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.

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. Electronic supplementary material The online version of this article (10.1186/s40478-019-0697-3) contains supplementary material, which is available to authorized users.

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.

No abstract

No abstract

Purpose: Medulloblastoma, a most 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. Therefore, the role of miR-148a in medulloblastoma biology was investigated. Experimental Design: MiR-148a was expressed either in a transient manner using synthetic mimic or in a stable doxycycline inducible manner using a lentiviral vector. Effect of miR-148a expression on the growth and malignant behavior of medulloblastoma cell lines was investigated. CpG Methylation status of region upstream to pri-miR-148a was determined by performing bisulphite sequencing of this region in medulloblastoma cell lines and tissues. Results: Expression of miR-148a to the levels comparable to that in the WNT subgroup tumors was found to inhibit proliferation, clonogenic potential and in vitro invasion potential of medulloblastoma cells. In vivo tumorigenicity of medulloblastoma cells Daoy, D425 and D283, as seen by either generating subcutaneous xenografts or orthotopic xenografts in immunodeficient mouse, was found to be reduced significantly upon miR-148a expression. NRP1, ROCK1 and DNMT1 were identified as direct targets of miR-148a with NRP1 being novel 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. NRP1expression in medulloblastomas was found to be associated with poor survival, with little or no expression in majority of the WNT tumors. This observation is consistent with high miR-148a expression and low incidence of metastasis and excellent survival of the WNT subgroup tumors. miR-148a is known to be down-regulated as a result of promoter hypermethylation in colon, lung, breast, head and neck carcinomas and melanoma. Bisulphite sequencing of the region upstream to pri-miR-148a revealed presence of CpG methylation in four medulloblastoma cell lines Daoy, D283, D341 and D425 and non WNT medulloblastoma tissues, while it was found to be un-methylated in WNT medulloblastomas. Conclusions: The tumor suppressive effect of miR-148a expression in medulloblastoma cells accompanied by the down-regulation of NRP1, ROCK1 and DNMT1 makes miR-148a an attractive therapeutic agent for the treatment of medulloblastomas. Preliminary findings provided by bisulphite sequencing suggest that under-expression of miR-148a may be as a result of CpG methylation of upstream region to pri-miR-148a sequence. Citation Format: Kedar Narsinha Yogi, Epari Sridhar, Naina Goel, Rakesh Jalali, Atul Goel, Aliasgar Moiyadi, Rahul Thorat, Pooja Panwalkar, Atul Khire, Archya Dasgupta, Prakash Shetty, Neelam Shirsat. miR-148a functions as tumor-suppressor microRNA in medulloblastoma cell lines by targeting Neuropilin1. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B41.