Transcriptional activation of the miR-17-92 miRNA cluster by MYC/MYCN transcription factors occurs in multiple tumor entities, including neuroblastoma (Hayashita et al., 2005
; Mestdagh et al., 2009a
; O'Donnell et al., 2005
). Although the oncogenic nature of miR-17-92 activation is well established, the underlying targets and signaling cascades that are deregulated remain largely elusive. In addition, studies aimed at determining miR-17-92 targets have focused on individual members of the cluster, despite the observation that the entire cluster is activated (Mestdagh et al., 2009b
; O'Donnell et al., 2005
). Here we have used an unbiased proteomics approach to identify miR-17-92 targeted pathways in a neuroblastoma tumor model. Direct quantitative measurement of protein expression is preferred over the more straightforward mRNA profiling as a high-throughput method for miRNA target identification (Baek et al., 2008
; Selbach et al., 2008
Computational analysis of miR-17-92 seeds in the 3’UTR of transcripts from proteins supported the expected enrichment of direct miR-17-92 targets within the list of down regulated proteins detected using mass spectrometry. Moreover, a proportional relationship between seed frequency and fold downregulation was noted. This relationship not only holds for multiple seeds from an individual miR-17-92 miRNA but also for multiple seeds from different miR-17-92 miRNAs, suggesting cooperation between individual miRNAs from the cluster towards target protein repression. MiR-17-92 miRNAs have indeed been shown to function in a cooperative and additive manner amongst others in the regulation of PTEN by miR-17 and miR-19 (Xiao et al., 2008
). Our results further indicate that miR-19a/miR-19b and miR-17/miR-20a sites significantly co-occur in the 3’UTR of transcripts from several downregulated proteins. As these co-occurring sites were not observed for every possible combination of individual miR-17-92 miRNAs, we hypothesize that in neuroblastoma, the miRNA components of the miR-17-92 cluster can regulate target expression either individually or in certain combinations with additive effects. However, miR-17-92 function might be highly context and cell-type specific as miR-19 was shown to be both necessary and sufficient to promote MYC-induced lymphomagenesis in the Eµ-myc mouse B-cell lymphoma model (Olive et al., 2009
While the fraction of downregulated proteins was enriched for seeds of miR-17/miR-20a, miR-19a/miR-19b and miR-92a, enrichment for the miR-18a seed was not detected. Strikingly, miR-18a seeds rarely occur as the only seed(s) in the 3’UTR of a downregulated target and showed little or no correlation to protein fold change. Although this suggests that miR-18a is not substantially contributing to target deregulation, it does not imply that miR-18a lacks functionality, as miR-18a has been shown to regulate important cancer genes such as CTGF in colon cancer and estrogen receptor-α (ESR1) in neuroblastoma (Dews et al., 2006
; Loven et al.
). Interestingly, we found miR-18a to regulate both SMAD2 and SMAD4, 2 key components of the TGFβ-signaling cascade, suggesting that miR-18a substantially contributes to pathway deregulation by regulating a selected set of target genes.
When all cluster components were combined, we identified a large number of targeted proteins belonging to diverse cancer-related pathways. Notably, estrogen receptor signaling was also among the targeted pathways. The fact that we identified such a wide variety of functions in neuroblastoma cells suggests that miR-17-92 pleiotropy is not only related to different targets in different cell types but also occurs within cell types. The molecular basis for this observation likely lies within the multiple components of the cluster and the complex interplay between them.
Mir-17-92-directed regulation of the TGFβ-responsive genes CDKN1A
in neuroblastoma cells has been described by previously (Fontana et al., 2008
). In gastric cancer, members of the miR-106b-25 cluster have also been shown to target CDKN1A
(Petrocca et al., 2008
). Here we comprehensively demonstrate that miR-17-92 dampens TGFβ-signaling in a multifaceted way by acting both upstream and downstream of pSMAD2/SMAD4, further underscoring its ability to regulate multiple components of the same pathway. This ability to simultaneously target the components of the signaling cascade as well as the downstream effectors through multiple miRNAs, allows for tight control of the TGFβ-transcriptional program. Moreover, it offers the cells enormous flexibility and plasticity for regulation of different subsets of TGFβ-target genes. In neuroblastoma, enhanced TGFβ-signaling, through increased TGFBR2 expression, results in reduced cell growth in vitro
and disables the ability of the cells to form tumors in vivo
(Turco et al., 2000
). Instead, cells assume a terminally differentiated neuronal phenotype and display increased expression of axonal growth-associated protein (GAP43) and neurofilaments (Turco et al., 2000
). Treatment of neuroblastoma cells with TGFβ1 induces a similar phenotype (Scarpa et al., 1996
). In addition, retinoic acid (RA) induces differentiation of neuroblastoma cells, known to down regulate MYCN, accompanied by the increased expression of TGFβ1, TGFBR1, TGFBR2 and TGFBR3, resulting in the induction of a negative autocrine TGFβ1 growth regulatory loop (Cohen et al., 1995
). We have shown that aggressive neuroblastoma tumors evade the cytostatic TGFβ-pathway through miR-17-92 directed targeting of key components of the pathway as well as downstream effectors. Reactivation of TGFβ-signaling through miR-17-92 inhibition could be a promising therapeutic approach as it would not only result in reactivation of TGFBR2 expression but also relieve the direct miR-17-92-mediated repression of TGFβ responsive genes.