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

Abstract

MYCN, a proto-oncogene normally expressed in the migrating neural crest, is in its amplified state a key factor in the genesis of human neuroblastoma (NB). However, the mechanisms underlying MYCN-mediated NB progression are poorly understood. Here, we present a MYCN-induced miRNA signature in human NB involving the activation and transrepression of several miRNA genes from paralogous clusters. Several family members derived from the miR-17 approximately 92 cluster, including miR-18a and miR-19a, were among the up-regulated miRNAs. Expression analysis of these miRNAs in NB tumors confirmed increased levels in MYCN-amplified samples. Specifically, we show that miR-18a and miR-19a target and repress the expression of estrogen receptor-alpha (ESR1), a ligand-inducible transcription factor implicated in neuronal differentiation. Immunohistochemical staining demonstrated ESR1 expression in human fetal sympathetic ganglia, suggesting a role for ESR1 during sympathetic nervous system development. Concordantly, lentiviral restoration of ESR1 in NB cells resulted in growth arrest and neuronal differentiation. Moreover, lentiviral-mediated inhibition of miR-18a in NB cells led to severe growth retardation, outgrowth of varicosity-containing neurites, and induction of neuronal sympathetic differentiation markers. Bioinformatic analyses of microarray data from NB tumors revealed that high ESR1 expression correlates with increased event-free survival in NB patients and favorable disease outcome. Thus, MYCN amplification may disrupt estrogen signaling sensitivity in primitive sympathetic cells through deregulation of ESR1, thereby preventing the normal induction of neuroblast differentiation. Collectively, our findings demonstrate the molecular consequences of abnormal miRNA transcription in a MYCN-driven tumor and offer unique insights into the pathology underlying MYCN-amplified NB.

Fig. 1.

Identification of miRNA expression in Tet21N cells with high MYCN levels. (A) Relative transcription of array-identified miRNAs in Tet21N cells with high versus low MYCN expression (P-value cutoff = P < 0.05). The color code identifies miRNAs bearing the same seed sequence. Asterisks (*) indicate miRNAs (green = miR-18a; red = miR-17; blue = miR-19a; and black = miR-199a-5p) validated using Northern blot in (B). (B) Northern blot analysis of Tet21N cells with high and low MYCN levels showing differential expression levels of miR-17, miR-18a, miR-19a, and miR-199a-5p. Splicesomal U5 snRNA served as a loading control. (C) qPCR analysis of mir-17, miR-18a, and miR19a in MYCN-amplified (red triangles) and nonamplified (blue triangles) primary NB tumors. Results are shown as fold-change compared to the internal control small nucleolar U48 RNA.

Fig. 2.

Suppression of proliferation and neuronal differentiation following down-regulation of miR-18 in NB cells. (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). 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 lentiviral constructs expressing scramble or anti-miR-18a. (Upper) Phase contrast images. (Lower) GFP expression for identification of the transduced cells. (C) qPCR analysis of the neuronal differentiation markers SCG10, GAP43, and NPY in SK-N-BE(2) cells transduced with lenti-scramble or lenti-anti-miR-18a.

Fig. 3.

miR-18a and miR-19a negatively regulate ESR1 expression via its 3′-UTR. (A) Schematic representation of the human 3′-UTR of ESR1 indicating potential miR-18a and miR-19a binding sites. Triangles indicate possible binding sites for other miRNAs. Asterisks below the ESR1 3′-UTR display predicted poly(A) sites using a support vector machine as described in (43). The evolutionary conservation, shown below the ESR1 3′-UTR, was generated using the UCSC Genome Browser (human genome May 2004 assembly) (B) Histogram indicating the levels of luciferase activity in HEK-293 cells transfected with the miRNAs indicated together with the wild-type 3′-UTR (3′-ESR1-wt) reporter or with a mutant derivative (3′-ESR1-mut). Data shown are means of quintuplicate experiments and error bars represent standard deviations. Asterisks denote significant differences between indicated samples (*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 transfection with miRNA precursors or scramble control as indicated. Mock denotes untransfected cells and β-actin was used as loading control.

Fig. 4.

ESR1 is repressed by MYCN-induced miRNAs and contributes to neural differentiation of NB cells. (A) Expression of ESR1 protein in Tet21N cells following short-term (+Dox 48 h) and long-term (+Dox 1 week) MYCN repression. (B) ESR1 expression following MYCN induction of MYC3 cells during 24, 48, and 72 h. (C) MYCN expressing Tet21N cells (−Dox) were transduced with lenti-scramble or lenti-anti-miR-18a followed by analysis for ESR1. (D) ESR1 expression in SK-N-BE(2) cells transduced with lenti-ESR1 or lenti-empty vector control. (A–D) Western blot analysis, β-actin served as loading control. (E) Cell proliferation (as measured by percent-EdU incorporation) after FACS analysis of lenti-ESR1 and control (lenti-empty vector) transduced cells (asterisks denote significant differences between indicated samples; *** P < 0.001 Student’s t-test for unpaired data). (F) Morphology of SK-N-BE(2) cells stably transduced with lenti-ESR1 or lenti-empty vector.Upper panel shows phase contrast images, while the lower panel shows GFP expression for identification of the transduced cells.

Fig. 5.

ESR1 is expressed during human fetal neuronal development and correlates to increased event-free survival in NB. (A) Immunohistochemical demonstration of ESR1 protein in developing sympathetic ganglia in an abdominal human fetal (week 9) cross-section. Fetal sympathetic ganglia were identified by their location and by tyrosine hydroxylase (TH) positivity. Adjacent sections were stained with two different dilutions (1:5 and 1:10) of the anti-ESR1 antibody demonstrating cytoplasmic and nuclear staining. An ESR1 positive ductal breast carcinoma in situ (DCIS) was used as a positive staining control (anti-ESR1 antibody dilution 1:50). (B) ESR1 mRNA levels, as analyzed using gene expression microarrays (28) were correlated to event-free survival for a group of 251 NB patients. Patients were divided into two groups based on ESR1 expression levels above (red line) or below (green line) cohort median (Left). The two groups showed a significant difference in event-free survival (P = 0.04, log-rank test). Patients categorized into four equal-size quartile groups based on ESR1 expression showed a significant correlation between increasing ESR1 expression and improved event-free survival (P = 0.002, log-rank test) (Right).