A subset of medulloblastomas, the most common brain tumor in children, is hypothesized to originate from granule neuron precursors (GNPs) in which the sonic hedgehog (SHH) pathway is over-activated. MXD3, a basic helix-look-helix zipper transcription factor of the MAD family, has been reported to be upregulated during postnatal cerebellar development and to promote GNP proliferation and MYCN expression. Mxd3 is upregulated in mouse models of medulloblastoma as well as in human medulloblastomas. Therefore, we hypothesize that MXD3 plays a role in the cellular events that lead to medulloblastoma biogenesis. In agreement with its proliferative role in GNPs, MXD3 knock-down in DAOY cells resulted in decreased proliferation. Sustained overexpression of MXD3 resulted in decreased cell numbers due to increased apoptosis and cell cycle arrest. Structure-function analysis revealed that the Sin3 interacting domain, the basic domain, and binding to E-boxes are essential for this activity. Microarray-based expression analysis indicated up-regulation of 84 genes and down-regulation of 47 genes. Potential direct MXD3 target genes were identified by ChIP-chip. Our results suggest that MXD3 is necessary for DAOY medulloblastoma cell proliferation. However, increased level and/or duration of MXD3 expression ultimately reduces cell numbers via increased cell death and cell cycle arrest.

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The transcription factor MXD3 is an atypical member of the MYC/MAX/MXD transcriptional network and has been previously shown to be an important regulator of cell proliferation. MXD3 has been shown to be overexpressed and to be required for medulloblastoma and acute lymphoblastic leukemia cell proliferation. In this study we leveraged datasets from The Cancer Genome Atlas to examine MXD3 across several cancers. We find that MXD3 transcripts are significantly overexpressed in ~72% of the available datasets. The gene itself is not frequently altered, while the promoter appears to be hypomethylated. We examine the possibility that aberrant regulation of the MXD3 message is the cause of abnormal MXD3 expression across cancers. Specifically, we looked at MXD3 alternative splicing in glioblastoma multiforme (GBM) and find notable functional differences between the splice variants. The 3′UTR confers differential message stability. Furthermore, the different coding sequences lead to different protein stabilities and localizations. Altogether, these data extend our knowledge of MXD3 in the context of human cancers while characterizing a previously unstudied splice variant of MXD3.

BackgroundMXD3 is a basic-helix-loop-helix-leucine-zipper transcription factor involved in cellular proliferation. In previous studies we demonstrated that knock-down of MXD3 in the human medulloblastoma cell line DAOY resulted in decreased proliferation. Surprisingly, overexpression of MXD3 in DAOY cells also decreased proliferation and increased cell death, suggesting that persistent expression of MXD3 triggers an apoptotic response, perhaps as a fail-safe mechanism. To investigate this apparent paradox in detail we developed a tamoxifen inducible system to analyze the temporal effects of MXD3 in the proliferation and transcriptional response of DAOY cells upon acute induction compared with long-term expression of MXD3.ResultsWe find that acute induction of MXD3 initially promotes cell cycle progression as assessed by a transient increase in bromodeoxyuridine incorporation. However, persistent induction of MXD3 ultimately results in decreased proliferation based on cell counts. Finally, with microarray expression profiling and gene ontology analysis we identify several major pathways enriched in response to acute (immune response, apoptosis, cell cycle) versus persistent (cell adhesion) MXD3 activation.ConclusionsIn this study, we demonstrate that acute MXD3 activation results in a transient increase in cell proliferation while persistent activation of MXD3 eventually results in an overall decrease in cell number, suggesting that the time course of MXD3 expression dictates the cellular outcome. Microarray expression profiling and gene ontology analysis indicate that MXD3 regulates distinct genes and pathways upon acute induction compared with persistent expression, suggesting that the cellular outcome is specified by changes in MXD3 transcriptional program in a time-dependent manner.

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