR) showed a reduction in luciferase activity, indicating that MYCN is not a direct target of mir-34a. There was a significant reduction in luciferase activity when synthesized oligonucleotides representing the putative E2F3 3-UTR target site was used (pgl3_e2f3 oligo). This effect was abolished when a sequence containing a mutated seed site was used (pgl3_e2f3 mut). (b) Effect of mir-34a expression on endogenous E2F3 levels. The SK-N-AS cell line was transfected with 10 nm mir-34a precursor molecule or a negative control precursor mirna. E2F3 protein levels were determined by Western blot of nuclear extracts isolated on day 3 post-transfection (Supplementary methods). Relative to the negative control mirna, mir-34a expression significantly reduced the levels of E2F3. (c) Effect of ATRA on mir-34a expression following treatment and differentiation of NB SK-N-BE cell line. The graph shows relative mir-34a expression using quantitative realtime PCR, as described in Figure 2. The cells treated with 5 or 10 mm ATRA displayed morphological changes such as neuritic extensions that were indicative of a differentiated state. MiR-34a expression increased 2 to 2.5-fold in SK-N-BE cells. It is important to note that SK-N-BE cells have hemizygous loss of the 1p region, so that presumably the fold induction of mir-34a would be twofold higher in cells having two copies of this locus. (d) Effect of ATRA treatment on endogenous E2F3 levels. Western blot analysis of E2F3 was performed on SK-N-BE nuclear extracts following 6 days of ATRA treatment. Differentiated SK-N-BE cells show a marked reduction in E2F3 levels. (Figure 3b). The maximum increase in caspase 3/7 activity in NGP and SK-N-AS cells occurred at day 5 post-transfection. Endogenous mir-34a expression, which is B50 fold higher in Kelly than in the other two cell lines (Figure 2), might explain why mir-34a has an earlier effect in this cell line. Computational predictions for human mrna targets of mir-34a indicated numerous potential candidates, as detailed at miranda (http://microrna.sanger.ac.uk/ sequences), TargetScan (http://genes.mit.edu/targetscan/) and PicTar (http://pictar.bio.nyu.edu/). Two of the more significant putative targets in the context of NB were MYCN and E2F3, which are conserved between humans and rodents. To validate whether mir-34a directly recognizes the 3 0 -UTR of these transcripts, we cloned oligonucleotides representing the presumed target sites (Supplementary Table 1) into the 3 0 -UTR of the luciferase gene and luciferase levels were compared in SK-N-AS cells cotransfected with one of these constructs and a pre-mir-34a or a negative control RNA. As a positive control, a luciferase construct was created that contains the complete complementary sequence to mir-34a. The overexpression of mir-34a had no effect on either the oligonucleotide construct representing the MYCN 3 0 UTR or on a construct containing 625 bp of the MYCN 3 0 UTR, indicating that MYCN is not a mir-34a target. In contrast, overexpression of mir-34a reduced the luciferase activity from the reporter construct containing the E2F3 site and the positive control (Figure 4a), whereas no effect was observed with a construct containing a mutated E2F3 seed site (Figure 4a). This effect was specific because there was no change in luciferase reporter activity when a negative control scrambled mirna was cotransfected with either reporter construct (Figure 4a). These results suggest that the complementary site in the E2F3 mrna is a target of mir-34amediated post-transcriptional gene silencing. Western blot analysis of SK-N-AS cells transfected with mir-34a showed a significant reduction in E2F3 protein in cells

overexpressing mir-34a, confirming that mir-34a targets E2F3 mrna (Figure 4b) All trans-retinoic acid (ATRA) induces the terminal differentiation of NB cells (Thiele et al., 1985). To test whether mir-34a is induced during the differentiation process, we analysed the expression level of mir-34a in SK-N-BE cells treated with ATRA. ATRA induced the dose-dependent expression of mir-34a following differentiation (Figure 4c). As predicted, Western blot analysis showed a significant reduction of E2F3 protein in SK-N-BE cells treated with ATRA relative to untreated cells (Figure 4d). This result suggests that the drop in E2F3 levels following ATRA treatment may be owing to the increase in expression of mir-34a. In summary, we demonstrate that mir-34a has tumor suppressor activity when ectopically expressed in NB cell lines through induction of a caspase 3/7 apoptotic References Attiyeh EF, London WB, Mosse YP, Wang Q, Winter C, Khazi D et al. (2005). Chromosome 1p and 11q deletions and outcome in neuroblastoma. N Engl J Med 353: 2243 2253. Brodeur GM. (2003). Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer 3: 203 216. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM. (1984). Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 224: 1121 1124. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E et al. (2002). Frequent deletions and down-regulation of micro- RNA genes mir15 and mir16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 99: 15524 15529. Calin GA, Ferracin M, Cimmino A, Di Leva G, Shimizu M, Wojcik SE et al. (2005). A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med 353: 1793 1801. Chan JA, Krichevsky AM, Kosik KS. (2005). MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res 65: 6029 6033. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT et al. (2005). Real-time quantification of micrornas by stem-loop RT-PCR. Nucleic Acids Res 33: e179. Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M, Shimizu M et al. (2005). mir-15 and mir-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci USA 102: 13944 13949. Ejeskar K, Krona C, Caren H, Zaibak F, Li L, Martinsson T et al. (2005). Introduction of in vitro transcribed ENO1 mrna into neuroblastoma cells induces cell death. BMC Cancer 5: 161. Esquela-Kerscher A, Slack FJ. (2006). Oncomirs - micrornas with a role in cancer. Nat Rev Cancer 6: 259 269. Fong CT, Dracopoli NC, White PS, Merrill PT, Griffith RC, Housman DE et al. (1989). Loss of heterozygosity for the short arm of chromosome 1 in human neuroblastomas: correlation with N-myc amplification. Proc Natl Acad Sci USA 86: 3753 3757. Guo Y, Chen Y, Ito H, Watanabe A, Ge X, Kodama T et al. (2006). Identification and characterization of lin-28homolog B (LIN28B) in human hepatocellular carcinoma. Gene 384: 51 61. pathway. MiR-34a may have an antiproliferative effect, in part, through targeting the E2F3 transcription factor. Recently, it has been shown that mir-17-5p and mir-20a, which are part of the mir-17 cluster on chromosome 13, act as tumor suppressors by targeting and reducing E2F1 levels (O Donnell et al., 2005). Similar to mir-34a, the region harboring the mir-17 cluster is hemizygously deleted in some human tumors (Lin et al., 1999). This same region, however, is amplified in diffuse large B-cell lymphoma samples (Calin et al., 2005; He et al., 2005). Thus, as suggested previously (O Donnell et al., 2005), mirnas may exert a tumor suppressor or oncogenic effect depending upon the cell type in which they are expressed. In this regard, it is interesting to note that E2F family members can have both cell proliferative and proapoptotic effects in different cellular contexts (Johnson and Degregori, 2006). He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S et al. (2005). A microrna polycistron as a potential human oncogene. Nature 435: 828 833. Janoueix-Lerosey I, Novikov E, Monteiro M, Gruel N, Schleiermacher G, Loriod B et al. (2004). Gene expression profiling of 1p35-36 genes in neuroblastoma. 23: 5912 5922. Johnson DG, Degregori J. (2006). Putting the Oncogenic and Tumor Suppressive Activities of E2F into Context. Curr Mol Med 6: 731 738. Lin YW, Sheu JC, Liu LY, Chen CH, Lee HS, Huang GT et al. (1999). Loss of heterozygosity at chromosome 13q in hepatocellular carcinoma: identification of three independent regions. Eur J Cancer 35: 1730 1734. Miska EA. (2005). How micrornas control cell division, differentiation and death. Curr Opin Genet Dev 15: 563 568. Mosse YP, Greshock J, Margolin A, Naylor T, Cole K, Khazi D et al. (2005). High-resolution detection and mapping of genomic DNA alterations in neuroblastoma. Genes Chromosomes Cancer 43: 390 403. O Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. (2005). c-myc-regulated micrornas modulate E2F1 expression. Nature 435: 839 843. Schleiermacher G, Peter M, Michon J, Hugot JP, Vielh P, Zucker JM et al. (1994). Two distinct deleted regions on the short arm of chromosome 1 in neuroblastoma. Genes Chromosomes Cancer 10: 275 281. Spitz R, Hero B, Ernestus K, Berthold F. (2003). Deletions in chromosome arms 3p and 11q are new prognostic markers in localized and 4 s neuroblastoma. Clin Cancer Res 9: 52 58. Stallings RL, Nair P, Maris JM, Catchpoole D, McDermott M, O Meara A et al. (2006). High-resolution analysis of chromosomal breakpoints and genomic instability identifies PTPRD as a candidate tumor suppressor gene in neuroblastoma. Cancer Res 66: 3673 3680. Thiele CJ, Reynolds CP, Israel MA. (1985). Decreased expression of N-myc precedes retinoic acid-induced morphological differentiation of human neuroblastoma. Nature 313: 404 406. Valentijn LJ, Koppen A, van Asperen R, Root HA, Haneveld F, Versteeg R. (2005). Inhibition of a new differentiation pathway in neuroblastoma by copy number 5021

5022 defects of N-myc, Cdc42, and nm23 genes. Cancer Res 65: 3136 3145. Van Roy N, Laureys G, Cheng NC, Willem P, Opdenakker G, Versteeg R et al. (1994). 1;17 translocations and other chromosome 17 rearrangements in human primary neuroblastoma tumors and cell lines. Genes Chromosomes Cancer 10: 103 114. Vandesompele J, Baudis M, De Preter K, Van Roy N, Ambros P, Bown N et al. (2005). Unequivocal delineation of clinicogenetic subgroups and development of a new model for improved outcome prediction in neuroblastoma. J Clin Oncol 23: 2280 2299. Wang Q, Diskin S, Rappaport E, Attiyeh E, Mosse Y, Shue D et al. (2006). Integrative genomics identifies distinct molecular classes of neuroblastoma and shows that multiple genes are targeted by regional alterations in DNA copy number. Cancer Res 66: 6050 6062. Supplementary Information accompanies the paper on the website (http://www.nature.com/onc).