Tep1 Regulates Yki Activity in Neural Stem Cells in Drosophila Glioma Model

Abstract

Glioblastoma Multiforme (GBM) is the most common form of malignant brain tumor with poor prognosis. Amplification of Epidermal Growth Factor Receptor (EGFR), and mutations leading to activation of Phosphatidyl-Inositol-3 Kinase (PI3K) pathway are commonly associated with GBM. Using a previously published Drosophila glioma model generated by coactivation of PI3K and EGFR pathways [by downregulation of Pten and overexpression of oncogenic Ras] in glial cells, we showed that the Drosophila Tep1 gene (ortholog of human CD109) regulates Yki (the Drosophila ortholog of human YAP/TAZ) via an evolutionarily conserved mechanism. Oncogenic signaling by the YAP/TAZ pathway occurs in cells that acquire CD109 expression in response to the inflammatory environment induced by radiation in clinically relevant models. Further, downregulation of Tep1 caused a reduction in Yki activity and reduced glioma growth. A key function of Yki in larval CNS is stem cell renewal and formation of neuroblasts. Other reports suggest different upstream regulators of Yki activity in the optic lobe versus the central brain regions of the larval CNS. We hypothesized that Tep1 interacts with the Hippo pathway effector Yki to regulate neuroblast numbers. We tested if Tep1 acts through Yki to affect glioma growth, and if in normal cells Tep1 affects neuroblast number and proliferation. Our data suggests that Tep1 affects Yki mediated stem cell renewal in glioma, as reduction of Tep significantly decreases the number of neuroblasts in glioma. Thus, we identify Tep1-Yki interaction in the larval CNS that plays a key role in glioma growth and progression.

Keywords: Drosophila; Hippo pathway; Tep; Tep1; Yki; glioma; neural stem cell.

FIGURE 1

Loss of Tep1 reduces the mitotic index in glioma. Comparisons of PH3 positive cell numbers in the central brain region of Drosophila third instar larval brain (see, schematic in A””) are shown for the following genotypes: wild type control repo-Gal4>GFP (A–A”, referred to as repo-GFP in all subsequent panels and figures), and glioma from repo-GFP>Pten^RNAi; UAS-Ras^V12 (B–B”), and repo-GFP>Pten^RNAi; UAS-Ras^V12; UAS-Tep1^RNAi (C–C”). Glial cells are marked by GFP (green, gray) and PH3 (red, gray). (D) Quantification of number of PH3 positive cells in the central brain is shown in the graph. Unpaired 2-tailed T-test with n = 5, 95% confidence was performed using GraphPad Prism 5, p < 0.05. Yellow ROI boxes indicate area in which PH3 positive cells are counted. All images were scanned at identical magnification [20×, scale bars, 100 μm = 62px].

FIGURE 2

Loss of Tep1 suppresses ectopic Neuroblasts that drive glioma growth. Panels show comparisons of Miranda (red) and Prospero (blue) positive cells in the central brain of Drosophila larval CNS from third instar larvae are shown for the following genotypes (A–C”’) GFP (green, glia), Mira (red, gray) and Pros (blue, gray) expression in third-instar larval CNS from repo-GFP (A–A”’), repo-GFP>UAS-Pten^RNAi; UAS-Ras^V12 (B–B”’), repo-GFP>UAS-Pten^RNAi; UAS-Ras^V12; UAS-Tep1^RNAi (C–C”’). (D) Quantification of brain lobe size was done using the magnetic lasso tool in Photoshop to create an ROI for measuring pixel values. At least five independent biological replicates for each genotype were compared. (E) Quantification of number of Mira positive cells in the central brain is shown in the graph. For (D) and (E) unpaired 2-tailed T-test with n = 5, 95% confidence was performed using GraphPad Prism 5, p < 0.05. Yellow ROI boxes indicate area in which Mira +ve cells are counted. All images were scanned at identical magnification [20×, scale bars, 100 μm = 62px].

FIGURE 3

Yki reporter activity in glioma brains. A comparison of Yki reporter activity is shown. (A–F”) show expression of ex-lacZ (ß-gal, red) in larval brains. Glia are marked by GFP (green) in the following genotypes: repo-GFP (A–A”), repo-GFP>UAS-Pten^RNAi (B–B”), repo-GFP>UAS-Tep^RNAi (C–C”), repo-GFP>UAS-Ras^V12 (D–D”), repo-GFP>UAS-Pten^RNAi; UAS-Ras^V12 (E–E”), repo-GFP>UAS-Pten^RNAi; UAS-Ras^V12; UAS-Tep1^RNAi (F–F”). Brain lobes are imaged at 20× magnification with scale bars 100 μm = 62px. Yellow lines represent the ROI within which Yki reporter activity is compared.

FIGURE 4

Increased survival in glioma. (A–F”) Expression of Diap1 (red) and glial cells marked with GFP (green) in third-instar larval CNS from repo-GFP (A–A”), repo-GFP>UAS-Pten^RNAi (B–B”), repo-GFP>UAS-Tep^RNAi (C–C”), repo-GFP>UAS-Ras^V12 (D–D”), repo-GFP>UAS-Pten^RNAi; UAS-Ras^V12 (E–E”), repo-GFP>UAS-Pten^RNAi; UAS-Tep^RNAi/UAS-Ras^V12 (F–F”). Brain lobes are imaged at 20× magnification with scale bars 100 μm = 62px.

FIGURE 5

Downregulation of Yki reduces glioma growth and neuroblast number in larval CNS. (A–D”) panels show a comparison of neuroblast number in the central brain region (the yellow line marks the ROI in one brain lobe for each genotype). In all panels glia are marked by GFP (green, gray), and neuroblasts by Mira expression (blue, gray). The genotypes are: repo-GFP (A–A”), repo-GFP>UAS-Yki^RNAi (B–B”), repo-GFP>UAS-Pten^RNAi; UAS-Ras^V12; UAS-Yki^RNAi (C–C”), and repo-GFP>UAS-Pten^RNAi; UAS-Ras^V12; UAS-Yki^RNAi/UAS-Tep^RNAi (D–D”). (E) Quantification of number of Mira positive cells in the central brain region is shown for the indicated genotypes. Unpaired 2-tailed T-test with n = 5, 95% confidence was performed using GraphPad Prism 8, p < 0.05. Yellow ROI boxes indicate area in which Mira positive cells are counted. All images were scanned at identical magnification [20×, scale bars, 100 μm = 62px].