Page 110 - Molecular features of low-grade developmental brain tumours

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two important pathways of tumour growth and cell survival.
In the present study, high-throughput sequencing of both coding and non-coding
transcriptome was performed on 19 SEGAs and 8 periventricular control tissue. We identified substantial gene expression changes in SEGAs compared to periventricular control tissue. These gene expression changes appear to be independent of the TSC1/TSC2 mutation or other clinical information available. Pathway enrichment analysis identified 116 pathways enriched in SEGA compared to control tissue, including immune system, extracellular matrix organization, metabolism, transmission across chemical synapses and the MAPK family signaling cascades. Several of the enriched pathways found in our study are related to the biological processes found in previous transcriptome based SEGA studies 23,28. Differential expression of genes related to the immune system has also been identified in cortical tubers through the use of RNA-Seq and microarrays 23,69,70. Multiple studies of TSC animal models and TSC human tissue, including prenatal TSC lesions have documented dysregulation of inflammation related pathways, such as immune response, suggesting that this biological process is more conserved across TSC pathology rather than a SEGA specific process 69,71-74. One of the enriched pathways found in this study was the MAPK pathway. Previous studies focusing on TSC2 mutated tubers and SEGAs in which the TSC2 protein is still present documented the presence of MAPK/ERK activation 25-27. In accordance with these studies we show that ERK activation is present in tubers and SEGA and that the activation of ERK seen in SEGAs seems to be independent of TSC1/TSC2 mutation and LOH. Therefore, it could be of interest to further investigate the MAPK/ERK activation in other TSC related lesions.
Current treatment of SEGAs is limited to surgical removal and mTORC1 inhibitors, including rapamycin and everolimus 33-39. In recent TSC clinical trials, it was shown that responses to mTORC1 inhibitors can be variable and that lesions tend to relapse after cessation of treatment 34,38-43. A possible explanation for this could be that inhibition of mTORC1 leads to the disruption of the negative feedback on the MAPK/ERK pathway resulting in MAPK/ ERK activation 75,76. Furthermore, MAPK/ERK activation can result in TSC2 phosphorylation and thereby increase mTORC1 activation, indicating that these two pathways are intrinsically linked 25-27. Only two patients included in the present study were treated with mTORC1 inhibitors, indicating that the ERK activation seen can not be explained by mTORC1 inhibition.
Previous studies have shown that inhibiting MAPK/ERK activity decreased the proliferation of Tsc2-/- MEF mouse cells, SEGA cells and tumour growth in mice heterozygous for TSC2 28,77,78. In accordance with these studies, we show that inhibiting ERK in a primary human derived SEGA culture using the ERK inhibitor U0126 decreased the proliferation in a similar manner to treatment with rapamycin as a mTORC1 inhibitor alone. In contrast to previous research, we did not observe differences between rapamycin and combined therapy with rapamycin and the ERK inhibitor U0126 in SEGA cells from one SEGA-derived cell culture 28,77. A previous study by Mi et al., 2009 identified that combined treatment of rapamycin and ERK inhibitors was more efficient in inhibiting the proliferation of TSC2 deficient cells then treatment with rapamycin or ERK inhibitors alone after 3 days of treatment, but not over shorter time periods 77. Furthermore, Tyburczy et al., 2010 showed that suppression of both