Introduction
Brain tumors are the second most common cancers in children and the leading cause of cancer-related mortality in this age group [
40]. Medulloblastoma, a highly malignant tumor of the posterior fossa region of the brain, is the single most common pediatric malignant brain tumor. Genome-wide expression profiling studies have identified four core molecular subgroups of medulloblastomas: WNT, SHH, Group 3 and Group 4 that are not only distinct in their underlying genetic alterations but also differ in clinical characteristics like age, gender related incidence, incidence of metastasis and overall survival rates [
36]. WNT subgroup medulloblastomas that are characterized by the activation of the canonical WNT signaling pathway have excellent (> 95%) long term survival [
44]. SHH subgroup medulloblastomas having activated Sonic Hedgehog signaling expression profile, have intermediate survival rates with those harboring mutation in the
TP53 tumor suppressor gene or amplification of
MYCN oncogene having poor survival [
44]. The two non-WNT, non-SHH subgroups have some overlap in their expression profiles with a number of transcription factors involved in neural development being overexpressed in both the subgroups [
37]. The two subgroups are distinguished based on the preferential expression of proliferation related genes, retina-specific genes in the Group 3 tumors and neuronal differentiation related genes in the Group 4 tumors [
37]. Group 3 tumors have the worst survival rates among all the four subgroups while Group 4 tumors have intermediate survival rate. NRL and CRX, the two retina-specific transcription factors have been found to be master regulators of photoreceptor signaling program in the Group 3 medulloblastomas [
9]. MYC amplifications are restricted to Group 3 [
38]. Structural variants leading to aberrant induction of
GFI1/GFI1B oncogenes and
MYCN amplifications are found in both Group 3 and Group 4. Pathway analysis of recurrent genetic alterations have found overrepresentation of genes involved in the TGFβ and Notch signaling pathway in Group 3 and chromatin modifiers in Group 4 [
34].
Surgery followed by radiation therapy and chemotherapy is the standard multimodal treatment for medulloblastoma [
1]. Long term sequelae of the intense treatment include neurocognitive impairment, endocrine dysfunction, psychiatric, developmental deficits and in some cases secondary malignancies [
17]. Accurate risk stratification of medulloblastomas is therefore necessary to spare the children having low risk of recurrence from excessive treatment to the developing brain. On the other hand, survival of high risk medulloblastoma cases can be improved by more aggressive treatment. Considerable heterogeneity exists in each of the three non-WNT subgroups for which molecular markers are necessary so that accurate risk stratification can be done for effective treatment with least side effects [
44].
MicroRNAs are small non-coding molecules that have been shown to regulate a wide array of cell functions, ranging from cell proliferation, differentiation, cell death and stress resistance. Since the first report of miR-15/miR-16 deletion in B cell chronic lymphocytic leukemia, large number of studies have reported microRNA dysregulation in cancer including medulloblastoma [
5,
56]. We have earlier reported differential expression profiles of microRNAs in the molecular subgroups of medulloblastomas [
13]. Further, we have developed an assay based on the microRNA profile that has 97% accuracy for molecular classification of medulloblastomas and is particularly useful for formalin-fixed, paraffin-embedded (FFPE) tumor tissues [
19]. In the present study, miR-204 expression was analyzed in 260 medulloblastomas from an Indian cohort and in 763 medulloblastomas from the MAGIC (Medulloblastoma Advanced Genomics International Consortium) cohort [
2]. A subset of Group 3 / Group 4 medulloblastomas having low expression of miR-204 was found to have significantly poor survival. The role of miR-204 expression in medulloblastoma biology was investigated by restoring miR-204 expression in established medulloblastoma cell lines and studying its effect on growth and malignant behavior of medulloblastoma cells.
Discussion
In a large scale study on 3312 tumors and 1107 non-malignant tissues contributed by 51 different cancer types, miR-204-211 family was found to be the top deleted microRNA family in cancer, suggesting its crucial role as a tumor suppressive miRNA in multiple cancer types [
55]. In the present study, miR-204 was found to be differentially expressed in the four core molecular subgroups of medulloblastomas, with almost all SHH and a subset of Group 3/Group 4 tumors showing downregulation of miR-204 expression. Despite the complexity of the heterogeneity and overlap present in the copy number variations, methylation profiles and somatic mutation profiles in the Group 3 / Group 4 medulloblastomas [
2], miR-204 expression levels identify a subset of these tumors having poor survival in the Indian as well as in the large MAGIC cohort. This finding is consistent with lower expression of miR-204 correlating with poor survival in breast cancer [
23], non-small cell lung cancer [
15], and neuroblastoma [
47]. Integrated genomic studies have identified novel molecular subtypes within the four core subgroups of medulloblastomas [
2,
49]. Among the three Group 3 subtypes, subtype 3γ was found to have the worst 5 year survival rate of 41.9% as compared to that of subtype 3α and subtype 3β at 66.2 and 55.8% respectively [
2]. Group 3γ having the worst survival showed the least miR-204 expression among the 3 subtypes of Group 3. The Group 4 subtypes do not show significant difference in their overall survival [
2]. MiR-204 expression on the other hand, identified a subset of Group 4 medulloblastomas having significantly poor survival of 59.7% as compared to 80% of the ‘miR-204 high’ subset. Group 3 poor prognostication markers like
MYC amplification and isochrome 17q do not have prognostication value in Group 4 [
44]. FSTL5 immunopositivity serves as a marker for poor prognostication in both Group 3 and Group 4 medulloblastomas [
45]. Adult Group 4 patients have also been reported to have poor survival rates [
46]. Loss of chromosome 11 or gain of chromosome 17 identify a small subset of Group 4 patients who have excellent survival [
51]. Biology underlying these cytogenetic alterations is however, not understood. Thus, low miR-204 expression serves as a marker of poor prognosis in Group 4 that has paucity of markers for prognostication. Integrated genomic analysis is expensive as well as technically demanding and thus cannot be used in routine clinical practice for risk stratification. MiR-204, a single microRNA on the other hand, can be easily combined with the Nanostring assay that classifies medulloblastomas into the four molecular subgroups [
39]. Furthermore, miR-204 due its small size resists degradation during formalin fixation and thus would be a reliable marker even in poor quality FFPE tissues.
Downregulation of miR-204 expression with poor survival is consistent with its tumor-suppressive effect in medulloblastoma cell lines. Restoration of miR-204 expression in multiple established Group 3 medulloblastoma cell lines was found to inhibit their anchorage-independent growth, invasion potential and tumorigenicity. Tumor suppressive effect of miR-204 in the
MYC amplified Group 3 cell lines is remarkable since other microRNAs downregulated in medulloblastoma like miR-206 for instance, fail to inhibit tumorigenicity of these cell lines [
41]. MiR-204 has been shown to inhibit invasion and tumorigenicity of various cancer cells including glioma, colorectal cancer, endometrial cancer and cervical cancer cells [
3,
27,
57,
58]. Thus, the tumor suppressive role of miR-204 in medulloblastoma cells is consistent with its role in other cancers.
MiR-204 has been reported to target a number of genes including
RAB22A, FOXC1, EZR, BCL2L2, M6PR, BCL2, MCL1, FOXA1, FOXM1, EPHB2 [
22,
42,
50,
52,
57]. Transcriptome sequencing / real time RT-PCR / western blot analysis showed downregulation of
RAB22A, M6PR, EZR, EPHB2, upon miR-204 expression in medulloblastoma cells as well.
IGF2R was identified and validated as a novel target of miR-204. MiR-204 expression in medulloblastoma cells resulted in downregulation of both M6PR and IGF2R that mediate transport of lysosomal enzymes from the Golgi apparatus to lysosomes [
26]. Furthermore, reduction in the levels of lysosomal enzymes Cathepsin B and Cathepsin D upon miR-204 expression in medulloblastoma cells suggests impairment of the lysosomal degradation pathway. Autophagy brings about p62/SQSTM1 mediated degradation of its cargo by lysosomal degradation pathway [
12]. MiR-204 is known to target LC3B, a crucial mediator of autophagy [
28]. In the present study as well, miR-204 expression in medulloblastoma cells resulted in reduction in the LC3B flux and increase in the levels of p62/SQSTM1 indicating autophagy inhibition. Autophagy has been shown to play role in tumor promotion by sustaining survival in stress, by reducing oxidative stress and, maintaining metabolic homeostasis [
14]. Inhibition of tumor growth upon miR-204 expression is consistent with these reports on the role of autophagy in tumor promotion. Autophagy has also been reported to promote invasion by activating Epithelial Mesenchymal Transition of hepatocellular carcinoma cells [
48], by promoting secretion of factors like IL6, MMP2 [
24] and by activating the MAP kinase signaling pathway in glioblastoma cells [
8]. Consistent with the inhibition of invasion capacity of medulloblastoma cells upon miR-204 expression, downregulation of miR-204 expression was found to be associated with higher incidence of metastasis at diagnosis in Group 3 / Group 4 medulloblastomas. Thus, poor survival of Group 3 / Group 4 medulloblastomas having low miR-204 expression is likely due to their higher invasive capacity and higher malignant potential.
Several microRNAs whose expression is deregulated in medulloblastoma are known to play role in embryonic brain development [
56]. MiR-9 and miR-124a that play crucial role in the onset of neurogenesis by targeting transcription factors like SOX9, FOXG1 and MEIS1, are downregulated in medulloblastoma [
6]. MiR-9 and miR-199b-5p target HES1, thereby silence Notch signaling pathway at the onset of neuronal differentiation [
7,
10]. Low expression of miR-9 and miR-199b-5p has been found to correlate with poor survival in medulloblastoma and their expression in medulloblastoma cell lines promotes growth arrest [
7,
10]. MiR-17-92 cluster microRNAs are overexpressed predominantly in the SHH subgroup medulloblastomas [
35]. Knock-out of this microRNA cluster brings about reduction in size of cerebellum and inhibits medulloblastoma formation in
Ptch knock-out mouse model of SHH subgroup medulloblastomas indicating role of these microRNAs in normal development and tumorigenesis [
33]. MiR-204 has been reported to play crucial role in lens and retinal development by targeting MEIS2 transcription factor in Medaka fish [
4]. MiR-204 expression has been found to be upregulated during aging in mouse hippocampus and target Ephrin B2 that plays role in axon guidance [
31]. MiR-204 has also been reported to control neuronal migration and cortical morphogenesis in mouse embryos presumably by targeting Doublecortin that is known to play role in neuronal migration [
54]. Effect of miR-204 on invasive capacity of medulloblastoma cells is consistent with the role of miR-204 in neuronal migration. Thus, miR-204 appears to play role in both normal brain development and tumorigenesis like several other miRNAs that are known to be deregulated in medulloblastoma.
Delineating the molecular mechanism underlying downregulation of miR-204 expression would suggest ways to increase its expression, thereby improving survival rate of medulloblastoma patients. Group 3 medulloblastoma cells treated with HDAC inhibitors showed modest 2 to 4 fold increase in the miR-204 expression levels. Treatment with HDAC inhibitors has been reported to inhibit medulloblastoma cell growth in several studies [
20,
29,
43]. Thus, HDAC inhibitors appear to have therapeutic potential in the treatment of medulloblastoma.