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Medulloblastoma (MB), the most common malignant pediatric brain tumor, is a leading cause of childhood related morbidity and mortality. Large-scale transcriptional profiling and mutational analyses have facilitated the stratification of medulloblastoma into four primary subgroups, i.e. SHH (sonic hedgehog), WNT (wingless), and non-SHH/WNT groups 3 and 4. The most frequent cytogenetic abnormality in medulloblastoma, i.e. i17q, distinguishes the non-SHH/WNT subgroup. Haploinsufficiency of 17p13.3 is reported in up to 50% of human MB cases. Included within this locus is miR-1253, which is exclusively expressed in the brain and an important regulator of bone morphogenic proteins that play a critical role in cerebellar development. Recently, two oncogenic targets of miR-1253, i.e. TGIF2 and ALX4, were identified in SHH medulloblastoma. Based upon these observations, we hypothesized that miR-1253 may be a putative tumor suppressor gene that undergoes epigenetic silencing in pediatric medulloblastoma. We first discovered reduced expression of miR-1253 in 24 pediatric medulloblastoma specimens and in 7 medulloblastoma cell lines. We then learned that miR-1253 silencing is accomplished via hypermethylation; expectedly, de-methylation of miR-1253, resulted in the recovery of expression with a subsequent decline in MB cell proliferation. MiR-1253 restoration was further concomitant with activation of apoptotic pathways and cell cycle arrest at G(0)/G(1) phase. Moreover, miR-1253 overexpression led to a reduction in cell proliferation, colony formation, migration and invasive potential of MB tumor cell lines. Using high throughput RNA sequencing analysis and luciferase reporter assay, we further identified several oncogenic targets of miR-1253, including CDK-6 and CD276. Taken together, these data strongly support a tumor suppressive role for miR-1253. This would be the first time such an effect has been attributed to miR-1253 in the context of medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric tumor of the central nervous system (CNS) and a leading cause of childhood morbidity and mortality. Haploinsufficiency of 17p13.3 is reported in up to 50% of human MB cases. Included within this locus is miR-1253, which is exclusively expressed in the brain and an important regulator of bone morphogenic proteins that play a critical role in cerebellar development. Recently, two oncogenic targets of miR-1253, i.e. TGIF2 and ALX4, were identified in SHH medulloblastoma. Based upon these observations, we hypothesized that miR-1253 may be a putative tumor suppressor gene that undergoes epigenetic silencing in pediatric medulloblastoma. We first discovered reduced expression of miR-1253 in 24 clinical specimens and in 7 medulloblastoma cell lines. We then learned that miR-1253 silencing is accomplished via hypermethylation; expectedly, de-methylation of miR-1253 using 5-AzaC, resulted in recovery of expression with a subsequent decline in MB cell proliferation. MiR-1253 restoration was further concomitant with activation of apoptotic pathways and cell cycle arrest at G(0)/G(1) phase. Moreover, miR-1253 overexpression led to a reduction in cell proliferation, colony formation, migration and invasive potential of MB tumor cell lines. Taken together, these data suggest that miR-1253 may possess tumor suppressor qualities, i.e. 1) loss of expression via epigenetic silencing and 2) negative trophic effects on tumor cell growth and migration. This would be the first time such an effect has been attributed to miR-1253 in the context of medulloblastoma.

Medulloblastoma (MB), the most common malignant pediatric brain tumor, is divided into four primary subgroups, i.e. SHH, WNT, and non-SHH/WNT groups 3 and 4. The most frequent cytogenetic abnormality in MB, i.e. i17q, distinguishes the non-SHH/WNT subgroups. Haploinsufficiency of 17p13.3 is reported in up to 50% of non-SHH/WNT MB cases and has been correlated with poor prognosis. At the terminal end of this locus is miR-1253, which is exclusively expressed in the brain and an important regulator of bone morphogenic proteins that play a critical role in cerebellar development. Recently, miR-1253 was identified as a tumor suppressor gene in non-small cell lung cancer (NSCLC) and osteosarcoma. Two oncogenic targets of miR-1253, i.e. TGIF2 and ALX4, have recently been identified in SHH medulloblastoma. Based upon these observations, we hypothesized that miR-1253 may be a tumor suppressor gene in pediatric medulloblastoma. Starting with an expression analysis of miR-1253, we found deregulation in 28 pediatric clinical samples (5 SHH, 10 Grp 3, 13 Grp 4) and 7 medulloblastoma cell lines. Moreover, the locus is hypermethylated in Grp 3 and Grp 4 MB. Expectedly, de-methylation using 5-AzaC resulted in rapid recovery of expression with a concurrent decline in cell proliferation, a property abrogated using an miR-1253 inhibitor. Overexpression of miR-1253 in tumor cell lines led to a reduction in cell proliferation, colony formation, migration and invasiveness, concomitant with activation of apoptotic pathways and reduced stemness. In subcutaneously transplanted HDMB03 cells overexpressing miR-1253, tumor size was noted to be significantly lower. Using high throughput RNA sequencing analysis and luciferase reporter assay, we further identified 2 putative oncogenic targets of miR-1253, i.e. CDK6 and CD276. Taken together, these data purport that in pediatric medulloblastoma, miR-1253 is epigenetically silenced by hypermethylation and possesses tumor suppressor qualities by downregulating its oncogenic targets CDK6 and CD276.

Medulloblastoma (MB), the most frequent malignant pediatric brain tumor is divided into four subgroups, i.e. wingless-type (WNT), sonic hedgehog (SHH), Group 3, and Group 4. Among them, haploinsufficiency of chromosome 17p13.3 is most commonly seen in Group 3 and 4 tumors, which are concurrently associated with the poorest prognosis. Recent studies have revealed the importance of microRNAs (miRNAs) in regulating posttranscriptional gene expression in MB tumorigenesis. In this study, we sought to identify the role of miR-212, which resides on chromosome 17p13.3, in the development of non-SHH/WNT medulloblastoma. RNA expression analysis showed significantly reduced expression of miR-212 in MB tumor cell lines and in ex vivo non-SHH/WNT MB tumor samples (n=16). To further elucidate its putative tumor suppressor role, miR-212 was over-expressed in non-SHH/WNT tumor cell line, HDMB03, followed by functional assays of tumor cell behavior. In these studies, significantly reduced cell proliferation, colony formation, migration and invasion were noted. Additionally, reduced levels of pAKT, a marker of cell proliferation, was confirmed in miR-212-overexpressing HDMB03 cells. Subsequent cell cycle analysis revealed increased G(0)/G(1) cell cycle arrest with a concurrent reduction in the corresponding cell cycle regulatory proteins, i.e. CDK6 and cyclinD1. Myc amplification, which is facilitated by phosphorylation of serine-62 (p-c-Myc-S62), is a characteristic feature of Group 3 medulloblastoma and a marker of poor prognosis. Decreased levels of p-c-Myc-S62 (active form favoring proliferation) with a complimentary increase in p-c-Myc-T58 (inactive form favoring apoptosis) results in degradation of c-Myc protein and cellular apoptosis, a trend recapitulated in miR-212-overexpressing HDMB03 cells. In addition, the pro-apoptotic binding partners of c-Myc, i.e. Bin-1 and P19(ARF), were noted to be upregulated in miR-212 over-expressed HDMB03 cells, further favoring apoptosis. That c-myc may also serve as a target of miR-212 is currently being studied. These results substantiate miR-212 as a tumor suppressor gene in non-SHH/WNT medulloblastoma.