Abstract
Medulloblastoma (MB) is the most common malignant primary pediatric brain cancer. Among the most aggressive subtypes, Group 3 and Group 4 originate from stem/progenitor cells, frequently metastasize, and often display the worst prognosis, yet we know the least about the molecular mechanisms driving their progression. Here, we show that the transcription factor orthodenticle homeobox 2 (OTX2) promotes self‐renewal while inhibiting differentiation in vitro and increases tumor initiation from MB stem/progenitor cells in vivo. To determine how OTX2 contributes to these processes, we employed complementary bioinformatic approaches to characterize the OTX2 regulatory network and identified novel relationships between OTX2 and genes associated with neuronal differentiation and axon guidance signaling in Group 3 and Group 4 MB stem/progenitor cells. In particular, OTX2 levels were negatively correlated with semaphorin (SEMA) signaling, as expression of 9 SEMA pathway genes is upregulated following OTX2 knockdown with some being potential direct OTX2 targets. Importantly, this negative correlation was also observed in patient samples, with lower expression of SEMA4D associated with poor outcome specifically in Group 4 tumors. Functional proof‐of‐principle studies demonstrated that increased levels of select SEMA pathway genes are associated with decreased self‐renewal and growth in vitro and in vivo and that RHO signaling, known to mediate the effects of SEMA genes, is contributing to the OTX2 KD phenotype. Our study provides mechanistic insight into the networks controlled by OTX2 in MB stem/progenitor cells and reveals novel roles for axon guidance genes and their downstream effectors as putative tumor suppressors in MB.
Highlights
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We discovered that when Group 3 and Group 4 MB cells are grown in stem cell-enriched conditions, orthodenticle homeobox 2 (OTX2) KD results in a decrease in both self-renewal capacity or stem cell function and cell growth 11
To explore whether the decrease in self-renewal capacity results in increased differentiation, we evaluated the expression of the classic neuronal differentiation markers, βIII-tubulin and MAP2, following OTX2 KD by qPCR and/or immunofluorescence staining (IF). βIII-tubulin or TUJI and MAP2 transcript levels were strongly upregulated in D283 tumorspheres following OTX2 KD by qPCR (Figure 4.2A-B)
Summary
Glutamatergic progenitors arise from the dorsal telencephalon and thalamus of the human brain 118. Studies suggest a genetic program suppresses GABAergic differentiation during specification of glutamatergic progenitors. Conditional Otx knockout mice exhibit an increase in GABAergic differentiation and a loss of glutamatergic differentiation in the thalamus 119. Puelles et al have observed that in the absence of Otx, GABAergic fate increases alongside achaete-scute family BHLH transcription factor 1 (Mash1) expression, while Neurogenin 2 (Ngn2) expression is repressed 119 concomitant with activation of the GABAergic transcription factors Pax, Pax, and Lhx 119. Analysis of cell cycle activity revealed increases in the frequency of cells in the S and M phases in OTX2- progenitors. These data underscore the notion that the regulatory role of OTX2 is both temporally and cell-type dependent
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