Page 107 - Molecular features of low-grade developmental brain tumours

THE CODING AND NON-CODING TRANSCRIPTIONAL LANDSCAPE OF SEGA
(ERK1), RAF1 and BRAF, as well as with proteins related to the mTORC1 pathway, including RAPTOR, MTOR, TSC1, TSC2 (Figure 2g). Furthermore, most of these interacting proteins in the wider network were found to be differentially expressed in the RNA-Seq data and/or belonged to at least one of the enriched pathways (Figure 2g).
ERK is activated in SEGA tissue and co-expressed with LAMTOR1
We next determined whether ERK was activated in SEGA compared to periventricular control tissue by evaluating the phosphorylation of ERK1/ERK2 (pERK). Western blotting showed higher ERK phosporylation in SEGA samples (n=6; including 3 SEGAs with evidence of LOH) compared to control (n=4; Mann-Whitney U test, p-value<0.01; Figure 3a, b). Furthermore, no difference in pERK was seen between TSC1 (n=3) and TSC2 (n=3) mutated SEGAs. Activity of ERK was also seen in tubers (n=4) and AML (n=1) but not in control cortex (n=1) and control kidney tissue (n=1; Figure 3c). LAMTOR1 was present in SEGA samples but not detected in control tissue (Mann-Whitney U test, p-value<0.01; Figure 3a, b). Although variable, weak expression of LAMTOR1 was seen in cortex control, two tubers and kidney control but not in the AML (Figure 3c). The cellular distribution of LAMTOR1-LAMTOR5 was assessed in SEGA (n=6) and periventricular tissue (n=5) using immunohistochemistry (Figure 3d,e). In control tissue a low to weak expression of LAMTOR1-LAMTOR5 was seen mainly in the ependymal lining of lateral ventricles (red arrows). Furthermore, expression of LAMTOR1-LAMTOR5 was seen in neurons of cortex tissue (Supplementary Figure 4, arrowheads). Both pERK and pS6 were not detected in control tissue (Figure 3d,e, Supplementary Figure 4). In SEGA high expression of LAMTOR1-LAMTOR5 was seen in giant cells (Figure 3d,e; arrows and insets). Furthermore, LAMTOR1-LAMTOR5 were co-expressed in cells with pERK (mainly nuclear) and pS6 (mainly cytoplasmic).
Inhibition of ERK with U0126 decreases the proliferation of primary SEGA cells
We further evaluated the role of ERK on the cell cycle progression of SEGA cells using flow cytometric cell cycle analysis in one SEGA-derived cell culture (TSC1 mutated). The viability of cells was not altered after treatment with the ERK inhibitor U0126, rapamycin or the combination of U0126 and rapamycin (Figure 4a). The percentage of cells in the S-phase was decreased after treatment with U0126 (p-value<0.01) or rapamycin (p-value<0.01) compared to control (DMSO; Figure 4a,b). Combining U0126 with rapamycin also decreased the proliferation of SEGA cells (p-value<0.01) compared to control but not compared to U0126 or rapamycin alone. Inhibition of ERK activity by U0126 was confirmed by western blotting as well as the inhibition of mTORC1 pathway by rapamycin using pS6 as a readout (Supplementary Figure 3).
The small non-coding RNA landscape of SEGAs
Small RNA-Seq was performed to detect miRNAs and other small non-coding RNAs in the same cohort of SEGA and control samples that were used for RNA-Seq. After quality assessment and filtering, ~6 million reads remained, of which 83% were successfully mapped
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