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 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.

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.