Neuroblastoma (NB) is a tumour of the sympathetic nervous system. It is considered an embryonal cancer and most cases occur in children and adolescents. It is estimated that about 15,000 new cases of paediatric cancers are diagnosed yearly in Europe, with approximately 8–10% corresponding to neuroblastomas, the incidence of which is 1.8 cases per million. Neuroblastoma accounts for 15% of all cancer-related deaths in children, and is the embryonal tumour with the lowest 5-year relative survival.1 The primary site is often one of the adrenal glands, but it may also originate in nervous tissues of the cervical region, thorax, abdomen and pelvis. Patients with NB are assigned to different risk groups based on their clinical and pathological characteristics, such as stage of disease (INSS), age at diagnosis, MYCN oncogene amplification, tumour histology (Shimada classification) and DNA ploidy.

While the survival of low- and intermediate-risk patients is excellent, high-risk patients have a poor prognosis and require intensive chemotherapy regimens. In spite of the intensive regimens, more than 60% of children with NB are not cured.2,3

The treatment sequence applied to high-risk neuroblastoma involves four phases: induction therapy, local control, consolidation, and treatment of residual disease with biologic therapy.4 The induction regimen consists of a combination of anthracyclines, alkylators, platinum compounds and topoisomerase II inhibitors known as COJEC (cisplatin, vincristine, carboplatin, etoposide and cyclophosphamide). It is usually administered in eight cycles at 10-day intervals. The aim of this period of intensive chemotherapy is to reduce the size of the primary tumour (local control) to facilitate its surgical resection.5 After surgery, treatment enters the consolidation phase with dose-intensive myeloablative chemotherapy (busulfan, melphalan), followed by autologous hematopoietic stem cell transplantation. When the patient has recovered, local radiotherapy is administered at the primary tumour site and metastatic foci.

Last of all, biologic agents are used to treat residual disease, such as antibodies against specific NB antigens (such as anti-GD2) and 13-cis-retinoic acid as a differentiating agent, alone or combined with IL-2, which helps activate the immune system.

Despite sequential and combined treatments, 60% of the patients experience recurrence and develop metastases.2,5 The progression of high-risk NB is associated to development of drug resistance, usually to multiple drugs (multidrug resistance [MDR]). Multidrug resistance is usually due to various cellular factors and cell signalling pathways, and is one of the major barriers to successful chemotherapy. Some elements that are typical of high-risk NB contribute to MDR, such as increased oncogene expression (for instance, of MYCN), increased signalling by tyrosine kinase receptors (BDNF-TrkB) or altered function in tumour suppressor genes (such as p53).6–9 There is also evidence of altered expression or activation patterns of crucial elements in apoptosis signalling pathways.10,11 Furthermore, acquired resistance to chemotherapy may be caused by increased drug excretion from cells due to overexpression of membrane transporters, such as those in the ABC family.12,13

Given the multiple mechanisms that can lead to resistance to conventional therapies in NB, it is unlikely that treatments addressing a single target will suffice to treat tumours successfully. Therefore, we should strive to identify molecules capable of affecting multiple cell processes to improve response to treatment.

The field of epigenetics comprehends various mechanisms of gene expression that involve the structural modification of chromatin (for instance, DNA methylation or histone acetylation) and post-transcriptional regulation by noncoding RNAs (such as microRNAs). Changes in epigenetic patterns have been observed in NB tumour cells, including alterations in histone acetylation or aberrant methylation of promoter regions of DNA in specific genes such as caspase-814 or in large-scale blocks in chromosomes.15 These changes are directly related with patient survival,16 tumour metastatic potential17 and drug resistance.18

Thus, epigenetic therapies are emerging as an alternative to conventional treatment and aim to reverse epigenetic changes that may be contributing to the aggressiveness of tumours. Histone deacetylase inhibitors (for instance, valproic acid) are an example of the “epigenetic drugs” that are being researched; they are currently undergoing clinical trials and have shown beneficial effects in patients with minimal side effects.19

Parallel to the study of drugs that modulate chromatin structure or function, there is growing interest in the therapeutic use of post-transcriptional regulators such as noncoding RNAs. The most promising candidates are microRNAs (miRNAs), small noncoding RNAs (18–25 nucleotides) that regulate gene expression through direct interaction with the 3′UTR region of target molecules, inhibiting their translation or promoting their degradation.

MicroRNA genes can be located in intergenic regions, in introns (also known as mirtrons) or in exons of coding or noncoding genes. They can be transcribed by RNA polymerase II–III into simple units from their own promoter, or into more complex molecules containing two or more miRNAs (known as clusters) from a common promoter. The primary transcript (known as pri-miRNA) contains a cap structure at the 5′ end and a poly(A) tail at the 3′ end. This molecule folds into a hairpin structure that is recognised by the Drosha and DGCR8 enzymes, which cleave the ends of the hairpin generating a shorter precursor (∼70–80 nucleotides) called pre-miRNA. This pre-miRNA is exported to the cytoplasm by the action of the XPO5 enzyme and processed by the DICER1 endonuclease, giving rise to the functional double-stranded molecule (18–25 nucleotides). Finally, the two strands are separated and incorporated to the RNA-induced signalling complex (RISC), a multiprotein complex whose function is to bring miRNAs to their target mRNA. Both strands (5p and 3p) may be functionally relevant, independently of their abundance and stability.

Generally, the mature form of the miRNA binds the 3′UTR region of its target molecules by means of a sequence of 7 or 8 nucleotides at the 5′ end of the miRNA known as the seed sequence that is highly complementary to the 3′UTR sequence of the target. Once the RISC–miRNA is positioned on the target, inhibition of translation and/or degradation of the mRNA take place. In very specific cases, miRNAs may exert their regulatory function on expression by binding the 5′UTR region.20

There is a growing number of preclinical trials of miRNA-based therapies that have demonstrated improved, additive or similar effects to conventional treatments. Their use may offer a number of technical advantages:

• 1.

  MicroRNAs are regulatory elements that may affect multiple mRNAs simultaneously, therefore affecting different components of a single molecular signalling pathway or even different pathways. This minimises the possibility of compensation by redundant pathways or different proteins of the same family.

• 2.

  Unlike mRNAs, mature miRNAs are already functional gene products and do not require additional transcriptional regulation to carry out their functions.

• 3.

  Lastly, miRNAs are stable in frozen tissues and in formalin-fixed and paraffin-embedded (FFPE) tissue samples. This allows their extraction from clinical biopsies and their quantification by standardised methods such as quantitative PCR at any point during a patient's treatment.

Lin et al. were the first researchers that observed that the expression profile of a subset of miRNAs could be used to classify high- and low-risk patients with a high sensitivity and specificity.21 To date, numerous miRNAs have been identified that regulate different oncogenic properties in NBs. In this section, we will highlight the miRNAs that have shown therapeutic effects in preclinical models (Table 1).

The use of miRNAs in clinical practice is already a reality. In the field of oncology, the first molecule that has entered clinical testing is MRX34. This compound mimics miR-34, a miRNA that functions as a tumour suppressor whose expression is reduced in multiple tumours.

One limitation of these compounds is their administration by the intravenous route and devoid of any encapsulation, so that their distribution concentrates mostly in the liver, where they are very effective but are also metabolised and excreted rapidly, limiting their therapeutic effect in less perfused tissues. Therefore, more research is needed to improve the biodistribution of miRNAs, possibly by their encapsulation in nanoparticle vesicles manufactured with biocompatible materials, which result to greater stability and longer circulation in the bloodstream, allowing more time for their accumulation in tumour tissues and thus enhancing their antitumour efficacy.

This study has been funded by the Instituto de Salud Carlos III (CP11/00052, PI14/00561, RD12/0036/0016) and co-funded by the European Regional Development Fund (ERDF), Generalitat de Catalunya (2014-SGR-660) and the Marie Curie Career Integration Grants (n° 618301).

The authors have no conflict of interests to declare.