MYCN-regulated microRNAs repress estrogen -α (ESR1) expression and neuronal differentiation in human neuroblastoma

Jakob Lovéna, Nikolay Zinina, Therese Wahlströma, Inga Müllera, Petter Brodina, Erik Fredlundb, Ulf Ribackea,1, Andor Pivarcsic, Sven Påhlmanb, and Marie Henrikssona,2

aDepartment of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden; bCenter for Molecular Pathology, Department of Laboratory Medicine, CREATE Health, Lund University, University Hospital MAS, SE-205 02 Malmö, Sweden; and cDepartment of Medicine, Dermatology and Venereology Unit, Center for Molecular Medicine, Karolinska University Hospital, SE-171 76 Stockholm, Sweden

Communicated by George Klein, Karolinska Institutet, Stockholm, Sweden, November 26, 2009 (received for review March 31, 2009) MYCN, a proto-oncogene normally expressed in the migrating repression is not fully understood (5). A direct link between neural crest, is in its amplified state a key factor in the genesis of MYCN expression and the transformed phenotype has been human neuroblastoma (NB). However, the mechanisms underlying established in a range of studies, including a transgenic model in MYCN-mediated NB progression are poorly understood. Here, we which targeted MYCN overexpression in migrating neural crest present a MYCN-induced miRNA signature in human NB involving cells results in NB (2, 6). However, despite considerable pro- the activation and transrepression of several miRNA from gress, NB development linked to MYCN amplification remains to paralogous clusters. Several family members derived from the be fully elucidated. miR-17∼92 cluster, including miR-18a and miR-19a, were among MicroRNAs (miRNAs), small noncoding RNAs that neg- the up-regulated miRNAs. Expression analysis of these miRNAs in fi NB tumors confirmed increased levels in MYCN-amplified samples. atively regulate expression through sequence-speci c base ′ ′ Specifically, we show that miR-18a and miR-19a target and repress pairing with the 3 -untranslated region (3 -UTR) of cognate the expression of -α (ESR1), a ligand-inducible mRNA targets (7), have recently been shown to play crucial implicated in neuronal differentiation. Immu- roles in processes associated with development, differentiation, nohistochemical staining demonstrated ESR1 expression in human homeostasis, and cancer (8–10). Aberrant expression and dys- fetal sympathetic ganglia, suggesting a role for ESR1 during sym- regulation of miRNAs occur in a wide range of neoplasias and pathetic nervous system development. Concordantly, lentiviral their role in tumor initiation, development, and progression is restoration of ESR1 in NB cells resulted in growth arrest and neuro- becoming increasingly evident (11, 12). Several studies have nal differentiation. Moreover, lentiviral-mediated inhibition of reported deregulated miRNA expression in NB, including miR-18a in NB cells led to severe growth retardation, outgrowth miRNAs derived from the miR-17∼92 cluster (13). Abnormal of varicosity-containing neurites, and induction of neuronal sym- regulation of mRNAs identified downstream of NB-associated pathetic differentiation markers. Bioinformatic analyses of micro- array data from NB tumors revealed that high ESR1 expression miRNAs include the Bcl-2 interacting mediator of cell death BIM correlates with increased event-free survival in NB patients and ( ), the neurotrophin receptor tropomyosin-related kinase C Cip1/Waf1/Sdi1 favorable disease outcome. Thus, MYCN amplification may disrupt (trkC), the p21 cyclin-dependent kinase inhibitor estrogen signaling sensitivity in primitive sympathetic cells (p21), and the potent transcriptional inducer of cell-cycle pro- through deregulation of ESR1, thereby preventing the normal gression, (13–15). Thus, owing to miRNA misexpression, induction of neuroblast differentiation. Collectively, our findings perturbations in critical pathways linked to proliferation, apop- demonstrate the molecular consequences of abnormal miRNA tosis, and cell cycle progression have been shown to play a transcription in a MYCN-driven tumor and offer unique insights central role in the development of NB. into the pathology underlying MYCN-amplified NB. In this study, we have investigated molecular mechanisms underlying MYCN-induced NB progression. Our findings show oncogene | embryonic development | pediatric tumor | transcription that hsa-miR-18a and hsa-miR-19a (hereafter referred to as miR- factor | 18a and miR-19a), from the oncogenic miR-17∼92 cluster, target and subsequently repress the expression of estrogen receptor-α euroblastoma (NB) represents a remarkably heterogeneous (ESR1). We propose that ESR1 represents a previously unde- pediatric cancer derived from precursor cells of the sym- N scribed MYCN target in NB and demonstrate a unique oncogenic pathetic ganglionic lineage, with clinical behavior ranging from circuitry in which the repression of ESR1 through MYCN-regu- spontaneous regression to rapid progression and death (1, 2). Despite the frequent display of multiple genetic defects, lated miRNAs may play a fundamental role in NB tumorigenesis. including chromosomal gains and losses, aneuploidy, and amplification of chromosomal material, few established molec- ular genetic markers associate with disease outcome. Amplifi- Author contributions: J.L. and M.H. designed research; J.L., N.Z., T.W., I.M., and A.P. MYCN ≈ performed research; S.P. contributed new reagents/analytic tools; J.L., P.B., E.F., U.R., cation of the locus, present in 20 to 30% of all cases, S.P., and M.H. analyzed data; and J.L., S.P., and M.H. wrote the paper. represents the most important genetic aberration, and is strongly The authors declare no conflict of interest. MYCN related to poor clinical diagnosis (3). is expressed in the Freely available online through the PNAS open access option. migrating neural crest and encodes a phosphoprotein that – – Data deposition: Protocols, as well as raw and processed data for the miRNA microarrays, belongs to the network of helix loop helix are publicly available at www.ebi.ac.uk/arrayexpress: E-MEXP-2006. transcriptional regulators, which play key roles in governing cell 1Present address: Department of Immunology and Infectious Diseases, Harvard School of growth, apoptosis, and differentiation (4). Transcriptional acti- Public Health, Boston, MA 02115. vation is mediated by binding of the Myc/Max dimer to the 2To whom correspondence should be addressed. E-mail: [email protected]. consensus E-box sequence CA(C/T)GTG in target gene pro- This article contains supporting information online at www.pnas.org/cgi/content/full/ MEDICAL SCIENCES moters while the mechanism of Myc-mediated transcriptional 0913517107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0913517107 PNAS | January 26, 2010 | vol. 107 | no. 4 | 1553–1558 Downloaded by guest on September 24, 2021 Results MYCN-amplified cell lines and tumors compared to the non- Expression Analysis of miRNAs in Tet21N Cells Reveals Specific miRNA MYCN amplified samples (Fig. 1C and Fig. S1 C and D). In Seed-Sequence Signatures. We used miRCURY Locked Nucleic addition, qPCR analysis for miR-18a and miR-19a showed that Acid (LNA) arrays capable of assaying 558 human-specific there is an overall correlation between all three methods (Fig. S1 E F miRNAs to identify MYCN-regulated miRNAs in Tet21N NB and ). cells with inducible MYCN expression. In doxycycline (Dox)-free medium, Tet21N cells express exogenous MYCN in levels similar MYCN Coordinates Transcriptional Regulation of miRNA Paralogs from Three Distinct Loci. It was evident from our array analyses to common NB cell lines (16). We observed consistent up- that the majority of up-regulated miRNAs originated from three regulation of several MYC-associated miRNAs previously iden- distinct clusters/loci mapping to 13 (miR-17∼92), tified (17, 18), including miRNAs from the oncogenic miR-17∼92 chromosome 7 (miR-106b∼25), and chromosome X (miR- cluster (e.g., miR-17, miR-18a, and miR-19a) (19) and its paralogs 106a∼363 A A A )(Fig. S1 ). Inspection of the genomic regions sur- on chromosome 7 and chromosome X (Fig. 1 , Fig. S1 , and rounding the miRNA clusters revealed several putative MYCN Table S1). Interestingly, we also noticed a robust down-regulation A E-box binding motifs. Using ChIP assays, we demonstrate that of miRNAs in Tet21N cells with high MYCN expression (Fig. 1 MYCN and Max associate with E-box sequences upstream of all and Table S1). Sequence comparisons of the up-regulated polycistron-derived miRNAs identified (Fig. S2), including the miRNAs uncovered four sets of seed signatures based on the host gene of the miR-106b∼25 cluster, MCM7. Furthermore, ′ sequence identity of nucleotides 2 to 8 from the 5 -end of the acetylation of histone H4 (α-AcH) was markedly enhanced in B fi mature miRNA (Fig. S1 ). By Northern blotting, we veri ed that these regions (Fig. S2), suggesting that these positions represent MYCN overexpression indeed resulted in a differential miRNA regions of transcriptionally active chromatin (20). expression pattern (Fig. 1B). Analyses of several NB cell lines and tumor specimens revealed that expression levels of miRNAs from Transient Knockdown of miR-18a and miR-19a Impedes Cell the oncogenic miR-17∼92 cluster were consistently higher in Proliferation in MYCN-Amplified NB Cells. Next, we investigated putative target genes of the miRNAs encoded by the identified polycistrons. Because overexpression of the miR-17∼92 cluster

1.5 strongly augments NB tumorigenesis (13), we focused on A miRNAs derived from this locus, thereby narrowing our target 1.0 * predictions. Furthermore, although the oncgenic contribution of * * miR-17 and miR-20a has been described (13), the biological 0.5 significance of miR-18a and miR-19a overexpression in NB has not been investigated in detail. We therefore examined high0 low the occurrence of (GO) terms associated with

-0.5 miR-18a and miR-19a target predictions from TargetScanHu-

2 man 5.1. The top significant (P < 0.01) target categories were

* (log MYCN /MYCN ) -1.0 * dominated by processes associated with cell cycle-related Relative microRNA transcription Relative mechanisms, morphogenesis, and metabolism (Fig. S3A). Nota- -1.5 bly, the miR-18a target predictions were significanlty enriched with GO categories associated with the cell cycle, cell division, B and cell growth. To test our GO analysis experimentally, we targeted miR-18a and miR-19a using LNA knockdown oligo- MYCNMYCN Low High MYCNMYCN Low High MYCNMYCN Low High MYCNMYCN Low High nucleotides in MYCN-amplified Kelly cells followed by EdU labeling and subsequent cell cycle analysis by FACS. Treatment with LNA-18a resulted in a robust decline in cell proliferation miR-17 miR-18a miR-19a miR-199a-5p (7.5 ± 2.9%, P = 0.0087) compared to cells transfected with LNA scramble control (32.8 ± 5.9%) as measured by EdU incorpo- U5 snRNA U5 snRNA U5 snRNA U5 snRNA ration (Fig. 2A). Inhibition of miR-19a also suppressed cell ± P = miR-17 miR-18a miR-19a proliferation, although to a slightly lesser extent (9.8 4.3%, C 5 5 5 0.0199). Together, these data suggest that miR-18a and miR-19a 4 4 4 overexpression provide MYCN-amplified cells with a prolif- 3 3 3 erative advantage by deregulating messages linked to cell cycle 2 2 2 progression. 1 1 1 Fold change Fold change Fold change 0 0 0 Stable Knockdown of miR-18a Results in Differentiation of MYCN-

(miR-17/U48 expression) (miR-17/U48 d d (miR-18a/U48 expression) (miR-18a/U48 ie ed expression) (miR-19a/U48 ie Amplified NB Cells. ifi lif lified To analyze the effect of long-term silencing pl p p mplif m am -a of miR-18a and miR-19a, we transduced SK-N-BE(2) cells with N- fi Non-amplified Non-am Non lentiviral vectors encoding speci c anti-sense microRNA MYCN-amplified MYCN-a MYC sequences designed to inhibit their function. At first, we Fig. 1. Identification of miRNA expression in Tet21N cells with high MYCN observed that SK-N-BE(2) cells infected with lenti-anti-miR-18a levels. (A) Relative transcription of array-identified miRNAs in Tet21N cells and lenti-anti-miR-19a grew noticeably slower than cells trans- with high versus low MYCN expression (P-value cutoff = P < 0.05). The color duced with lenti-scramble control. Interestingly, after prolonged code identifies miRNAs bearing the same seed sequence. Asterisks (*) indi- propagation, the cells that expressed anti-miR-18a underwent a cate miRNAs (green = miR-18a; red = miR-17; blue = miR-19a; and black = noticeable morphological differentiation, manifested by out- miR-199a-5p) validated using Northern blot in (B). (B) Northern blot analysis growth of varicosity-containing neurites that terminated in visible of Tet21N cells with high and low MYCN levels showing differential B expression levels of miR-17, miR-18a, miR-19a, and miR-199a-5p. Splicesomal growth cones (Fig. 2 ). In contrast, no apparent change in U5 snRNA served as a loading control. (C) qPCR analysis of mir-17, miR-18a, morphology was observed in scramble control or in anti-miR-19a and miR19a in MYCN-amplified (red triangles) and nonamplified (blue tri- transduced cells (Fig. 2B and Fig. S3B). We further observed a angles) primary NB tumors. Results are shown as fold-change compared to dramatic increase (4- to 6-fold) in expression of a set of neuronal the internal control small nucleolar U48 RNA. sympathetic differentiation markers [superior cervical ganglia-10

1554 | www.pnas.org/cgi/doi/10.1073/pnas.0913517107 Lovén et al. Downloaded by guest on September 24, 2021 A Kelly Cells * LNA Scramble LNA-18a LNA-19a 50 ** 105 105 105 40

104 104 104 30

103 103 103 20

2 2 2 Anti-EdU 10 Alexa 647 10 10 10 0 0 0 Proliferation 0 0 50K 100K 150K 200K 250K 0 50K 100K150K 200K 250K 0 50K 100K 150K 200K 250K

Propidium Iodide Propidium Iodide Propidium Iodide (%EdU incorporation)

LNA-18aLNA-19a

LNA-Scramble Lenti-Scramble Lenti-Anti-miR-18a B C Lenti-Scramble Lenti-Anti-miR-18a

SCG10 Fig. 2. Suppression of proliferation and neuronal differ- entiation following down-regulation of miR-18 in NB cells. GAP43 (A) Cell proliferation as measured by EdU incorporation of Kelly cells transfected with LNA-inhibitors for miR-18a (LNA-18a), miR-19a (LNA-19a), or control (LNA-scramble). NPY One representative FACS plot from four independent experiments is shown; the graph is the summary of all four experiments. (B) SK-N-BE(2) cells were transduced with

GFP Phase Contrast 0 2 4 6 8 lentiviral constructs expressing scramble or anti-miR-18a. Relative expression (Upper) Phase contrast images. (Lower) GFP expression for identification of the transduced cells. (C) qPCR analysis of the neuronal differentiation markers SCG10, GAP43,and SK-N-BE(2) Cells NPY in SK-N-BE(2) cells transduced with lenti-scramble or lenti-anti-miR-18a.

(SCG10), growth-associated 43 (GAP43) and neuro- introduced target site mutations abolished the ability of miR-18a peptide Y (NPY)] (21) in miR-18a-depleted cells compared with to regulate the ESR1 3′-UTR in these settings (Fig. 3B). MiR- scramble control (Fig. 2C), indicating that miR-18a may repre- 19a also reduced 3′-ESR1–wt luciferase activity, albeit to a lesser sent a unique, key regulator of neuronal differentiation in cells degree (∼40%). Cotransfection of miR-18a and miR-19a pre- derived from the sympathetic ganglionic lineage. cursors also resulted in a strong repression of 3′-ESR1–wt activity, even when the amount of each miRNA precursor was miR-18a and miR-19a Negatively Regulate ESR1 Expression via miRNA reduced by half (Fig. 3B). Taken together, these data suggest Elements in Its 3′-UTR. To investigate the mechanisms through that ESR1 expression may be negatively regulated via both miR- which miR-18a and miR-19a induces growth arrest and sub- 18a and miR-19a 3′-UTR miRNA binding sites. sequent differentiation, we examined predicted overlapping To assess miRNA-mediated repression of endogenous ESR1, target sets of these miRNAs using three algorithms: Target- we overexpressed miR-18a and miR-19a precursor molecules in ScanHuman 5.1 (22), PicTar (23), and EIMMo (24). Several MCF-7 cells. Ectopic expression of miR-18a resulted in a sub- interesting genes were predicted to be targets of miR-18a and stantial down-regulation (∼90%) of ESR1 protein levels within 24 miR-19a, many of which are involved in neural or cancer- h of transfection, which was maintained throughout the time- associated processes (Table S2). One gene in particular, ESR1, course, indicating that miR-18a binding sites are highly efficient in scored well in all algorithms used and was selected for further conferring immediate and long-lasting repression of ESR1 (Fig. analysis. Inspection of the 3′-UTR of ESR1 revealed two 3C). Forced miR-19a expression also resulted in reduced ESR1 potential miR-18a target sites that generate a palindrome-like levels 24 h posttransfection, although the degree of inhibition was miR-18 target region composed of two closely positioned 8mer less pronounced when compared to miR-18a (Fig. 3C). Over the seed-matched sites (Fig. S4A). Moreover, the ESR1 3′-UTR next 48 h, ESR1 protein levels continued to drop until reaching a harbors two highly conserved binding sites for miR-19a in the level comparable to miR-18a transfected cells. qPCR analysis distal 3′-end of its UTR (Fig. 3A). The presence of multiple demonstrated that the observed dampening of ESR1 protein cluster-related miRNA binding sites indicates that MYCN- coincided with a time-dependent ESR1 transcript reduction, dependent regulation of ESR1 can be achieved through the indicative of mRNA destabilization, the major mode of repression concomitant expression of miR-17∼92-derived miRNAs. for highly suppressed miRNA targets (25) (Fig. S4C). To demonstrate that miR-18a and miR-19a directly regulate ESR1 expression, we transfected a ESR1 3′-UTR (3′-ESR1-wt) ESR1 Is Repressed by MYCN-Induced miRNAs and Contributes to luciferase reporter construct (Fig. S4B) together with miR-18a Neuronal Differentiation of NB Cells. Long-term MYCN repression precursors into HEK-293 cells and noticed an ∼60% reduction in in Tet21N cells revealed a significant recovery of ESR1 protein luciferase activity compared to cells transfected with the scram- over time, demonstrating that reduction of miR-18a and miR- ble control (Fig. 3B). Conversely, no reduction in luciferase 19a expression through the prolonged dampening of MYCN activity was detected with the miR-18a seed-modified ESR1 3′- protein levels alleviates translation and protein maturation of UTR (3′-ESR1-mut) reporter construct, demonstrating that the ESR1 (Fig. 4A and Fig. S5A). In MYCN3 cells, which contain a MEDICAL SCIENCES

Lovén et al. PNAS | January 26, 2010 | vol. 107 | no. 4 | 1555 Downloaded by guest on September 24, 2021 A ‘miRNA domain’ 1 ‘miRNA domain’ 2 A BC

ScrambleAnti-miR-18a 2x miR-18 miR-19 miR-19 ESR1 3’UTR (4.3 kb) - Dox + Dox 48+ Doxhr 1 week - Dox+ Dox 24+ Dox hr 48+ Dox hr 72 hr - Dox - Dox *** MYCN Conservation MYCN MYCN ESR1 ESR1 ESR1 3’-ESR1-wt 3’-ESR1-mut β-actin β-actin β-actin Tet21N cells Tet21N cells Tet21N B *** MYCN3 cells (MYCN Tet-Off) (MYCN Tet-Off) 120 *** 120 (MYCN Tet-On) *** 100 100 80 80 D E F 60 60 GFP-sorted cells GFP-analysed cells Lenti-Empty Vector Lenti-ESR1 40 40 20 20 0 0 20 Lucifease Activity (%)

Lucifease Activity (%) *** 15

miR-18a miR-19a miR-18a Scramble Scramble Lenti-EmptyLenti-ESR1 Vector 10

ESR1 Phase Contrast 5 miR-18a + miR 19a β-actin Proliferation

(%EdU incorporation) 0 C GFP Mock miR-18amiR-19aScrambleMock miR-18amiR-19aScrambleMock miR-18amiR-19aScramble Lenti-ESR1 ESR1 Lenti-Empty Vector

-actin Fig. 4. ESR1 is repressed by MYCN-induced miRNAs and contributes to MCF-7 cells neural differentiation of NB cells. (A) Expression of ESR1 protein in Tet21N 24 hours 48 hours 72 hours cells following short-term (+Dox 48 h) and long-term (+Dox 1 week) MYCN repression. (B) ESR1 expression following MYCN induction of MYC3 cells Fig. 3. miR-18a and miR-19a negatively regulate ESR1 expression via its 3′- during 24, 48, and 72 h. (C) MYCN expressing Tet21N cells (−Dox) were UTR. (A) Schematic representation of the human 3′-UTR of ESR1 indicating transduced with lenti-scramble or lenti-anti-miR-18a followed by analysis for potential miR-18a and miR-19a binding sites. Triangles indicate possible ESR1. (D) ESR1 expression in SK-N-BE(2) cells transduced with lenti-ESR1 or binding sites for other miRNAs. Asterisks below the ESR1 3′-UTR display lenti-empty vector control. (A–D) Western blot analysis, β-actin served as predicted poly(A) sites using a support vector machine as described in (43). loading control. (E) Cell proliferation (as measured by percent-EdU incor- The evolutionary conservation, shown below the ESR1 3′-UTR, was gen- poration) after FACS analysis of lenti-ESR1 and control (lenti-empty vector) erated using the UCSC Genome Browser ( May 2004 assem- transduced cells (asterisks denote significant differences between indicated bly) (B) Histogram indicating the levels of luciferase activity in HEK-293 cells samples; *** P < 0.001 Student’s t-test for unpaired data). (F) Morphology of transfected with the miRNAs indicated together with the wild-type 3′-UTR SK-N-BE(2) cells stably transduced with lenti-ESR1 or lenti-empty vector. (3′-ESR1-wt) reporter or with a mutant derivative (3′-ESR1-mut). Data shown Upper panel shows phase contrast images, while the lower panel shows GFP are means of quintuplicate experiments and error bars represent standard expression for identification of the transduced cells. deviations. Asterisks denote significant differences between indicated sam- ples (*P < 0.05; **P < 0.01; *** P < 0.001 Student’s t test for unpaired data). (C) Western blot showing downregulation of ESR1 in MCF-7 cells following After prolonged propagation, we observed a robust ESR1- transfection with miRNA precursors or scramble control as indicated. Mock dependent morphological shift, marked by extensive neurite denotes untransfected cells and β-actin was used as loading control. outgrowth that did not occur in control cells (Fig. 4F). Thus, reintroduction of ESR1 expression in NB cells resulted in a pronounced cell cycle arrest and morphological changes indica- doxycycline-inducible (Tet-On) MYCN transgene (Fig. S5B), tive of neuronal differentiation. MYCN induction resulted in ESR1 protein loss over time (Fig. B fi 4 ), and was accompanied by signi cantly increased levels of ESR1 Is Expressed During Human Fetal Neuronal Development and Is C miR-18a and miR-19a (Fig. S5 ). Finally, we analyzed the effect Associated with a Favorable Disease Outcome. To establish the of robust knockdown of miR-18a on ESR1 protein levels in physiological relevance of ESR1 expression during normal MYCN expressing Tet21N cells. Infection with lentiviral par- development of the sympathetic nervous system, we stained for ticles encoding anti-miR-18a resulted in ESR1 protein recovery ESR1 in abdominal cross-sections from a 9-week-old human in miR-18a-depleted Tet21N cells, despite high MYCN levels fetus. Expression of tyrosine hydroxylase was used as a marker (Fig. 4C). Collectively, our data provide a link between MYCN for symphathetic ganglia (21) (Fig. 5A). We could indeed dem- protein expression and subsequent modulation of an identified onstrate nuclear, but also cytoplasmic, ESR1 expression in downstream target, ESR1, via the regulation miR-17∼92 cluster- human fetal sympathetic ganglia (Fig. 5A), indicating that ESR1 derived miRNAs. has a role during human neuronal development. Furthermore, Next, we analyzed the functional role of ESR1 expression in ESR1 expression analysis of the primary NB tumors used in Fig. NB cells. Even though it is well-established that ESR1-mediated 1 demonstrated an inverse relationship between MYCN and signaling contributes to proliferation of breast cancer cells (26), ESR1 levels (Fig. S6A). None of the MYCN amplified tumors it has been reported that ligand-dependent activation of ESR1 in showed strong ESR1 expression, while the majority of non- neuronal cells results in growth arrest and differentiation (27). amplified tumors expressed moderate to high levels of ESR1.By To reaffirm this finding, we used lentiviral constructs expressing querying clinical microarray data from several sources, including ESR1 cDNA and transduced SK-N-BE(2) cells. Western blot the GeneSapiens database, we could establish that ESR1 is analysis of GFP-positive cells confirmed an enhanced expression expressed at low but detectable levels in NB specimens (28–30). of ESR1 at the protein level (Fig. 4D), and subsequent analysis Analysis of a NB microarray data set representing 251 children of EdU incorporation indicated that re-establishment of ESR1 (28), demonstrated a weak but significant negative correlation expression in SK-N-BE(2) cells resulted in a twofold decrease in between MYCN and ESR1 (r = −0.26, P < 0.001, n = 251) an proliferation (8.5 ± 0.3% EdU incorporation) compared to observation that was strengthened when analyzing the MYCN empty vector control (14.1 ± 0.2% EdU incorporation) (Fig. 4E). amplified cases only (r = −0.37, P < 0.04, n = 33) (Fig. S6B).

1556 | www.pnas.org/cgi/doi/10.1073/pnas.0913517107 Lovén et al. Downloaded by guest on September 24, 2021 MYCN fi A TH ESR1 (1:5) ESR1 (1:10) ESR1 (1:50) retinoic acid-induced differentiation of the -ampli ed cell line SK-N-BE (14). All together, this implies that specific changes

20X in miRNA expression levels may promote tumorigenic behavior of MYCN-amplified NB. Our findings illustrate that the direct reprogramming of the miRNA transcriptome allows MYCN to modulate additional, fi ESR1 40X previously unidenti ed downstream targets, such as .We demonstrate that MYCN activation of miR-18a and miR-19a results in the negative regulation of ESR1 expression. Both of the miR-18a and miR-19a target sites are located within highly con- Developing human fetal sympathetic ganglia DCIS (9 weeks) served genomic regions, suggesting that these sites are under selective pressure to preserve their sequence, and presumably B ESR1 expression ESR1 expression quartile their functionality, across evolution. The presence of multiple 100 100 cluster-related miRNA binding sites indicates that MYCN- 80 80 dependent regulation of ESR1 can be achieved through the con- 60 60 comitant expression of miR-17∼92-derived miRNAs. Recently, 40 40 several c-Myc-regulated miRNAs derived from the miR-17∼92 Above median 20 20 1st 3rd miR-106a∼363 Below median and the clusters (including miR-18 and miR-19 2nd 4th 0 0 family members) were shown to negatively regulate ESR1 Event-free survival (%) Event-free survival (%) 0 1000 2000 3000 4000 5000 0 1000 2000 3000 4000 5000 expression in an estradiol-dependent manner in breast cancer cells (36), reinforcing the notion of a complex genetic circuitry Follow-up time (days) Follow-up time (days) involving Myc , miR-17∼92-derived miRNAs, and ESR1. Fig. 5. ESR1 is expressed during human fetal neuronal development and Furthermore, we have demonstrated that inhibition of miR- correlates to increased event-free survival in NB. (A) Immunohistochemical 18a and miR-19a expression results in a pronounced cell cycle demonstration of ESR1 protein in developing sympathetic ganglia in an arrest, suggesting that overexpression of these miRNAs in abdominal human fetal (week 9) cross-section. Fetal sympathetic ganglia MYCN-amplified NB may interfere with normal induction of fi were identi ed by their location and by tyrosine hydroxylase (TH) positivity. growth arrest and the subsequent onset of differentiation. Ele- Adjacent sections were stained with two different dilutions (1:5 and 1:10) of vated levels of miR-18a, and in part miR-19a, may therefore the anti-ESR1 antibody demonstrating cytoplasmic and nuclear staining. An MYCN ESR1 positive ductal breast carcinoma in situ (DCIS) was used as a positive provide -driven tumors with a proliferative advantage by staining control (anti-ESR1 antibody dilution 1:50). (B) ESR1 mRNA levels, as deregulating messages linked to these processes. The GO cate- analyzed using microarrays (28) were correlated to event- gories associated with predicted miR-18a and miR-19a targets free survival for a group of 251 NB patients. Patients were divided into two showed significant overlap with biological processes that are groups based on ESR1 expression levels above (red line) or below (green line) overrepresented in the Myc target gene network (37), implying cohort median (Left). The two groups showed a significant difference in that MYCN-responsive miRNAs act to synergise with the bio- event-free survival (P = 0.04, log-rank test). Patients categorized into four logical function of MYCN on a genome-wide level. Notably, we fi equal-size quartile groups based on ESR1 expression showed a signi cant found that stable, long-term inhibition of miR-18a in MYCN correlation between increasing ESR1 expression and improved event-free amplified NB cells resulted in robust morphological and bio- survival (P = 0.002, log-rank test) (Right). chemical differentiation. It is tempting to speculate that miR-18a may be a promising therapeutic target for MYCN-amplified NB Furthermore, Kaplan-Meier survival analysis showed that high tumors. Likewise, the morphological changes observed following expression of ESR1 correlated to an increased event-free survival the restoration of ESR1 in NB cells together with our Kaplan- (P = 0.04, log-rank test, n = 251) (Fig. 5B). This relationship Meier survival analysis of 251 NB patients suggest that ESR1- became more apparent when dividing the 251 patients into four positive NBs may represent a potential subgroup of tumors quartile groups based on increasing ESR1 expression: patients amenable to estrogen treatment. with low expression of ESR1 (first quartile; blue line) had a sig- To our knowledge, ESR1 represents a unique MYCN target in nificantly impaired event-free survival compared to patients with NB and is one of the key estrogen-receptor subtypes known to mediate the pleiotropic effects, including differentiation, of comparatively high ESR1 expression (fourth quartile; red line)(P estrogen during the development and maturation of the nervous = 0.002, log-rank test, n = 251) (Fig. 5B). In light of these data, system. Our demonstration of ESR1 expression in human fetal ESR1 may represent a previously unidentified prognostic marker MYCN fi sympathetic ganglia suggests that ESR1 plays an important role in -ampli ed NB patients. during the development of the sympathetic nervous system. Discussion Furthermore, ESR1 has been reported to induce growth arrest and differentiation of NB cells following estradiol treatment The biological impact of deregulated miRNA expression in NB fi – (27). Our data support this nding, as we observed ESR1- has just begun to emerge (14, 15, 18, 31 34), reinforcing the mediated neuronal differentiation of SK-N-BE(2) cells. The importance of miRNA biology in NB-associated tumorigenesis. importance of estrogen and its receptors during neural devel- Here, we describe a MYCN-mediated miRNA signature involving opment is recapitulated in embryonic neuronal stem cells, which the activation and down-regulation of several miRNA genes from undergo differentiation in response to estradiol exposure (38). paralogous clusters. In line with previous reports, we show that Clearly, estrogen signaling plays a pivotal role in neuroendocrine MYCN transcriptionally activates oncogenic miRNAs from the biology, which if impaired, may have detrimental consequences miR-17∼92 cluster and its paralogs miR-106a∼363 and miR- for normal developmental processes, including proper matura- 106b∼25. This preferential selection of miRNAs bearing the same tion and differentiation of progenitor cells derived from the seed sequence suggests that MYCN regulates downstream targets sympathetic nervous system. During human fetal life, the through the coordinated control of specific miRNA families. androgenic precursors dehydroepiandrosterone sulfate and 16α- Importantly, our array data show that activation of MYCN also hydroxyde-dehydroepiandrosterone sulfate of fetal and maternal leads to extensive suppression of miRNAs, several of which have adrenal origin are hydrolyzed in the placenta with the sub- tumor-suppressive like functions (14, 35). The expression of many sequent formation of estrogens (39, 40). Like other steroid of these miRNAs was recently reported to increase upon trans- hormones, estrogens easily pass through the placental barrier MEDICAL SCIENCES

Lovén et al. PNAS | January 26, 2010 | vol. 107 | no. 4 | 1557 Downloaded by guest on September 24, 2021 and enter the fetal adrenomedullary circulation (41). As a result, Materials and Methods fi immature sympathetic cell groups that are in suf cient contact Tet21N, a derivative of the SH-EP human NB cell line containing a doxycycline- with the circulation may undergo differentiation in response to repressible (Tet-Off) MYCN gene, was cultured as previously described (16). estrogen, given that they express estrogen receptors. Sympathetic To repress MYCN expression, doxycycline was added at a final concentration neuroblasts are frequently seen in developing human fetal of 1 μgmL−1. Chromatin immunoprecipitation assays and Western blots adrenal glands, which have a well-developed network of capil- were performed as described (44, 45). Cell culture, miRNA microarray laries, venules, and large veins intended to provide surrounding hybridization and data processing, qPCR, Northern blot, luciferase assays, cells with appropriate environmental cues, such as estrogens. flow cytometry analysis, transfections, immunohistochemistry, gene Likewise, the small intensely fluorescent cells that reside in the expression data analysis, lentiviral transductions, as well as antibodies, sympathetic ganglia are closely associated with fenestrated constructs, and primers are described in SI Materials and Methods. capillaries and may also require molecular signals provided by the fetal circulation to further promote their development (42). ACKNOWLEDGMENTS. We thank M. Schwab and J. Shohetfor cells, S. Beckman, Hence, the steroidogenic activity of the fetal adrenal gland can E. Johansson, and E. Pivarcsi for excellent technical assistance, L.-G. Larsson for critical reading of the manuscript, M. Corcoran, J.-Å. Gustafsson, C. Williams, impart differentiation cues necessary for the elements of the and members of our laboratory for stimulating discussions. J.L. and I.M. were sympathetic ganglionic lineage to acquire a fully differentiated supported by Karolinska Institutet and N. Z. by a stipend from the Cancer phenotype. Aberrant regulation of ESR1 expression in primitive Research Institute (New York) and Concern Foundation (Los Angeles, CA). M.H. sympathetic cells by MYCN-driven miRNAs may interfere with is a recipient of the Senior Investigator Award from the Swedish Cancer Society. This work was supported by funding from the Swedish Childhood Can- the normal induction of neuroblast differentiation and thus cer Foundation and the Swedish Cancer Society (S.P and M.H), the Swedish Foun- represents a unique tumorigenic mechanism that may play a dation for Strategic Research (S.P), the Hedlund Foundation and Karolinska fundamental role in NB etiology. Institutet (M.H).

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