Pi3K-mTOR signaling and AMOG expression in epilepsy-associated glioneuronal tumors

Abstract

Gangliogliomas (GGs) and dysembryoplastic neuroepithelial tumors (DNTs) represent the most frequent type of neoplasms in pediatric medically intractable epilepsy. Several data suggest a pathogenetic relationship between GGs and other glioneuronal malformations of cortical development (MCDs), including activation of the Pi3K-mTOR signaling pathway. To further reveal these pathogenetic similarities, we investigated immunocytochemically the expression of phosphorylated (p)-PDK1, p-AKT, p-mTOR, p-4E-BP1, p-eIF4G, p-p70S6K and p-S6, the effector proteins ERM (ezrin/radixin/moesin) and the pathway regulator AMOG (adhesion molecule on glia) in both GGs and DNTs. Components of the Pi3K-mTOR signaling pathway were observed in a higher percentage of neuronal cells in GGs compared with control cortex. In DNTs, the expression of these components was low and comparable with the expression in control samples. Strong immunoreactivity for ERM was observed in GGs, but not in DNTs. Additionally, AMOG was strongly expressed within GGs (but not in DNTs) in CD34-positive precursor cells. These findings support the previously suggested pathogenic relationship between GG and MCDs concerning activation of the Pi3K-mTOR signaling pathway and suggest a different pathogenetic origin for DNTs. The strong expression of AMOG within the precursor cells of GG may represent an additional marker for the diagnostic evaluation of these glioneuronal lesions.

Figure 1

Schematic representation of the Pi3K‐mTOR signaling pathway. Ligand binding to insulin receptors or growth factor receptors trigger phosphatidylinositol‐3 kinase (Pi3K), which in turn activates the phosphoinositide‐dependent protein kinase 1 (PDK1) by phosphorylation. Akt is phosphorylated and activated by phosphorylated (p)‐PDK1 or by the adhesion molecule on glia (AMOG) independently of PDK1 and Pi3K. P‐Akt inactivates the tumor suppressor tuberin (TSC2) by phosphorylation which results in the indirect activation of the mammalian target of rapamycin (mTOR). Downstream phosphorylation of the eukaryotic initiation factor 4E binding protein 1 (4E‐BP1) releases the eukaryotic initiation factor 4E (eIF4E). eIF4E interacts with p‐eukaryotic initiation factor 4G (eIF4G) to activate cap‐dependent mRNA translation which enhances cell size and cell proliferation. Cell size and proliferation is also regulated by phosphorylation of the ribosomal protein S6 kinase (p70S6K) and its downstream effector ribosomal protein S6. The ERM proteins (ezrin, radixin and moesin) interact with hamartin (TSC1) and regulate cell adhesion and migration. Components of the pathway examined in this study are shaded in grey.

Figure 2

Histopathological features of glioneuronal tumors. Panels A–D: Representative photomicrographs of ganglioglioma (GG). A. Hematoxylin/Eosin (HE) staining of GG showing the mixture of neuronal cells, lacking uniform orientation (arrows) and glial cells. B. NeuN staining detects the neuronal component (nuclear staining) of GG. C. GFAP immunoreactivity showing the astroglial tumor component. D. Prominent CD34 (precursor cell marker) immunoreactivity within GG. E,F. Representative photomicrographs of dysembryoplastic neuroepithelial tumor (DNT). E. HE staining of DNT showing a typical heterogeneous cellular composition, with floating neurons (arrow) surrounded by a prominent population of oligodendroglia‐like cells. F. NeuN staining detects the neuronal component of DNT. Insert in F: GFAP detects few astrocytes between the GFAP‐negative oligodendroglia‐like cells. Scale bar in A: A–C, E, F: 40 µm; D: 80 µm. Abbreviations: GFAP = glial fibrillary acidic protein; NeuN = neuronal nuclear protein.

Figure 3

P‐PDK1, p‐AKT and p‐mTOR immunoreactivity in normal cortex and glioneuronal tumors. In histologically normal cortex (CTX), significant expression of phosphorylated (p)‐PDK1 (A), p‐AKT (E) and p‐mTOR (I) is not observed. Dysembryoplastic neuroepithelial tumor (DNT) specimens showing no significant expression of phosphorylated (p)‐PDK1 (B), p‐AKT (F) and mTOR (J). Arrows indicate negative dysplastic neurons within DNT. Ganglioglioma (GG) specimens showing consistent expression of phosphorylated (p)‐PDK1 (C–D), p‐AKT (G–H) and p‐mTOR (K–L). Arrows indicate the positive dysplastic cells within the tumors. Scale bar in L: A, C, E, G, I, K: 80 µm; B, D, F, H, J, L: 40 µm. Abbreviations: (p)‐PDK1 = phosphorylated‐phosphoinositide‐dependent protein kinase 1; p‐mTOR =  phosphorylated‐mammalian target of rapamycin.

Figure 4

p‐4E‐BP1, p‐eIF4G, p‐p70S6K and p‐S6 immunoreactivity in normal cortex and glioneuronal tumors. Histologically normal cortex (CTX) with no significant expression of phosphorylated (p)‐4E‐BP1 (A), p‐eIF4G (D), p‐p70S6K (G) and p‐S6 (K). In dysembryoplastic neuroepithelial tumors (DNT), significant expression of p‐4E‐BP1 (B), p‐eIF4G (E), p‐p70S6K (H) and pS6 (L) is not observed (arrows indicate negative dysplastic neurons). Sporadically, p‐S6 positive neurons are observed in DNT (inset in L) as well as in histologically normal cortex (data not shown). Representative ganglioglioma (GG) specimen showing expression of p‐4E‐BP1 in dysplastic neurons (C: arrow and inset). p‐eIF4G immunoreactivity in GG (F: arrows indicate positive dysplastic cells). GG specimens showing p‐p70S6K immunoreactivity for both p‐p70S6K‐Thr389 (I) and ‐Thr229 (J) in dysplastic cells. GG specimens showing consistent expression of p‐S6 (M and N: arrows indicate positive cells with different morphology). Scale bar in N: A–G, J–M: 80 µm; H: 60 µm; I, N: 30 µm. Abbreviations: p‐4E‐BP1 = phosphorylated‐eukaryotic initiation factor 4E binding protein 1; p‐eIF4G =  phosphorylated‐eukaryotic initiation factor 4G; p‐p70S6K = phosphorylated‐ribosomal protein S6 kinase; p‐S6 = phosphorylated‐ribosomal protein S6.

Figure 5

Ezrin, radixin and moesin (ERM) immunoreactivity in normal cortex and glioneuronal tumors. A. In histologically normal cortex (CTX), ERM immunoreactivity (IR) is restricted to endothelial cells (arrow). B. Dysembryoplastic neuroepithelial tumor (DNT) showing endothelial ERM IR (arrow) and ERM expression is not observed in dysplastic neurons (arrowheads). C,D. Dysplastic cells in ganglioglioma (GG, arrows) show ERM IR. Scale bar in D: A–C: 40 µm; D: 25 µm.

Figure 6

AMOG immunoreactivity patterns in normal cortex and glioneuronal tumors. A,B. AMOG immunoreactivity (IR) in histologically normal brain. A diffuse moderate staining is observed in cerebral cortex (CTX; A), while in the white matter (WM; B) AMOG is detected in the perivascular astrocytes. C. AMOG IR in few astrocytes in a dysembryoplastic neuroepithelial tumor (DNT). Panels D–H: AMOG IR in ganglioglioma (GG). D. Strong and diffuse AMOG IR within GG with multiple cell clusters. E. Higher magnification of a region with diffuse AMOG IR. F. AMOG‐immunoreactive cell cluster (clustered‐bushy pattern). G. Solitary IR pattern of AMOG, co‐localization of AMOG (brown) with CD34 (pink) is shown in inset. H,I. Sequential sections show cells immunoreactive for AMOG (H; arrows) as well as CD34 (I, arrows). J–L. Double‐labeling of AMOG (green, J) with GFAP (red, K) shows absence of co‐localization (merged image, L). M–O: double‐labeling of AMOG (green, M) with p‐S6 (red, N) show co‐localization (merged image, O) in dysplastic cells. Scale bar in O: A, D: 200 µm; B: 60 µm; C: 40 µm; E: 80 µm; F–G; 30 µm; H–I: 100 µm; J–L: 30 µm, M–O: 20 µm. Abbreviations: AMOG = adhesion molecule on glia; GFAP = glial fibrillary acidic protein; p‐S6 = phosphorylated‐ribosomal protein S6.