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Medulloblastoma (MB), the most common malignant pediatric brain tumor, is a leading cause of childhood mortality. Of the four primary subgroups, patients with group 3 tumors have the poorest prognosis. Loss of chromosome 17p is a high-risk feature associated with poor outcomes in group 3 tumors. We recently elucidated the tumor suppressive properties of a novel miR, miR-1253, on the terminal end of 17p. In further exploring its anti-neoplastic effects, we discovered that miR-1253 can disrupt iron homeostasis, causing oxidative stress and inducing lipid peroxidation. These concurrent events are capable of triggering an iron-mediated form of cell death called ferroptosis. Notably, our in silico interrogation of ferroptosis regulator genes (FRGs) in group 3 tumors revealed high expression of genes associated with iron transport and glutathione metabolism. These included mitochondrial iron transporters and GPX4, a critical regulator of ferroptosis. Restoration of miR-1253 expression in group 3 cell lines resulted in specific downregulation of ABCB7, an iron-sulfur cluster exporter, and GPX4. Consequently, cytosolic and mitochondrial labile iron pools rose, glutathione levels declined, and mitochondrial oxidative stress and lipid peroxidation were induced. These events were recapitulated by ABCB7 knockdown and potentiated cell death. Treating miR-1253-expressing cancer cells with cisplatin, a group 3 MB chemotherapeutic agent with ferroptotic properties, further elevated oxidative stress, depleted glutathione levels, and augmented lipid peroxidation, with added inhibitory effects on cell viability and colony formation. Treatment with a ferroptosis inhibitor (ferrostatin-1) lead to recovery from the cytotoxic effects of this combination therapy. Our studies highlight a novel mechanism for group 3 MB pathogenesis via ferroptosis regulation and provide a proof-of-concept for exploiting group 3 MB tumor vulnerability to iron imbalance as a novel treatment strategy.

Medulloblastoma (MB), the most common malignant pediatric brain tumor and a leading cause of childhood mortality, is stratified into four primary subgroups, i.e. SHH (sonic hedgehog), WNT (wingless), and non-SHH/WNT groups 3 and 4, the latter representing high-risk MB. Haploinsufficiency of 17p13.3, which houses the tumor suppressor gene miR-1253, characterizes high-risk tumors. Despite improvements in targeted therapies, a limited proportion of these patients survive the disease. Capitalizing on the tumor suppressive properties of miRNAs as adjuncts to chemotherapy provides a promising alternative to current therapeutic strategies. In this study, we explored the potentiating effects of miR-1253 on cisplatin cytotoxicity in group 3 MB. First, in silico and in vitro analyses revealed an upregulation of ABCB7, a mitochondrial iron transporter and putative target of miR-1253, in MB cell lines and group 3 MB tumors. Overexpression of miR-1253 resulted in downregulation of ABCB7 and GPX4, a critical ferroptosis regulator, which consequently increased labile mitochondrial iron pool and, in turn, mitochondrial ROS (mtROS). Complementarily, we demonstrated, using CRISPR knockdown of ABCB7, ferroptosis induction with downregulation of GPx4 expression, liberation of free iron, mtROS generation and lipid peroxidation. Cisplatin is reported as an inducer of both apoptosis and ferroptosis-mediated cancer cell death. Therapeutically, the combination of miR-1253 and cisplatin led to an additive effect on cell viability, colony formation, apoptosis, and ROS generation. In turn, treatment with mtROS inhibitor (MnTBAP) and ferroptosis inhibitor (Ferrostatin) lead to partial recovery from the cytotoxic effects of this combination therapy. These studies identify an miR-1253-induced ferroptosis pathway targeting the ABCB7/GPX4/mtROS axis in group 3 MB. They further provide proof-of-concept in using miR-based therapeutics to augment treatment efficacy of current chemotherapeutics in the treatment of high-risk tumors.

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.