The deubiquitylase (DUB) USP37 is a component of the ubiquitin system and controls cell proliferation by regulating the stability of the cyclin-dependent kinase inhibitor 1B, (CDKN1B/p27Kip1). The expression of USP37 is downregulated in human medulloblastoma tumor specimens. In the current study, we show that USP37 prevents medulloblastoma growth in mouse orthotopic models, suggesting that it has tumor-suppressive properties in this neural cancer. Here, we also report on the mechanism underlying USP37 loss in medulloblastoma. Previously, we observed that the expression of USP37 is transcriptionally repressed by the RE1 silencing transcription factor (REST), which requires chromatin remodeling factors for its activity. Genetic and pharmacologic approaches were employed to identify a specific role for G9a, a histone methyltransferase (HMT), in promoting methylation of histone H3 lysine-9 (H3K9) mono- and dimethylation, and surprisingly trimethylation, at the USP37 promoter to repress its gene expression. G9a inhibition also blocked the tumorigenic potential of medulloblastoma cells in vivo. Using isogenic low- and high-REST medulloblastoma cells, we further showed a REST-dependent elevation in G9a activity, which further increased mono- and trimethylation of histone H3K9, accompanied by downregulation of USP37 expression. Together, these findings reveal a role for REST-associated G9a and histone H3K9 methylation in the repression of USP37 expression in medulloblastoma. Implications: Reactivation of USP37 by G9a inhibition has the potential for therapeutic applications in REST-expressing medulloblastomas. Mol Cancer Res; 15(8); 1073–84. ©2017 AACR.

Overexpression of the oncoprotein Aurora A kinase occurs in multiple types of cancer, often early during cell transformation. To identify the mechanism(s) contributing to enhanced Aurora A protein expression, a comparison between normal human lung fibroblast and breast epithelial cells to nontumorigenic breast (MCF10A and MCF12A) and tumorigenic breast (MCF-7) and cervical cell lines (HeLa S3) was performed. A subset of these immortalized lines (MCF10A, MCF12A, and HeLa S3) exhibited increased levels of Aurora A protein, independent of tumorigenicity. The increase in Aurora A protein in these immortalized cells was not due to increased transcription/RNA stability, protein half-life, or cap-dependent translation. Assays utilizing monocistronic and dicistronic RNA constructs revealed that the 5′-leader sequence of Aurora A contains an internal ribosomal entry site (IRES), which is regulated in a cell cycle–dependent manner, peaking in G₂/M phase. Moreover, IRES activity was increased in the immortalized cell lines in which Aurora A protein expression was also enhanced. Additional studies indicated that the increased internal initiation is specific to the IRES of Aurora A and may be an early event during cancer progression. These results identify a novel mechanism contributing to Aurora A kinase overexpression. Implications: The current study indicates that Aurora A kinase contributes to immortalization and tumorigenesis. Mol Cancer Res; 11(8); 887–900. ©2013 AACR.

In medulloblastomas (MBs), the expression and activity of RE1-silencing transcription factor (REST) is increased in tumors driven by the sonic hedgehog (SHH) pathway, specifically the SHH-α (children 3 to 16 years) and SHH-β (infants) subgroups. Neuronal maturation is greater in SHH-β than SHH-α tumors, but both correlate with poor overall patient survival. We studied the contribution of REST to MB using a transgenic mouse model (RESTTG) wherein conditional NeuroD2-controlled REST transgene expression in lineage-committed Ptch1^+/− cerebellar granule neuron progenitors (CGNPs) accelerated tumorigenesis and increased penetrance and infiltrative disease. This model revealed a neuronal maturation context–specific antagonistic interplay between the transcriptional repressor REST and the activator GLI1 at Ptch1. Expression of Arrb1, which encodes β-arrestin1 (a GLI1 inhibitor), was substantially reduced in proliferating and, to a lesser extent, lineage-committed RESTTG cells compared with wild-type proliferating CGNPs. Lineage-committed REST^TG cells also had decreased GLI1 activity and increased histone H3K9 methylation at the Ptch1 locus, which correlated with premature silencing of Ptch1. These cells also had decreased expression of Pten, which encodes a negative regulator of the kinase AKT. Expression of PTCH1 and GLI1 were less, and ARRB1 was somewhat greater, in patient SHH-β than SHH-α MBs, whereas that of PTEN was similarly lower in both subtypes than in others. Inhibition of histone modifiers or AKT reduced proliferation and induced apoptosis, respectively, in cultured REST-high MB cells. Our findings linking REST to differentiation-specific chromatin remodeling, PTCH1 silencing, and AKT activation in MB tissues reveal potential subgroup-specific therapeutic targets for MB patients.

Approximately five out of 100,000 children from 0 to 19 years old are diagnosed with a brain tumor. These children are treated with medication designed for adults that are highly toxic to a developing brain. Those that survive are at high risk for a lifetime of limited physical, psychological, and cognitive abilities. Despite much effort, not one drug exists that was designed specifically for pediatric patients. Stagnant government funding and the lack of economic incentives for the pharmaceutical industry greatly limits preclinical research and the development of clinically applicable pediatric brain tumor models. As more data are collected, the recognition of disease sub-groups based on molecular heterogeneity increases the need for designing specific models suitable for predictive drug screening. To overcome these challenges, preclinical approaches will need continual enhancement. In this review, we examine the advantages and shortcomings of in vitro and in vivo preclinical pediatric brain tumor models and explore potential solutions based on past, present, and future strategies for improving their clinical relevancy.

Medulloblastoma is the most common malignant pediatric brain tumor. Current treatments including surgery, craniospinal radiation and high-dose chemotherapy have led to improvement in survival. However, the risk for recurrence as well as significant long-term neurocognitive and endocrine sequelae associated with current treatment modalities underscore the urgent need for novel tumor-specific, normal brain-sparing therapies. It has also provided the impetus for research focused on providing a better understanding of medulloblastoma biology. The expectation is that such studies will lead to the identification of new therapeutic targets and eventually to an increase in personalized treatment approaches.

Translational regulation of the dendritically localized mRNA encoding for the neurotrophin receptor TrkB has important ramifications for synaptic function. We examined whether the TrkB mRNA is translated through an internal initiation entry site (IRES). The human TrkB 5′ leaders are derived from the use of alternative promoters and alternative splicing, but all 5′ leaders share a common exon. Insertion of a full-length 5′ leader, as well as the common exon into the intercistronic region of a dicistronic luciferase construct, yielded luciferase activity generated from the second cistron that was either equivalent or higher than that observed from the encephalomyocarditis virus IRES. Moreover, inhibiting cap-dependent translation ex vivo and in in vitro lysates had only a minimal effect on the translation of mRNA containing the TrkB 5′ leader. Dissecting the 5′ leader showed that the IRES is located in the exon common to all TrkB 5′ leaders. Moreover, six regions ranging from 2 to 25 nt were identified that either promoted or inhibited IRES activity. Taken together, these results suggest that the 5′ leader of the human TrkB mRNA contains multiple cis-elements that regulate internal initiation of translation and that this mechanism may contribute significantly to the translation of the TrkB mRNA in neuronal dendrites.