Real-Time PCR Assays on Formalin-Fixed, Paraffin-Embedded Medulloblastomas.

Real-time PCR technology has been instrumental in contributing toward biomarker discovery, classification of tumors as well as risk stratification of patients. However, much of its success depends on the quality and quantity of the starting material used for RNA extraction. Clinical samples are most often provided as formalin-fixed and paraffin-embedded, wherein the RNA is extensively degraded, affecting sensitivity. Here, we describe a real-time PCR based assay developed for molecular subgrouping of medulloblastomas that is particularly useful for formalin-fixed, paraffin-embedded (FFPE) samples.

MiR-592 activates the mTOR kinase, ERK1/ERK2 kinase signaling and imparts neuronal differentiation signature characteristic of Group 4 medulloblastoma.

Medulloblastoma, a common malignant brain tumor in children, consists of four molecular subgroups WNT, SHH, Group 3 and Group 4. Group 3, Group 4 tumors have an overlap in their expression profiles and genetic alterations but differ significantly in their clinical characteristics, with Group 3 having the worst 5-year overall survival of <60%. MiR-592 is overexpressed predominantly in Group 4 tumors. MiR-592 expression reduced the anchorage-independent growth, invasion potential and tumorigenicity of Group 3 medulloblastoma cells. DEPTOR, an endogenous inhibitor of the mTOR kinase, and EML1 were identified as novel targets of miR-592. The miR-592 mediated decrease in the DEPTOR expression levels activated both mTORC1 and mTORC2 complex in medulloblastoma cells. However, the miR-592 expression also decreased the AKT kinase activity, likely to be due to the activation of the inhibitory feedback of the mTOR signaling. MiR-592 expression upregulated several neuronal differentiation-related genes, a characteristic of Group 4 medulloblastoma in Group 3 cell lines. The expression of miR-592 also upregulated the activity of ERK1/ERK2 kinases indicating activation of the MAPK signaling pathway. The inhibition of MAPK signaling by the ERK1/ERK2 inhibitor and mTOR signaling by rapamycin abrogated the miR-592-mediated upregulation of neuronal differentiation-related genes. Group 4 medulloblastomas showed higher activity of the mTOR and MAPK signaling compared to Group 3 tumors. Thus, miR-592 overexpression appears to be a driver event and a determining factor of Group 4 biology, which activates the mTOR and MAPK signaling pathways and thereby imparts its characteristic expression profile of neuronal differentiation-related genes.

Downregulation of CRX, a Group 3-specific oncogenic transcription factor, inhibits TGF-ß/activin signaling in medulloblastoma cells.

Medulloblastoma, the most common malignant brain tumor in children, consists of four molecular subgroups WNT, SHH, Group 3, and Group 4. Group 3 has the worst survival rate among the four subgroups and is characterized by the expression of retina-specific genes. CRX, the master regulator of the photoreceptor differentiation, is aberrantly expressed in Group 3 medulloblastomas. CRX expression increased the proliferation, anchorage-independent growth, invasion potential, and tumorigenicity of medulloblastoma cells indicating the oncogenic role of CRX in medulloblastoma pathogenesis. CRX knockdown resulted in the downregulation of expression of several retina-specific genes like IMPG2, PDC, RCVRN. and Group 3 specific genes like GABRA5, MYC, PROM1. Thus, CRX plays a major role not only in the expression of retina-specific genes but also in defining Group 3 identity. Increased expression of several pro-apoptotic genes upon CRX knockdown suggests that CRX could protect Group 3 medulloblastoma cells from cell death. Several negative regulators of the TGF-ß signaling pathway like SMAD7, PMEPA1, KLF2 were upregulated upon the CRX knockdown. Western blot analysis showed a decrease in the levels of (Phospho)-SMAD2, total levels of SMAD2, SMAD4, and an increase in the levels of SMAD7 indicating inhibition of the TGF-ß signaling pathway upon CRX knockdown. Copy number variations in several genes involved in the TGF-ß signaling pathway occur in a subset of Group 3 tumors. Autocrine TGF-ß/activin signaling has recently been reported to be active in a subset of Group 3 medulloblastomas. CRX knockdown resulting in the inhibition of the TGF-ß/activin signaling pathway demonstrates an interaction between the two Group 3 specific oncogenic pathways and suggests simultaneous targeting of both CRX and TGF-ß signaling as a possible therapeutic strategy.

Downregulation of ARID1B, a tumor suppressor in the WNT subgroup medulloblastoma, activates multiple oncogenic signaling pathways.

Medulloblastoma, a common pediatric malignant brain tumor, consists of four distinct molecular subgroups WNT, SHH, Group 3 and Group 4. Exome sequencing of 11 WNT subgroup medulloblastomas from an Indian cohort identified mutations in several chromatin modifier genes, including genes of the mammalian SWI/SNF complex. The genome of WNT subgroup tumors is known to be stable except for monosomy 6. Two tumors, having monosomy 6, carried a loss of function mutation in the ARID1B gene located on chromosome 6. ARID1B expression is also lower in the WNT subgroup tumors compared to other subgroups and normal cerebellar tissues that could result in haploinsufficiency. The short hairpin RNA-mediated knockdown of ARID1B expression resulted in a significant increase in the malignant potential of medulloblastoma cells. Transcriptome sequencing identified upregulation of several genes encoding cell adhesion proteins, matrix metalloproteases indicating the epithelial-mesenchymal transition. The ARID1B knockdown also upregulated ERK1/ERK2 and PI3K/AKT signaling with a decrease in the expression of several negative regulators of these pathways. The expression of negative regulators of the WNT signaling like TLE1, MDFI, GPX3, ALX4, DLC1, MEST decreased upon ARID1B knockdown resulting in the activation of the canonical WNT signaling pathway. Synthetic lethality has been reported between SWI/SNF complex mutations and EZH2 inhibition, suggesting EZH2 inhibition as a possible therapeutic modality for WNT subgroup medulloblastomas. Thus, the identification of ARID1B as a tumor suppressor and its downregulation resulting in the activation of multiple signaling pathways opens up opportunities for novel therapeutic modalities for the treatment of WNT subgroup medulloblastoma.

Autophagy inhibition impairs the invasion potential of medulloblastoma cells.

Medulloblastoma, a highly malignant pediatric brain tumor, consists of four distinct molecular subgroups called WNT, SHH, Group 3, and Group 4 that differ in their clinical characteristics with the WNT subgroup having excellent survival rate. About 1/3rd medulloblastomas have metastasis at the time of diagnosis suggesting, high invasion potential of these tumors. We have earlier reported that the tumor-suppressive role of miR-204 and miR-30a is accompanied by inhibition of autophagy in medulloblastoma cells. In the present study, we have investigated the role of autophagy in medulloblastoma biology. Autophagy was inhibited in the medulloblastoma cell lines belonging to the SHH, Group 3, and Group 4 using the shRNA mediated knockdown of ATG5, an upstream regulator of autophagy. The effect of autophagy inhibition was studied on the growth and malignant behavior of medulloblastoma cells. ATG5 knockdown resulted in the autophagy inhibition in medulloblastoma cells as judged by the reduction in the flux of LC3B, a marker for autophagy. Autophagy inhibition did not result in a significant difference in the proliferation and anchorage-independent growth of the medulloblastoma cells. On the other hand, autophagy inhibition brought about a substantial reduction in the invasion potential of all three medulloblastoma cell lines studied. The present study suggests a therapeutic potential for autophagy inhibitors in the treatment of medulloblastoma. Autophagy inhibitors could be effective in reducing the dose of craniospinal radiation, thereby leading to a significant reduction in the treatment-related side effects.

Restoration of miR-193a expression is tumor-suppressive in MYC amplified Group 3 medulloblastoma.

Medulloblastoma, a highly malignant pediatric brain tumor, consists of four molecular subgroups, namely WNT, SHH, Group 3, and Group 4. The expression of miR-193a, a WNT subgroup-specific microRNA, was found to be induced by MYC, an oncogenic target of the canonical WNT signaling. MiR-193a is not expressed in Group 3 medulloblastomas, despite MYC expression, as a result of promoter hypermethylation. Restoration of miR-193a expression in the MYC amplified Group 3 medulloblastoma cells resulted in inhibition of growth, tumorigenicity, and an increase in radiation sensitivity. MAX, STMN1, and DCAF7 were identified as novel targets of miR-193a. MiR-193a mediated downregulation of MAX could suppress MYC activity since it is an obligate hetero-dimerization partner of MYC. MYC induced expression of miR-193a, therefore, seems to act as a feedback inhibitor of MYC signaling. The expression of miR-193a resulted in widespread repression of gene expression that included not only several cell cycle regulators, WNT, NOTCH signaling genes, and those encoding DNA replication machinery, but also several chromatin modifiers like SWI/SNF family genes and histone-encoding genes. MiR-193a expression brought about a reduction in the global levels of H3K4me3, H3K27ac, the histone marks of active chromatin, and an increase in the levels of H3K27me3, a repressive chromatin mark. In cancer cells having high MYC expression, MYC brings about transcriptional amplification of all active genes apart from the induction of its target genes. MiR-193a, on the other hand, brought about global repression of gene expression. Therefore, miR-193a has therapeutic potential in the treatment of not only Group 3 medulloblastomas but possibly other MYC overexpressing aggressive cancers as well.

Downregulation of miR-204 expression defines a highly aggressive subset of Group 3/Group 4 medulloblastomas.

Genome-wide expression profiling studies have identified four core molecular subgroups of medulloblastoma: WNT, SHH, Group 3 and Group 4. Molecular markers are necessary for accurate risk stratification in the non-WNT subgroups due to the underlying heterogeneity in genetic alterations and overall survival. MiR-204 expression was evaluated in molecularly classified 260 medulloblastomas from an Indian cohort and in 763 medulloblastomas from the MAGIC cohort, SickKids, Canada. Low expression of miR-204 in the Group 3 / Group 4 tumors identify a highly aggressive subset of tumors having poor overall survival, in the two independent cohorts of medulloblastomas. Downregulation of miR-204 expression correlates with poor survival within the Group 4 as well indicating it as a valuable risk-stratification marker in the subgroup. Restoration of miR-204 expression in multiple medulloblastoma cell lines was found to inhibit their anchorage-independent growth, invasion potential and tumorigenicity. IGF2R was identified as a novel target of miR-204. MiR-204 expression resulted in downregulation of both M6PR and IGF2R that transport lysosomal proteases from the Golgi apparatus to the lysosomes. Consistent with this finding, miR-204 expression resulted in reduction in the levels of the lysosomal proteases in medulloblastoma cells. MiR-204 expression also resulted in inhibition of autophagy that is known to be dependent on the lysosomal degradation pathway and LC3B, a known miR-204 target. Treatment with HDAC inhibitors resulted in upregulation of miR-204 expression in medulloblastoma cells, suggesting therapeutic role for these inhibitors in the treatment of medulloblastomas. In summary, miR-204 is not only a valuable risk stratification marker in the combined cohort of Group 3 / Group 4 medulloblastomas as well as in the Group 4 itself, that has paucity of good prognostication markers, but also has therapeutic potential as indicated by its tumor suppressive effect on medulloblastoma cells.

Restoration of miR-30a expression inhibits growth, tumorigenicity of medulloblastoma cells accompanied by autophagy inhibition.

Medulloblastoma is a highly malignant pediatric brain tumor. About 30% patients have metastasis at diagnosis and respond poorly to treatment. Those that survive, suffer long term neurocognitive, endocrine and developmental defects due to the cytotoxic treatment to developing child brain. It is therefore necessary to develop targeted treatment strategies based on underlying biology for effective treatment of medulloblastoma with minimal side effects. Medulloblastomas are believed to be the result of deregulated nervous system development as evident from the role of WNT and SHH developmental signaling pathways in pathogenesis of medulloblastomas. MicroRNAs are known to play vital roles in nervous system development as well as in cancer. MicroRNA profiling of medulloblastomas identified miR-30 family members' expression to be downregulated in medulloblastomas belonging to the four known molecular subgroups viz. WNT, SHH, Group 3 and Group 4 as compared to that in normal brain tissues. Furthermore, established medulloblastoma cell lines Daoy, D283 and D425 were also found to underexpress miR-30a. Restoration of miR-30a expression using inducible lentiviral vector inhibited proliferation, clonogenic potential and tumorigenicity of medulloblastoma cells. MiR-30a is known to target Beclin1, a mediator of autophagy. MiR-30a expression was found to downregulate Beclin1 expression and inhibit autophagy in the medulloblastoma cell lines as judged by downregulation of LC3B expression and its turnover upon chloroquine treatment and starvation induced autophagy induction. MiR-30a therefore could serve as a novel therapeutic agent for the effective treatment of medulloblastoma by inhibiting autophagy that is known to play important role in cancer cell growth, survival and malignant behavior.

ETV/Pea3 family transcription factor-encoding genes are overexpressed in CIC-mutant oligodendrogliomas.

Oligodendrogliomas with combined loss of chromosome arms 1p and 19q are known to be particularly sensitive to chemotherapy, and the CIC gene located on 19q is known to be mutated in over 50% of the 1p/19q codeleted oligodendrogliomas. However, the role of CIC in the oligodendroglioma pathogenesis is not known. Exome sequencing of 11 oligodendroglial tumors identified 9 tumors with combined loss of 1p and 19q. Somatic mutations were found in the CIC and FUBP1 genes. Recurrent somatic mutations were also identified in the Notch signaling pathway genes NOTCH1 and MAML3, the chromatin modifying gene ARID1A and in KRAS. Comparison of the transcriptome profiles of CIC-mutant and CIC-wild type oligodendrogliomas from the study cohort as well as 65 1p/19q codeleted oligodendrogliomas from the TCGA cohort identified genes encoding the ETV transcription factor family to be significantly upregulated in the CIC-mutant tumors. Upregulation of a number of negative regulators of the receptor tyrosine kinase signaling pathway like Sprouty and SPRED family members in the CIC-mutant oligodendrogliomas is likely due to the constitutive activation of the pathway resulting from inactive CIC protein. Higher expression of the oncogenic ETV transcription factors in the CIC-mutant oligodendrogliomas may make these tumors more aggressive than the CIC-wild type tumors.

Tamoxifen-Induced Cell Death of Malignant Glioma Cells Is Brought About by Oxidative-Stress-Mediated Alterations in the Expression of BCL2 Family Members and Is Enhanced on miR-21 Inhibition.

High-grade gliomas are refractory to the current mode of treatment primarily due to their inherent resistance to cell death. Tamoxifen has been reported to inhibit growth and induce cell death of glioma cells in vitro, in an estrogen-receptor-independent manner. Delineating the molecular mechanism underlying tamoxifen-induced cell death of human glioma cells would help in identifying pathways/genes that could be targeted to induce tumor-cell-specific cell death. In the present study, tamoxifen was found to bring about autophagic cell death of human glioma cells that was accompanied by oxidative stress induction, JNK activation, downregulation of anti-autophagic BCL2 family members, viz. BCL2 and BCL-XL, and increased expression of the pro-autophagic members BCL-Xs and BAK. Oxidative stress induction appears to be primarily responsible for the tamoxifen-induced cell death since the cell death, JNK activation, and the alterations in the expression levels of BCL2 family members were abrogated on pretreatment with antioxidant vitamin E. MiR-21, an oncogenic miRNA, is known to be highly upregulated in malignant glioma. Inhibition of miR-21 activity was found to enhance tamoxifen-induced cell death of U87 MG malignant glioma cells. Tamoxifen treatment coupled with miR-21 inhibition could therefore be an effective strategy for the treatment of malignant gliomas.

MiR-148a, a microRNA upregulated in the WNT subgroup tumors, inhibits invasion and tumorigenic potential of medulloblastoma cells by targeting Neuropilin 1.

Medulloblastoma, a common pediatric malignant brain tumor consists of four molecular subgroups viz. WNT, SHH, Group 3 and Group 4. MiR-148a is over-expressed in the WNT subgroup tumors, which have the lowest incidence of metastasis and excellent survival among all medulloblastomas. MiR-148a was expressed either in a transient manner using a synthetic mimic or in a stable doxycycline inducible manner using a lentiviral vector in non-WNT medulloblastoma cell lines. Expression of miR-148a to levels comparable to that in the WNT subgroup tumors was found to inhibit proliferation, clonogenic potential, invasion potential and tumorigenicity of medulloblastoma cells. MiR-148a expression in medulloblastoma cells brought about reduction in the expression of NRP1, a novel miR-148a target. Restoration of NRP1 expression in medulloblastoma cells was found to rescue the reduction in the invasion potential and tumorigenicity brought about by miR-148a expression. NRP1 is known to play role in multiple signaling pathways that promote tumor growth, invasion and metastasis. NRP1 expression in medulloblastomas was found to be associated with poor survival, with little or no expression in majority of the WNT tumors. The tumor suppressive effect of miR-148a expression accompanied by the down-regulation of NRP1 makes miR-148a an attractive therapeutic agent for the treatment of medulloblastomas.

MiR-224 expression increases radiation sensitivity of glioblastoma cells.

Glioblastoma (GBM) is the most common and highly aggressive primary malignant brain tumor. The intrinsic resistance of this brain tumor limits the efficacy of administered treatment like radiation therapy. In the present study, effect of miR-224 expression on growth characteristics of established GBM cell lines was analyzed. MiR-224 expression in the cell lines as well as in primary GBM tumor tissues was found to be low. Exogenous transient expression of miR-224 using either synthetic mimics or stable inducible expression using doxycycline inducible lentiviral vector carrying miR-224 gene, was found to bring about 30-55% reduction in clonogenic potential of U87 MG cells. MiR-224 expression reduced clonogenic potential of U87 MG cells by 85-90% on irradiation at a dose of 6Gy, a dose that brought about 50% reduction in clonogenic potential in the absence of miR-224 expression. MiR-224 expression in glioblastoma cells resulted in 55-65% reduction in the expression levels of API5 gene, a known target of miR-224. Further, siRNA mediated down-regulation of API5 was also found to have radiation sensitizing effect on glioblastoma cell lines. Analysis of the Cancer Genome Atlas data showed lower miR-224 expression levels in male GBM patients to correlate with poorer survival. Higher expression levels of miR-224 target API5 also showed significant correlation with poorer survival of GBM patients. Up-regulation of miR-224 or down-regulation of its target API5 in combination with radiation therapy, therefore appear as promising options for the treatment of glioblastoma, which is refractory to the existing treatment strategies.

Real-time PCR assay based on the differential expression of microRNAs and protein-coding genes for molecular classification of formalin-fixed paraffin embedded medulloblastomas.

BACKGROUND: Medulloblastoma has recently been found to consist of 4 molecularly and clinically distinct subgroups: WNT, Sonce hedgehog (SHH), Group 3, and Group 4. Deregulated microRNA expression is known to contribute to pathogenesis and has been shown to have diagnostic and prognostic potential in the classification of various cancers. METHODS: Molecular subgrouping and microRNA expression analysis of 44 frozen and 59 formalin-fixed paraffin embedded medulloblastomas from an Indian cohort were carried out by real-time RT-PCR assay. RESULTS: The differential expression of 9 microRNAs in the 4 molecular subgroups was validated in a set of 101 medulloblastomas. The tumors in the WNT subgroup showed significant (P < .0001) overexpression of miR-193a-3p, miR-224, miR-148a, miR-23b, and miR-365. Reliable classification of medulloblastomas into the 4 molecular subgroups was obtained using a set of 12 protein-coding genes and 9 microRNAs as markers in a real-time RT-PCR assay with an accuracy of 97% as judged by the Prediction Analysis of Microarrays. Age at diagnosis, histology, gender-related incidence, and the relative survival rates of the 4 molecular subgroups in the present Indian cohort were found to be similar to those reported for medulloblastomas from the American and European subcontinent. Non-WNT, non-SHH medulloblastomas underexpressing miR-592 or overexpressing miR-182 were found to have significantly inferior survival rates, indicating utility of these miRNAs as markers for risk stratification. CONCLUSIONS: The microRNA based real-time PCR assay is rapid, simple, inexpensive, and useful for molecular classification and risk stratification of medulloblastomas, in particular formalin-fixed paraffin embedded tissues, wherein the expression profile of protein-coding genes is often less reliable due to RNA fragmentation.

Distinctive microRNA signature of medulloblastomas associated with the WNT signaling pathway.

AIM: Medulloblastoma is a malignant brain tumor that occurs predominantly in children. Current risk stratification based on clinical parameters is inadequate for accurate prognostication. MicroRNA expression is known to be deregulated in various cancers and has been found to be useful in predicting tumor behavior. In order to get a better understanding of medulloblastoma biology, miRNA profiling of medulloblastomas was carried out in parallel with expression profiling of protein-coding genes. MATERIALS AND METHODS: miRNA profiling of medulloblastomas was carried out using Taqman Low Density Array v 1.0 having 365 human microRNAs. In parallel, genome-wide expression profiling of protein-coding genes was carried out using Affymetrix gene 1.0 ST arrays. RESULTS: Both the profiling studies identified four molecular subtypes of medulloblastomas. Expression levels of select protein-coding genes and miRNAs could classify an independent set of medulloblastomas. Twelve of 31 medulloblastomas were found to overexpress genes belonging to the canonical WNT signaling pathway and carry a mutation in CTNNB1 gene. A number of miRNAs like miR-193a, miR-224/miR-452 cluster, miR-182/miR-183/miR-96 cluster, and miR-148a having potential tumor/metastasis suppressive activity were found to be overexpressed in the WNT signaling associated medulloblastomas. Exogenous expression of miR-193a and miR-224, two miRNAs that have the highest WNT pathway specific upregulation, was found to inhibit proliferation, increase radiation sensitivity and reduce anchorage-independent growth of medulloblastoma cells. CONCLUSION: Expression level of tumor/metastasis suppressive miRNAs in the WNT signaling associated medulloblastomas is likely to determine their response to treatment, and thus, these miRNAs would be important biomarkers for risk stratification within the WNT signaling associated medulloblastomas.