MicroRNA-542-5p as a novel tumor suppressor in neuroblastoma

doi:10.1016/j.canlet.2011.01.016

Cancer Letters

Volume 303, Issue 1, 1 April 2011, Pages 56-64

https://doi.org/10.1016/j.canlet.2011.01.016 Get rights and content

Abstract

Several studies have implicated the dysregulation of microRNAs in neuroblastoma pathogenesis, an often fatal paediatric cancer arising from precursor cells of the sympathetic nervous system. Our group and others have demonstrated that lower expression of miR-542-5p is highly associated with poor patient survival, indicating a potential tumor suppressive function. Here, we demonstrate that ectopic over-expression of this miRNA decreases the invasive potential of neuroblastoma cell lines in vitro, along with primary tumor growth and metastases in an orthotopic mouse xenograft model, providing the first functional evidence for the involvement of miR-542-5p as a tumor suppressor in any type of cancer.

Introduction

Mature, biologically active, miRNAs are ∼22 nucleotides in length and are involved in the regulation of gene expression at a post-transcriptional level. The targeting of specific sequences within the mRNA 3′ untranslated region (UTR) by miRNAs within the RNA induced silencing complex (RISC) results in either degradation of the mRNA or translational inhibition [1]. More recently, it has also become apparent that miRNAs can target sites that are in 5′ UTR or exonic positions, and that interaction of miRNAs with gene promoters can regulate gene activity at a transcriptional level [2], [3], [4]. Several studies have demonstrated a significant role for microRNAs in various forms of cancer, including a role in the pathogenesis of neuroblastoma [5], [6], [7], [8]. Neuroblastoma is a paediatric cancer derived from precursor cells of the sympathetic nervous system which display extreme heterogeneity in clinical behaviour, ranging from spontaneous regression to rapid progression and death due to disease [9].

A number of recurrent genomic abnormalities have been associated with aggressive disease course in neuroblastoma, including MYCN amplification, loss of 1p and 11q material, and gain of 1q and 17q, as reviewed by Stallings [10]. Recent miRNA expression profiling studies have demonstrated that miRNA expression has been dysregulated by these genomic aberrations and that the expression levels of specific miRNAs can be significantly associated with clinical outcome [6], [7], [11]. Moreover, in vivo and in vitro functional studies have further implicated a number of miRNAs, as having either oncogenic or tumor suppressor effects in neuroblastoma [12], [13], [14], [15], [16], [17], [18].

Recently, our research group [11] and others [6], [7] have demonstrated that miR-542-5p expression levels are very significantly inversely correlated with MYCN amplification and that low expression of this miRNA is highly associated with poor clinical outcome in neuroblastoma. However, functional studies confirming a tumor suppressive function have not been reported. Here, we provide the first in vitro and in vivo functional studies demonstrating the biological effects of this miRNA in neuroblastoma. To the best of our knowledge, this is also the first demonstration for a tumor suppressive function for this miRNA in any form of cancer.

Section snippets

Cell culture

Kelly and SKNAS cell lines were purchased from the European Collection of Animal Cell Cultures. Kelly and SKNAS cells were grown in EMEM and RPMI-1640, respectively, supplemented with 10% foetal bovine serum (FBS), 2 mM Glutamine and 2 mM penicillin and streptomycin (GIBCO® Invitrogen by Life Technologies Corp., Carlsbad, CA). NB1691 and SKNAS cell lines containing the luciferase plasmid were maintained in RPMI-1640 supplemented with FBS (10%), l-glutamine (1%) and 100 μg/mL Zeocin (InVivoGen, San

Association of miR-542-5p expression levels with patient event free (EFS) and overall (OS) survival and known predictive neuroblastoma disease risk factors

We previously profiled the expression of 449 miRNA loci in a cohort of 145 primary neuroblastoma tumors [11]. One of the most significantly associated miRNAs with patient survival was miR-542-5p, and in this report we provide a more in depth analysis of this miRNA with known disease risk factors and genetic subtypes. As expected, MYCN amplification was predictive of decreased EFS and OS in this data set (Fig. 1A and B). As indicated in Fig. 1C and D, patients with tumors that completely lacked

Discussion

Our group [11] and others [6], [7] have reported that very low expression of miR-542-5p in primary neuroblastoma tumors is highly correlated with poor patient survival, indicating that this miRNA might have a tumor suppressor function. Here, we provide the first functional evidence that miR-542-5p acts as a tumor suppressor in neuroblastoma by blocking cellular invasiveness and by decreasing primary tumor growth and metastasis in an orthotopic mouse xenograft model. Our finding that miR-542-5p

Conflict of interest

None declared.

Acknowledgements

This work was supported in part by a Science Foundation Ireland Short Term Travel Fellowship (AT), a Science Foundation Ireland Principal Investigator Award (07/IN.1/B1776)(RLS), the Children’s Medical and Research Foundation (RLS), the NIH (5R01CA127496) (RLS), the Assisi Foundation of Memphis (AMD), the US Public Health Service Childhood Solid Tumor Program Project Grant No. CA23099 (AMD), the Cancer Center Support Grant No. 21766 from the National Cancer Institute (AMD), and by the American

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Cited by (86)

Neuroblastoma
2023, Epigenetic Cancer Therapy, Second Edition
Neuroblastoma is a highly malignant pediatric cancer accounting for approximately 15% of all childhood cancer deaths. There is strong experimental evidence that patterns of genome-wide methylation and miRNA expression play a critical role in neuroblastoma pathogenesis. The purpose of this chapter is to review growing evidence of epigenetic changes contributing to aggressive disease development along with current and potential future epigenetic-based therapeutics.
The clinical utility of dysregulated microRNA expression in paediatric solid tumours
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However, it should be noted that a phase 1 clinical trial of a liposomal miR-34a mimic, MRX34, in adult patients with advanced solid tumours, was stopped early due to serious immune-related adverse events, so the clinical application of these in vitro and in vivo studies remains a key barrier to overcome [146]. Other miRNAs identified as potential TSGs in NB pathogenesis in functional experiments include miR-542-3p and miR-542-5p [147,148], miR-204 [149], miR-137 [94], miR-497 [96], miR-323a-5p and miR-342-5p [150], miR-15a-5p, miR-15b-5p and miR-16-5p [151], miR-26a-5p and miR-26b-5p [152], and miR-184 [153,154]. Conversely, miR-558 has been shown to have an oncogenic role as overexpression increased growth, invasion, metastasis and angiogenesis of NB cells via enhancing heparanase, an endogenous endoglycosidase that degrades heparan sulphate proteoglycans [155].
MicroRNAs (miRNAs) are short, non-protein-coding genes that regulate the expression of numerous protein-coding genes. Their expression is dysregulated in cancer, where they may function as oncogenes or tumour suppressor genes. As miRNAs are highly resistant to degradation, they are ideal biomarker candidates to improve the diagnosis and clinical management of cancer, including prognostication. Furthermore, miRNAs dysregulated in malignancy represent potential therapeutic targets. The use of miRNAs for these purposes is a particularly attractive option to explore for paediatric malignancies, where the mutational burden is typically low, in contrast to cancers affecting adult patients. As childhood cancers are rare, it has taken time to accumulate the necessary body of evidence showing the potential for miRNAs to improve clinical management across this group of tumours. Here, we review the current literature regarding the potential clinical utility of miRNAs in paediatric solid tumours, which is now both timely and justified. Exploring such avenues is warranted to improve the management and outcomes of children affected by cancer.
Nanotechnological based miRNA intervention in the therapeutic management of neuroblastoma
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Xiang et al. (2015) reported over-expression of miR-337-3p in NB to significantly inhibit the tumor growth, invasion, metastasis and angiogenesis, mediated via arrest of MMP-14 (matrix metalloproteinase 14) transcription [43]. Similarly, overexpression of miR-542-5p caused significant reduction of tumor in orthotropic models of NB, while that of miR-709 suppressed both transcriptional and post-transcriptional epigenetic silencing by forming complexes with H3K27me3 and AGO1 (Argonaute-1) of Egr2 promoter [44,45]. The down-regulation of MYCN and miR-17-5p level in NB cells and nude mice strongly augments p21 and BIM (at mRNA and protein level), by binding to 3′ UTR region, leading to 30% of cases with treatment success, i.e., complete inhibition of tumor invasion, metastasis, and angiogenesis along with stimulation of apoptosis [46].
Neuroblastoma (NB) is a widely diagnosed cancer in children, characterized by amplification of the gene encoding the MYCN transcription factor, which is highly predictive of poor clinical outcome and metastatic disease. microRNAs (a class of small non-coding RNAs) are regulated by MYCN transcription factor in neuroblastoma cells. The current research is focussed on identifying differential role of miRNAs and their interactions with signalling proteins, which are intricately linked with cellular processes like apoptosis, proliferation or metastasis. However, the therapeutic success of miRNAs is limited by pharmaco-technical issues which are well counteracted by nanotechnological advancements. The nanoformulated miRNAs unload anti-cancer drugs in a controlled and prespecified manner at target sites, to influence the activity of target protein in amelioration of NB. Recent advances and developments in the field of miRNAs-based systems for clinical management of NBs and the role of nanotechnology to overcome challenges with drug delivery of miRNAs have been reviewed in this paper.
Current challenges in the management of neuroblastoma
2019, Neuroblastoma: Molecular Mechanisms and Therapeutic Interventions
Neuroblastoma diagnostic criteria and therapeutic options have been identified and implemented within the oncology clinical setting as best possible and in a timely manner following the ever-changing advances in modern medical research technologies, as time progressed. Nonetheless, pediatric patients afflicted with this condition and their clinicians are still faced with a plethora of challenges on a daily basis that potentially thwart rapidly efficient and effective diagnosis, management, and treatment of this condition. This chapter serves as a humbling eye-opener on one of the recent, although most crucial, challenges hindering all those involved in neuroblastoma basic/translational research, namely, the influence of noncoding RNAs, with particular reference to the realm of microRNA (miRNA) and long noncoding RNA (lncRNA) theranostics.
Role of genetic and epigenetic alterations in pathogenesis of neuroblastoma
2019, Neuroblastoma: Molecular Mechanisms and Therapeutic Interventions
Neuroblastoma is a solid embryonal tumor of the sympathetic nervous system. The clinical course of patients with neuroblastoma is highly heterogeneous, from localized tumors and spontaneous regression to early metastasizing, rapid progression, and therapy resistance. This clinical heterogeneity can be attributed largely to the interaction of multiple genetic and epigenetic factors, including both sequence and copy number variants that occur in neuroblastoma cells. Recent methodological advances have enabled the detailed analysis of the neuroblastoma genome, leading to the identification of new prognostic markers and better patient stratification.
Amplification of MYCN gene and DNA ploidy have been used as the most reliable indicators of aggressive and treatment-resistant neuroblastoma, yet few of the high-risk tumors lack these features [1]. Candidate tumor suppressor genes have been identified on chromosomes 1p, 3p, and 11q that are deleted in some neuroblastomas. Despite extensive knowledge of somatically acquired genomic rearrangements in neuroblastoma and their association with the clinical tumor phenotype, very few data are available about the factors leading to these genetic events. While genetic alterations are almost impossible to reverse, epigenetic changes can dynamically respond to signals from physical, biological, and social environments [2]. New functional studies suggest that microRNAs regulate important genes involved in neuroblastoma pathogenesis [3].
Modulation of expression of miRNAs for therapeutic effects in human malignant neuroblastoma
2019, Neuroblastoma: Molecular Mechanisms and Therapeutic Interventions
The small non-coding RNAs are called microRNAs or miRNAs that play key roles in mRNA silencing as well as in post-transcriptional regulation of gene expression in eukaryotic cells. The finding of miRNAs as negative regulators of gene expression at post-transcriptional level have added a new layer to the fine regulation of cell signaling mechanisms in human health and disease. Many miRNAs are known to play key roles in tumor biology. The actions of miRNAs may produce oncogenic or tumor suppressive effects. A specific miRNA may act as an oncogenic miRNA or a tumor suppressor miRNA depending on the tumor. The pathogenesis of human malignant neuroblastoma, which is a deadly childhood tumor, is now known to be associated with upregulation of oncogenic miRNAs and down regulation of tumor suppressor miRNAs. Upregulation of oncogenic miRNAs drives the growth of human malignant neuroblastoma by avoiding cell cycle arrest, differentiation, and apoptotic death. On the other hand, the down regulation of tumor suppressor miRNAs promotes cell proliferation, hypoxia, autophagy, multidrug resistance, migration, invasion, angiogenesis, and metastasis in human malignant neuroblastoma. So, inhibition of expression of specific oncogenic miRNAs and promotion of expression of tumor suppressor miRNAs may provide novel therapeutic opportunities to induce cell cycle arrest, differentiation, and apoptosis in human malignant neuroblastoma cells in vitro as well as in vivo. The success of modulation of expression of miRNAs in preclinical models of human malignant neuroblastoma may eventually be translated to the clinics for successful treatment of neuroblastoma patients in the future.

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¹: These authors contributed equally to the work and are considered co-first authors.

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MicroRNA-542-5p as a novel tumor suppressor in neuroblastoma

Abstract

Introduction

Section snippets

Cell culture

Association of miR-542-5p expression levels with patient event free (EFS) and overall (OS) survival and known predictive neuroblastoma disease risk factors

Discussion

Conflict of interest

Acknowledgements

Mol. Cell

Cancer Genet. Cytogenet.

J. Pediatr. Surg.

Cell

MicroRNAs: small RNAs with a big role in gene regulation

Nat. Rev. Genet.

miR-148 targets human DNMT3b protein coding region

RNA

MicroRNA-373 induces expression of genes with complementary promoter sequences

Proc. Natl. Acad. Sci. USA.

Chromosomal and microRNA expression patterns reveal biologically distinct subgroups of 11q- neuroblastoma

Clin. Cancer Res.

MYCN/c-MYC-induced microRNAs repress coding gene networks associated with poor outcome in MYCN/c-MYC-activated tumors

Oncogene

Accurate prediction of neuroblastoma outcome based on miRNA expression profiles

Int. J. Cancer

Therapeutic targeting of miRNAs in neuroblastoma

Expert Opin. Ther. Targets

A developmental model of neuroblastoma: differentiating stroma-poor tumors progress along an extra-adrenal chromaffin lineage

Lab. Invest.