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, 143 (1), 131-149

The Coding and Non-Coding Transcriptional Landscape of Subependymal Giant Cell Astrocytomas

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The Coding and Non-Coding Transcriptional Landscape of Subependymal Giant Cell Astrocytomas

Anika Bongaarts et al. Brain.

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited neurocutaneous disorder caused by inactivating mutations in TSC1 or TSC2, key regulators of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. In the CNS, TSC is characterized by cortical tubers, subependymal nodules and subependymal giant cell astrocytomas (SEGAs). SEGAs may lead to impaired circulation of CSF resulting in hydrocephalus and raised intracranial pressure in patients with TSC. Currently, surgical resection and mTORC1 inhibitors are the recommended treatment options for patients with SEGA. In the present study, high-throughput RNA-sequencing (SEGAs n = 19, periventricular control n = 8) was used in combination with computational approaches to unravel the complexity of SEGA development. We identified 9400 mRNAs and 94 microRNAs differentially expressed in SEGAs compared to control tissue. The SEGA transcriptome profile was enriched for the mitogen-activated protein kinase (MAPK) pathway, a major regulator of cell proliferation and survival. Analysis at the protein level confirmed that extracellular signal-regulated kinase (ERK) is activated in SEGAs. Subsequently, the inhibition of ERK independently of mTORC1 blockade decreased efficiently the proliferation of primary patient-derived SEGA cultures. Furthermore, we found that LAMTOR1, LAMTOR2, LAMTOR3, LAMTOR4 and LAMTOR5 were overexpressed at both gene and protein levels in SEGA compared to control tissue. Taken together LAMTOR1-5 can form a complex, known as the 'Ragulator' complex, which is known to activate both mTORC1 and MAPK/ERK pathways. Overall, this study shows that the MAPK/ERK pathway could be used as a target for treatment independent of, or in combination with mTORC1 inhibitors for TSC patients. Moreover, our study provides initial evidence of a possible link between the constitutive activated mTORC1 pathway and a secondary driver pathway of tumour growth.

Keywords: SEGA; TSC; low grade glioma; sequencing.

Figures

Figure 1
Figure 1
The protein-coding transcriptome of SEGAs. (A) A principal component analysis (PCA) of the RNA-Seq data in SEGA (n = 19) and periventricular control tissue (n = 8) showing that the major source of variability in gene expression is the diagnosis. x-axis: the first principal component (PC); y-axis: the second PC. (B) Spearman’s rank correlation matrix of the RNA-Seq data showing separate clustering of SEGAs from control tissue. The scale bar indicates the strength of the correlation with 1 indicating a strong positive correlation (dark blue) and 0 indicating no correlation (dark red) between samples. (C) Volcano plot showing the DEGs (adjusted P < 0.05) between SEGAs and control tissue. A total of 4621 mRNAs were found to be overexpressed and 4779 under-expressed in SEGA compared to control tissue. (D) Spearman’s rank correlation of the fold changes from TSC1 mutated SEGAs compared to the fold changes from TSC2 mutated SEGAs showing a strong correlation (rho = 0.89, P < 0.001). The Venn diagram shows 5292 DEGs in common between TSC1 and TSC2 mutated SEGAs, 721 DEGs were specific for TSC1 mutated SEGAs and 2816 DEGs were specific for TSC2 mutated SEGAs. (E) Schematic overview using Cytoscape of pathways enriched in SEGA compared to control tissue. Geometric testing was used to determine if the amount of DEGs was significant (adjusted P < 0.02) per pathway. Lines indicate genes in common between pathways. (F) Graphical representation of overexpressed genes (red) and under-expressed genes (blue) in 25 enriched pathways containing the highest amount of DEGs.
Figure 2
Figure 2
The Ragulator complex in SEGAs. (A) Schematic overview showing the Ragulator complex dependent mTORC1/MAPK signalling. Crystal structure of the Ragulator complex revealed that LAMTOR1 holds together the LAMTOR2/3 and LAMTOR4/5 heterodimers and anchors the complex to the late endosomes/lysosomes (de Araujo et al., 2017). In the presence of amino acids and growth factors, the Ragulator complex can promote Rag GTPase- dependent mTORC1 activation and MEK1 dependent ERK activation via direct interaction of RagA-D and MEK1 with LAMTOR2/3 heterodimer (Teis et al., 2002; Bar-Peled et al., 2012). Furthermore, the Ragulator complex interacts with vacuolar H+-ATPase and neutral amino acid transporter SLC38A9 and thereby can promote mTORC1 activation in the presence of amino acids (Rebsamen et al., 2015; Wang et al., 2015). Rapamycin inhibits mTORC1, whereas U0126 inhibits the MEK1 dependent ERK activation. (BF) RT-qPCR of LAMTOR1 (B), LAMTOR2 (C), LAMTOR3 (D), LAMTOR4 (E) and LAMTOR5 (F) in SEGA (n = 19) compared to control tissue (n = 8), which together can form the Ragulator complex. Data are expressed relative to the expression observed in control tissue. *P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney U-test. (G) A String functional protein association network of the Ragulator complex (LAMTOR1–LAMTOR5) interactions identified by StringApp for Cytoscape. High confidence interactions were selected (0.7), identifying interactions with proteins related to the MAPK/ERK and mTORC1 pathway. The outer circle indicates overexpression (red) or under-expression (blue) on RNA level based on the RNA-Seq data. The inner circle indicates to which enriched pathways each protein-interactor belongs to.
Figure 3
Figure 3
ERK activation and LAMTOR1-LAMTOR5 protein expression in SEGAs. (A) Western blot showing pERK1/2 and LAMTOR1 expression in SEGA [n = 6; TSC1 mutated: sample 1–3 and TSC2 mutated: samples 4–6; loss of heterozygosity: samples 3, 4 and 6; loss of heterozygosity identified as described in Bongaarts et al. (2017)] but not in periventricular control tissue (n = 4). β-tubulin was used as a loading control. (B) Quantification of pERK1/2 and LAMTOR1 signals normalized to either total ERK1/2 or β-tubulin. **P < 0.01, Mann-Whitney U-test. (C) Western blot showing pERK1/2 and LAMTOR1 expression in SEGA (n = 4), cortical tubers (n = 4) and angiomyolipoma (AML; n = 1) but not in periventricular control tissue (n = 1: sample C1), cortex control (n = 1; sample C2) and normal kidney tissue (n = 1; sample C3). β-tubulin was used as a loading control. (D and E) Immunohistochemistry for pERK1/2 (red, D) or pS6 (red, E) together with LAMTOR1–LAMTOR5 (blue) on SEGA (n = 6) and periventricular control tissue (n = 5). Insets show a higher magnification of giant cells (indicated with arrows) in SEGA and red arrows indicate the ependymal lining of lateral ventricles. Scale bar = 200 μm; insets = 100 μm.
Figure 4
Figure 4
ERK inhibitor U0126 and rapamycin inhibit proliferation of SEGA cells in vitro. (A) Primary SEGA cells from one SEGA-derived cell culture were stimulated for 24 h with U0126 (5 μM), rapamycin (0.01 μM), a combination of rapamycin with U0126 (rapamycin + U0126) or DMSO (0.05%) as a control. Flow cytometry analysis was used to assay the viability with eFluor or the cell-cycle state using propidium iodide (PI) staining of SEGA cells (n = 5). (B) Quantification of the PI staining showed lesser cells in the S-phase in the U0126, rapamycin and the rapamycin + U0126 conditions compared to control (0.05% DMSO). Data are expressed relative to the control condition. **P < 0.01, Kruskal-Wallis test followed by Mann-Whitney U-test.
Figure 5
Figure 5
The small non-coding RNA landscape of SEGAs. (A) Volcano plot showing 72 under-expressed and 68 overexpressed small RNAs in SEGA (n = 19) compared to control tissue (n = 8; adjusted P < 0.05). (B) Pie chart showing the distribution of different small RNAs differentially expressed in SEGA compared to control. miRNA = microRNA; snRNA = small nuclear RNA; snoRNA = small nucleolar RNA; vtRNA = vault RNA. (C) Volcano plot showing 49 under-expressed and 45 overexpressed miRNAs in SEGA (n = 19) compared to control tissue (n = 8; adjusted P < 0.05). (D) Heat map showing 81/92 enriched pathways from GSEA that were enriched for validated targets of 45/94 of the differentially expressed miRNAs (Fisher’s exact test, adjusted P < 0.05). Pathways enriched for a specific miRNA are indicted with a green box. (E) Validation of selected differentially expressed miRNAs (miRNA-20a-5p, miRNA-34a-5p, miRNA-130b-3p and miRNA-181a-5p) in SEGA (n = 19) compared to control tissue (n = 8) using TaqManTM PCR. **P < 0.01, ***P < 0.001, Mann-Whitney U-test.
Figure 6
Figure 6
Relative expression of LAMTOR genes after transfection with miRNA-20a-5p mimic in foetal astrocytes. TaqManTM PCR of miRNA-20a-5p (A) and RT-qPCR of LAMTOR1 (B), LAMTOR2 (C), LAMTOR3 (D), LAMTOR4 (E) and LAMTOR5 (F) in foetal astrocytes transfected with miRNA-20a-5p mimic (miR20a) for 24h (n = 3 biological triplets and two technical duplicates). Data are normalized to Lipofectamine® (control). *P < 0.05, **P < 0.01, Mann-Whitney U-test.

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