MicroRNA expression patterns and signalling pathways in the development and progression of childhood solid tumours

A number of studies in recent times have indicated the frequent involvement of miRNAs in the progression and development of childhood brain tumours, and that this tumour has unique characteristics among rare tumour types. Brain tumours are among the most common cancers in children. The different tumour types and classifcations are generally based on cell structure, composition, growth rate of the tumour and a range of further characteristics. Central nervous system primitive neuro-ectodermal brain tumours (CNS-PNETs) are a heterogeneous group of CNS neoplasms. These tumours are composed of poorly differentiated neuroepithelial cells and, some variants are associated with aggressive clinical behaviour and poor outcome.

Chromosome 19q13.41 contains the largest human miRNA gene cluster (chromosome 19 miRNA cluster, C19MC); the miRNAs comprising this cluster have been identified as oncogenic [62]. C19MC may drive oncogenic processes in part by facilitating maintenance and transformation of a very early neural compartment [63, 64]. A particularly aggressive group of CNS-PNET tumours demonstrated C19MC amplification with high LIN28 expression. In addition, recent identification of Tweety family member 1 (TTYH1):C19MC gene fusions in embryonal tumours with multi-layered rosettes (ETMRs) have been identified and observed to be associated with very high expression of specific miRNAs. ETMRs are a rare and deadly form of paediatric brain tumours, and are characterized by high levels of amplification of C19MC. ETMRs, cell lines and xenografts all demonstrated specific DNA methylation signatures distinct from other tumours and normal tissues, very high overexpression of a previously uncharacterized isoform of DNMT3B, which originates from an alternative promoter, and which is interestingly only active in the first weeks of neural tube development. This research uncovers a potential oncogenic re-activation of an early developmental signalling program in ETMR via an epigenetic alteration mediated by the brain specific DNMT3B isoform, which has a known embryonic origin [65].

Pediatric pilocystic astrocytoma (PA) is a Word Health Organisation grade I glioma. It is a brain tumour, which often arises in the cerebellum or close to the brainstem in the hypothalamic region or the optic chiasm. However it is possible for it to occur in any location where astrocytes are present (cerebral hemispheres, and the spinal cord). While often these tumours are slow growing and benign, the neoplasms can be cystic and grow very large, causing multiple problems due to the increased intracranial pressure. miRNA profiling has been performed in a group of 43 PA tumours and 5 non-neoplastic brain controls. Differentially expressed miRNAs were identified between PA and normal brain tissue; 13 miRNAs were underexpressed in PA and 20 were over expressed compared to normal brain tissue (Table 1). An example of the importance of a few of these differentially expressed miRNAs is miR-124, which was underexpressed in PA tumours, confirming previous findings. miR-124 targets putative oncogenes, is enriched in brain tissue, and is also found to be downregulated in glioblastomas. miR-124 also has been shown to negatively affect glioblastoma proliferation and migration in vitro [66, 67]. Additionally, upregulation of miR-21 was shown in these tumours and elevation of this miRNA has also been seen in other tumour types, compared with normal tissues (Table 2). An important target of miR-21 is PTEN, a critical suppressor of the PI3K/Akt/mTOR pathway. Previously PTEN loss had been frequently identified in high-grade gliomas, and decreased levels had been shown in PAs with particularly aggressive histological features. An overall observation of miRNAs in PA is that there are sets of miRNAs underexpressed compared to controls, and alongside this there is increased expression and protein levels of putative oncogenes which may be vital in understanding the biology of PA [68, 69].

Ependymal tumours (ET) are the third most common group of brain tumours in children. These tumours comprise four entities; the most common are grade II and grade III, most of which are located in the posterior fossa, with infiltration into vital brain structures. Due to the difficult location of these tumours surgical resection is highly limited. Additionally there are currently no effective prognostic features aside from how accessible they are to surgical intervention. Childhood ependymomas usually don’t have genomic imbalances, further making it difficult to establish molecular prognostic features. Recently, however, three miRNAs were demonstrated to differentiate between grade II and III ependymomas. These include miR-17–5p, miR-19a–3p and miR-106b–5p, and the expression of these miRNAs was also significantly correlated with EZH2 expression (a suggested marker for PFA ependymomas). Survival analysis indicated overall and event free survivals were reduced with higher expression levels of miR-17–5p [70].

Medulloblastomas (MBs) are a common brain tumour in children, thought to originate in cerebellar granule neuron progenitors, which have failed to undergo normal cell cycle exit and differentiation. In MB miR-218 expression is downregulated, and low expression of this miRNA has been identified in other cancer types. In addition, reduced expression has been observed in glioma cells [71] (Table 1). Previous research has identified multiple targets of miR-218 that control pathways involved with the cell cycle, cell metabolism and motility. Of those identified, CDK6 is of interest as it has previously been identified as an adverse prognostic marker in MB because of its role in upregulating cell cycle progression and blocking differentiation [72]. The miR-17-92 cluster is also implicated in MB. Mouse MB models have demonstrated overexpression of several members of the miR-17-92 cluster, three of which, (miR-19a, miR-20, miR-92), were also overexpressed in human MBs with a constitutively activated Sonic Hedgehog signaling pathway, and not in other forms of the disease (Table 2). To explore whether the miR-17-92 cluster could be promoting MB formation, the expression of these miRNAs was enforced in granule neuron progenitors isolated from cerebella of postnatal day six mouse models (lnk4c−/−; ptch1+/−). These mice formed MBs in orthotopic transplants with complete penetrance, but in similarly engineered cells from lnk4−/−; p53−/− mice there was no MB formation. These findings point toward a possible functional connection between the miR-17-92 cluster, the Sonic Hedgehog signaling pathway, and the development of MBs in both mice and humans [73].

Neuroblastoma (NB) is an extracranial solid childhood tumour, and is the most common cancer in infants. The cancer is a neuroendocrine tumour, stemming from neural crest elements of the sympathetic nervous system; it is an ectodermally derived tumour, which reflects its developmentally derived origin [59]. While it frequently originates in one of the adrenal glands, it can also develop in nerve tissues in the neck, chest, abdomen or pelvis [74]. It is a unique and interesting cancer, as it is one of the few malignancies that can demonstrate spontaneous regression from an undifferentiated state to a benign cellular appearance; additionally, there is a large amount of heterogeneity observed in these tumours [75].

Recent work in this area suggests the dysregulation of miRNAs may play an important role in the pathogenesis of NB. For instance, the miR-17-5p-92 cluster of miRNAs, (miR-17-5p, miR-18a, miR-19a, miR-20a and miR-92), is found to be expressed at higher levels in NB cells lines exhibiting overexpression of MYCN [76]. Tumours with amplification of the MYCN transcription factor and/or loss of the distal chromosome 1p and gain of 17q are a major genetic subtype of metastatic NB that has a particularly poor prognosis (known as MNA). It has also been demonstrated that MYCN proteins c-Myc/N-Myc can bind directly to the promoter of the miR-17-5p-92 cluster, initiating transcription and resulting in the up-regulation of this group of miRNAs from a single transcription unit [77, 78]. A poor prognosis and therapy resistance is associated with MYCN amplified NBs. However, previous research has shown that therapy resistant NB can be abolished in vivo using antagomir-17-5p [76].

Components within the miRNA biogenesis pathway have also been implicated in NB, which globally affects miRNA expression. A group of 66 tumours were assessed for a panel of 162 miRNAs using quantitative PCR and low expression of DICER and DROSHA were identified in high-risk tumours. The low expression subsequently accounted for the overall global reduction of miRNA expression observed in the advanced disease state and correlated with a poor outcome for these patients [26]. A global reduction of miRNA expression can be detrimental, and conversely the presence of some miRNAs has been attributed to more positive outcomes, such as the tumour suppressor miR-34a, which is also a critical component of the p53 network [79, 80]. This miRNA has anti-proliferative effects and has been found to target MYCN. miR-34a is located on chromosome 1 and it is not surprising that lower levels are expressed in tumours with/or due to a 1p deletion [81]. This type of deletion is found most often in the MNA type of tumours; a functionally active miR-34a would help to negate the ill effects of the amplification of MYCN. miR-34a is also involved in the regulation of other genes associated with cell proliferation and apoptosis such as E2F3, BCL2, CCND1, and CDK6 [82, 83]. Since miR-34a plays many roles in controlling and regulating growth, it is obvious disruption of the function of this miRNA would be deleterious and could lead to unrestricted growth leading to cancer development, and indeed miR-34a has been described previously as a tumour suppressor [79, 84]. Another miRNA that has been reported as a potential tumour suppressor is miR-497; this is due to its action in targeting the key cell cycle regulator WEE1. Expression of miR-497 in MNA tumours is significantly lower than other tumour types, and high WEE1 levels (low miR-497) are significantly associated with poor overall survival and event free survival in NB (Table 1). Additionally, overexpression of miR-497 reduces cell viability and increases apoptosis in MNA cells, and in particular was found to be compatible with an enhanced response to cisplatin. The discovery of the complexity involved in miRNA control has begun to uncover new and promising therapeutic targets for high-risk NB patients [85].

Another common issue seen in childhood solid tumours is chemotherapy resistance; one study investigated drug resistance and expression of miRNAs, and identified several miRNAs of interest in NB chemoresistance models. Their results confirmed an up-regulation of the expression of miR-188-5p in all three of the chemoresistance models examined; miR-501-5p was found up-regulated, and miR-125b-1 was down-regulated in two of the models [86]. These types of investigations are valuable in identifying good miRNA candidates for in-depth exploration to look for novel drug development and perhaps RNAi based therapies for those individuals who are not responding to chemotherapy and may have more aggressive tumour types [87, 88].

Osteosarcoma (OS) is an aggressive malignant neoplasm of the bone that originates in primitive transformed mesenchymal cells [89]. This cancer begins in osteoblasts responsible for forming new bone and is common in adolescents. Bone formation is a dynamic process; orchestrating osteoblast differentiation, which requires precise mechanisms of control, and is particularly critical for both development and growth. OSs tend to occur at the sites of bone growth, and the proliferation occurring at these locations may make the osteoblastic cells more susceptible to acquiring mutations, or other events, which may induce the transformation of cells [90]. Therefore, often the ends of long bones (arms and legs) are affected, although formation in the knee is also a common event.

The molecular etiology of OS remains elusive; miRNAs have been investigated in association with many diseases due to their properties that may allow for widespread effects on development and disease progression. In prior investigations four miRNAs were found to be differentially expressed in OS cell lines compared to normal human osteoblast cell lines [91]. The miRNAs that were overexpressed in the OS cell lines included, miR-135b, which has previously been implicated in cancer and is associated with osteoblast differentiation, miR-150, miR-542-5p and miR-652 [92‐95]. Several miRNAs have been identified that may act as tumour suppressors in OS and are additionally found to be down-regulated in OS cell lines. These miRNAs, (miR-199a-3p, miR-127-3p and miR-370), assist with the suppression of oncogenic and anti-apoptotic proteins and also play a role in regulating proliferation of the osteoblasts [96]. As with NB, the miR-34 family is again highlighted as an important regulator of growth signals and acts as a tumour suppressor miRNA. These miRNAs are targeted by p53 and play a very important part of the tumour suppressor pathway [82, 97‐102].

In a recent investigation, a miRNA signature reflecting the pathogenesis of OS was described using surgically procured samples from human patients [103]. These samples highly expressed miR-181a, miR-181b and miR-181c and also showed reduced expression of miR-16, miR-29b and miR-142-5p. However, it is noteworthy this study also identified valuable pre-treatment biomarkers of metastasis and indicators of responsiveness to therapy. The biomarkers and indicators of metastatic development risk included a higher expression of miR-27a and miR-181c-3p in the pre-treatment samples. This characterised patients who went on to develop metastastic lesions. Furthermore, higher expression of miR-451 and miR-15b in pre-treatment samples correlated with positive responses to chemotherapy [103].

Wilms tumour (WT), or nephroblastoma is a solid tumour occurring in the kidneys of children. Embryonal renal neoplasms are thought to develop in nephrogenic rests (NRs) with morphological and molecular analogies to kidney development. Most commonly WTs are unilateral with a single tumour affecting the kidney. However multiple tumours can arise in one of the kidneys, or the cancer may occur bilaterally in both kidneys [104, 105].

In WT relatively few driver genes have been identified [106]. Recently both Sanger sequencing and whole exome sequencing have provided the means to identify mutations in critical elements of the miRNA processing machinery in WT patients. Several recent studies have uncovered miRNA biogenesis disruptions in WT, which provides an excellent starting point for research in this area [27, 28, 107]. Rakheja and colleagues investigated the presence of these potential mutations in 44 WT samples using whole-exome sequencing [28]. In addition to identifying novel mutations in MYCN, SMARCA4 and ARID1A, missense mutations were discovered in the miRNA processing enzymes DROSHA and DICER1. Further examination of tumour miRNA expression using in vitro processing assays and genomic editing of cell lines demonstrated that these mutations in DROSHA and DICER1 greatly influence miRNA processing and likely cause down regulation in mature miRNAs, which generate an altered miRNA expression profile in these WT patients. Further, there were distinct mechanisms also identified; DICER1 RNase IIIB mutations preferentially affected the processing of miRNAs derived from the 5’ arm of pre-miRNA hairpins. Conversely DROSHA RNase IIIB mutations were found to cause global inhibition of miRNA biogenesis through a dominant-negative mechanism. Mutations in these enzymes also impaired the expression of tumour suppressor miRNAs such as the let-7 family; this is an important regulator of genes such as MYCN, LIN28 as well as other previously identified WT oncogenes [28]. Downregulation of certain miRNAs in WT were also observed to have a major affect on critical pathways that are involved in kidney development, such as, TGF-β. For example Activin A receptor type 2B (ACVR2B), an important component of the TGF-β pathway, is often highly expressed in renal neoplasms; several miRNAs have been noted as downregulated and have been confirmed to directly target this gene (Table 1). This investigation was the first to implicate the TGF-β pathway in the pathogenesis of WT [108].

A number of up regulated miRNAs were also identified in WT; up-regulation of miR-17-5p, miR-18a, miR-19b, miR-92 and miR-20a was reported in WT compared to other kidney tumours or normal kidney tissues [109]. This is of interest, because the cluster (miR-17-5p-92) was also identified as being up-regulated in the subtype of NB known as MNA [76] (Table 3). As mentioned, these miRNAs are thought to contribute to tumour progression and metastasis in the later stages of the cancer [103, 110]. Other miRNAs found to be upregulated in WT have been investigated for being able to distinguish the different subtypes of WT and for giving an indication on severity of disease, or for influencing important factors such as endothelial to mesenchymal transitions (EMT) and chemosensitivity [111]. Another mechanism recently recognised in the development of WT is the aberrant regulation of miRNA Let-7 through overexpression of LIN28 [112]. Let-7 is a direct target of LIN28, and it is a potent regulator of stem cell self-renewal and differentiation. A murine model has shown that directed overexpression of both LIN28A and LIN28B in the renal lineage results in the formation of WTs [113]. Further investigation of LIN28 expression in mutant mice revealed that terminal differentiation only occurred in nephron progenitor cells after LIN28 had been completely withdrawn. It is plausible that the overexpression of LIN28 may cause an imbalance in proliferation and differentiation in these specific cells, rather than a complete blockage of differentiation, resulting in the formation of these types of tumours [113, 114].

Hepatoblastoma (HB) is a relatively rare disease, although it is the most common childhood liver cancer. It is often diagnosed as an asymptomatic abdominal mass [115]. The most commonly affected age group are infants between six months and three years of age. The majority of HB cases are sporadic. However, HB may occur in conjunction with a developmental syndrome such as Beckwith-Wiedemann Syndrome (BWS) and Familial Adenomatous Polyposis (FAP) [116, 117].

Since HB is a rare cancer, research is challenging and often relies on formalin fixed paraffin embedded (FFPE) samples, which are known to provide obstacles in the extraction of total RNA due to degradation. However, recent research has demonstrated miRNA expression is relatively stable and well preserved in these valuable archival samples which is an important factor for future research in this field [118, 119]. Previous studies provided evidence for a few prognostic miRNAs associated with HB, such as miR-492, which is a potential biomarker in metastatic HB. Overexpression of pleiomorphic adenoma gene 1 (PLAG1) has been characterised in HB, and has been demonstrated through RNA interference analysis combined with miRNA arrays to strongly influence miR-492. Additionally, it was revealed that miR-492 could originate from the coding sequence of the HB marker gene keratin 19 (KRT19). Significantly elevated levels of co-expressed KRT19 and miR-492 were identified in metastatic HB tumour samples [120]. This is of interest as metastatic HB is often associated with poor prognosis. Indeed, miR-492 and its associated targets may provide biomarkers or inform on targeted therapies [120].

Other potential prognostic miRNAs have been identified in a recent study, which showed that histological subtypes of a group of 20 tumour samples did not correlate with survival. However this investigation did identify that the levels of several miRNAs were independently prognostic for HB with significantly increased overall survival. These miRNAs included the high relative expression of miR-21 and low relative expression of miR-222 and miR-224 [121] (Tables 2 and 3). In a recent study miR-21 was examined for its potential role as a diagnostic/prognostic indicator in peripheral blood where it has been reported to be relatively stable within protective exosomes or nanovesicles. Exosomes, which can be released in large amounts from tumour cells due to a hypoxic environment and other internal changes, are of interest because a blood sample is a less invasive method for diagnosis and prognosis for the patient. Blood samples were prepared retrospectively in 32 Chinese hepatoblastoma patients and healthy controls; the blood samples were separated to isolate RNA directly from the exosomes present in the sample (which precipitate at the bottom), while also preparing exosome-depleted samples (using the supernatant of the same sample). The concentration of miR-21 in the exosomes prepared from blood of HB patients was significantly higher than in exosome-depleted supernatants and whole plasma. The expression of miR-21 detected in HB children was significantly higher in both the plasma and exosomes when compared to controls. Additionally, exosomal miR-21 is not only an independent predictor of event-free survival for patients, it also was more accurate in diagnosing HB than alpha-fetoprotein levels (AFP), the traditional means of HB diagnosis [122]. There were also miRNAs identified with unique expression levels in both fetal and embryonal subtypes in comparison with surrounding non-tumorous liver tissues. In the fetal subtype of samples, there was a lower expression compared to surrounding normal liver tissue in miRNAs miR-17-5p, miR-195, miR-210 and miR-214, while higher expression was observed in miR-221 (Tables 1, 2 and 3). In the embryonal subtype of HB a lower expression of miR-122-5p was demonstrated. Loss of miR-122-5p is also frequent in hepatocellular carcinoma (HCC) and has been correlated with migration, invasion, and in vivo tumorigenesis. miR-122-5p is also considered a differentiation marker for hepatocytes. Lower miR-122-5p expression levels indicated in the embryonal subtype is in agreement with the lower degree of differentiation found in early embryonal development. In a recent comprehensive investigation of 33 different types of childhood solid tumours and 20 control cases, serum miRNA profiles were examined. Four HB samples were included in this group of tumours and tumour specific serum miRNA profiles were determined for each specific tumour type. Using the panel of miRNAs identified for HB, (high overexpression of miR-483-3p, miR-205-5p, and miR-122-5p), a non-invasive differential diagnosis of a liver mass could be performed when compared to tumour mixed samples of neuroblastomas (MYCN-amplified and others) [25]. It is of interest that contradicting results from two studies identified miR-122-5p as being of interest, except at opposite ends of the spectrum, (one observing lower relative expression and the other stating the miRNA to be in the top ten of the highest relative overexpression) (Table 3). It is important to acknowledge that in serum miRNA profiling investigations only four HB samples were investigated, and in addition the authors pointed out that miR-122-5p was found in increased levels in non-HB samples in this study, such as in pancreatic pleiomorphic rhadomyosarcoma (RMS) presenting with obstructive jaundice [25]. Serum levels of miR-122-5p were increased, while the expression of miR-122-5p was reduced in liver tissue, which could be due to passive miRNA drainage from tumour cells, as has been observed in other tumours [123]. Expression of miR-122-5p has been identified as a non-specific marker of liver damage, and is increased in the serum of jaundiced patients [124]. These studies highlight the importance of identifying miRNA profiles (in tumour tissue and in serum), and shows that there may be specific miRNAs with unique roles in various types of childhood solid tumours.