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A drosophila model for EGFR-Ras and PI3K-dependent human glioma.

Read RD, Cavenee WK, Furnari FB, Thomas JB - PLoS Genet. (2009)

Bottom Line: This network acts synergistically to coordinately stimulate cell cycle entry and progression, protein translation, and inappropriate cellular growth and migration.In particular, we found that the fly orthologs of CyclinE, Cdc25, and Myc are key rate-limiting genes required for glial neoplasia.Moreover, orthologs of Sin1, Rictor, and Cdk4 are genes required only for abnormal neoplastic glial proliferation but not for glial development.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, California, United States of America. rread@salk.edu

ABSTRACT
Gliomas, the most common malignant tumors of the nervous system, frequently harbor mutations that activate the epidermal growth factor receptor (EGFR) and phosphatidylinositol-3 kinase (PI3K) signaling pathways. To investigate the genetic basis of this disease, we developed a glioma model in Drosophila. We found that constitutive coactivation of EGFR-Ras and PI3K pathways in Drosophila glia and glial precursors gives rise to neoplastic, invasive glial cells that create transplantable tumor-like growths, mimicking human glioma. Our model represents a robust organotypic and cell-type-specific Drosophila cancer model in which malignant cells are created by mutations in signature genes and pathways thought to be driving forces in a homologous human cancer. Genetic analyses demonstrated that EGFR and PI3K initiate malignant neoplastic transformation via a combinatorial genetic network composed primarily of other pathways commonly mutated or activated in human glioma, including the Tor, Myc, G1 Cyclins-Cdks, and Rb-E2F pathways. This network acts synergistically to coordinately stimulate cell cycle entry and progression, protein translation, and inappropriate cellular growth and migration. In particular, we found that the fly orthologs of CyclinE, Cdc25, and Myc are key rate-limiting genes required for glial neoplasia. Moreover, orthologs of Sin1, Rictor, and Cdk4 are genes required only for abnormal neoplastic glial proliferation but not for glial development. These and other genes within this network may represent important therapeutic targets in human glioma.

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Coactivation of EGFR and PI3K cell-autonomously promotes cell cycle entry.(A–F) Close-ups of late 3rd instar larval brain hemispheres. Anterior up; midline to left. 20 µm scale bars. (A–C) Glial cell nuclei are labeled with Repo (red). Glial cell bodies and membranes are labeled with CD8GFP (green) driven by repo-Gal4. In wild-type (A), surface glia (SG) that cover the brain form a single-cell layer of fairly flat cells whereas astrocyte-like cortex glia are often evenly spaced and form a honeycomb-like network of fine projections throughout the brain. Superficial cortex glia show stellate morphology (arrowheads), and cortex glia deeper in the brain sometimes show a radial morphology (asterisk). In repo>dEGFRλ;dp110CAAX brains (B,C) surface glia (SG) and superficial cortex glia (brighter GFP, arrowheads) form thick multicellular aggregations throughout the brain and often adopt a spindle-shaped morphology (arrowheads). In repo>dEGFRλ;dp110CAAX brains, deeper cortex glia (C) can still form fine projections, but more often lack these and create loose aggregates of abnormally shaped cells (asterisks). (D,E) BrdU labeling (red) of S-phase cells. The prominent replicative cells in normal brains (D) are neuroblasts (‘NB’) and optic lobe neural precursors (‘NP’), identified by their stereotyped position and/or staining for HRP (faint blue). The prominent replicative cells in repo>dEGFRλ;dp110CAAX brains (E) are glia (‘G’), which do not stain for HRP. 2 µm optical sections. (F–I) dCyclinE (F,G) and dCyclinB (H,I) expression (red) shown alone (left panels) and with Repo (blue nuclei) and CD8GFP (green, membranes/cytoplasm) glial markers (right in each panel). ‘G’ denotes representative glia, ‘NB’, representative neuroblasts. In wild-type (F, H), glia rarely expressed dCyclinE or dCyclinB, while neuroblasts (‘NB’) showed predominant expression. dCyclinE expression is primarily nuclear in dEGFRλ;dp110CAAX glia (G), visible as purple in overlay (right panel), although not all mutant glia express dCyclinE. Inset in (G) shows dCyclinE expression in polyploid glia (‘ppG’). (I) dCyclinB expression in dEGFRλ;dp110CAAX glia is primarily cytoplasmic, visible as yellow in overlay (right panel). ppG in repo>dEGFRλ;dp110CAAX brains do not express dCyclinB, as seen by the absence of yellow or purple in overlay. (F,G) 6.5 µm and (H,I) 5 µm optical projections. (J–M) 2 µm optical sections of larval brain hemispheres from late 3rd instar larvae approximately 130 hr AED, displayed at the same scale. Frontal sections, midway through brains. repo>dEGFRλ;dp110CAAX brains (K) show a dramatic increase in number of glial nuclei (red) relative to wild-type (J). Glial cell nuclei are decreased by co-overexpression of Dap (L) or Rbf1 (M) with dEGFRλ;dp110CAAX. Glial cell bodies and membranes (green, repo>CD8GFP reporter) appear relatively normal in (L) and (M) compared to (J). HRP counter-stain (blue) reveals neuropil at high intensity and neuronal cell bodies at low intensity. Genotypes: (A), (D), (F), (H), and (J) repo-Gal4 UAS-CD8GFP/+, (B), (C), (E), (G), (I), and (K) UAS-dEGFRλ UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/+, (L) UAS-dEGFRλ UAS-dp110CAAX/UAS-dap; repo-Gal4 UAS-CD8GFP/+, (M) UAS-dEGFRλ;UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/UAS-Rbf1.
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pgen-1000374-g002: Coactivation of EGFR and PI3K cell-autonomously promotes cell cycle entry.(A–F) Close-ups of late 3rd instar larval brain hemispheres. Anterior up; midline to left. 20 µm scale bars. (A–C) Glial cell nuclei are labeled with Repo (red). Glial cell bodies and membranes are labeled with CD8GFP (green) driven by repo-Gal4. In wild-type (A), surface glia (SG) that cover the brain form a single-cell layer of fairly flat cells whereas astrocyte-like cortex glia are often evenly spaced and form a honeycomb-like network of fine projections throughout the brain. Superficial cortex glia show stellate morphology (arrowheads), and cortex glia deeper in the brain sometimes show a radial morphology (asterisk). In repo>dEGFRλ;dp110CAAX brains (B,C) surface glia (SG) and superficial cortex glia (brighter GFP, arrowheads) form thick multicellular aggregations throughout the brain and often adopt a spindle-shaped morphology (arrowheads). In repo>dEGFRλ;dp110CAAX brains, deeper cortex glia (C) can still form fine projections, but more often lack these and create loose aggregates of abnormally shaped cells (asterisks). (D,E) BrdU labeling (red) of S-phase cells. The prominent replicative cells in normal brains (D) are neuroblasts (‘NB’) and optic lobe neural precursors (‘NP’), identified by their stereotyped position and/or staining for HRP (faint blue). The prominent replicative cells in repo>dEGFRλ;dp110CAAX brains (E) are glia (‘G’), which do not stain for HRP. 2 µm optical sections. (F–I) dCyclinE (F,G) and dCyclinB (H,I) expression (red) shown alone (left panels) and with Repo (blue nuclei) and CD8GFP (green, membranes/cytoplasm) glial markers (right in each panel). ‘G’ denotes representative glia, ‘NB’, representative neuroblasts. In wild-type (F, H), glia rarely expressed dCyclinE or dCyclinB, while neuroblasts (‘NB’) showed predominant expression. dCyclinE expression is primarily nuclear in dEGFRλ;dp110CAAX glia (G), visible as purple in overlay (right panel), although not all mutant glia express dCyclinE. Inset in (G) shows dCyclinE expression in polyploid glia (‘ppG’). (I) dCyclinB expression in dEGFRλ;dp110CAAX glia is primarily cytoplasmic, visible as yellow in overlay (right panel). ppG in repo>dEGFRλ;dp110CAAX brains do not express dCyclinB, as seen by the absence of yellow or purple in overlay. (F,G) 6.5 µm and (H,I) 5 µm optical projections. (J–M) 2 µm optical sections of larval brain hemispheres from late 3rd instar larvae approximately 130 hr AED, displayed at the same scale. Frontal sections, midway through brains. repo>dEGFRλ;dp110CAAX brains (K) show a dramatic increase in number of glial nuclei (red) relative to wild-type (J). Glial cell nuclei are decreased by co-overexpression of Dap (L) or Rbf1 (M) with dEGFRλ;dp110CAAX. Glial cell bodies and membranes (green, repo>CD8GFP reporter) appear relatively normal in (L) and (M) compared to (J). HRP counter-stain (blue) reveals neuropil at high intensity and neuronal cell bodies at low intensity. Genotypes: (A), (D), (F), (H), and (J) repo-Gal4 UAS-CD8GFP/+, (B), (C), (E), (G), (I), and (K) UAS-dEGFRλ UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/+, (L) UAS-dEGFRλ UAS-dp110CAAX/UAS-dap; repo-Gal4 UAS-CD8GFP/+, (M) UAS-dEGFRλ;UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/UAS-Rbf1.

Mentions: In repo>dEGFRλ;dp110CAAX brains, excess glia emerged in early larval stages and accumulated over 5–7 days. dEGFRλ;dp110CAAX glia severely disrupt the normal cellular architecture of the larval brain (Figure 1A and 1B and Figure 2A–C), lose normal stellate glial morphologies (Figure 2A–C), and generate multilayered aggregations of abnormal glia throughout the brain (Figure 2A–C); in these ways dEGFRλ;dp110CAAX glia are neoplastic [18]. Like neoplastic epithelial cells, dEGFRλ;dp110CAAX glia ectopically expressed an active form of the matrix metalloprotease dMMP1 (Figure S3), which can confer an invasive potential [19],[20], implying that abnormal dEGFRλ;dp110CAAX glia may be invasive within the brain. Unlike neoplastic epithelia, neoplastic neural cells, such as dEGFRλ;dp110CAAX glia, typically retain expression of genes that regulate neural cell fate, such as Repo [21],[22].


A drosophila model for EGFR-Ras and PI3K-dependent human glioma.

Read RD, Cavenee WK, Furnari FB, Thomas JB - PLoS Genet. (2009)

Coactivation of EGFR and PI3K cell-autonomously promotes cell cycle entry.(A–F) Close-ups of late 3rd instar larval brain hemispheres. Anterior up; midline to left. 20 µm scale bars. (A–C) Glial cell nuclei are labeled with Repo (red). Glial cell bodies and membranes are labeled with CD8GFP (green) driven by repo-Gal4. In wild-type (A), surface glia (SG) that cover the brain form a single-cell layer of fairly flat cells whereas astrocyte-like cortex glia are often evenly spaced and form a honeycomb-like network of fine projections throughout the brain. Superficial cortex glia show stellate morphology (arrowheads), and cortex glia deeper in the brain sometimes show a radial morphology (asterisk). In repo>dEGFRλ;dp110CAAX brains (B,C) surface glia (SG) and superficial cortex glia (brighter GFP, arrowheads) form thick multicellular aggregations throughout the brain and often adopt a spindle-shaped morphology (arrowheads). In repo>dEGFRλ;dp110CAAX brains, deeper cortex glia (C) can still form fine projections, but more often lack these and create loose aggregates of abnormally shaped cells (asterisks). (D,E) BrdU labeling (red) of S-phase cells. The prominent replicative cells in normal brains (D) are neuroblasts (‘NB’) and optic lobe neural precursors (‘NP’), identified by their stereotyped position and/or staining for HRP (faint blue). The prominent replicative cells in repo>dEGFRλ;dp110CAAX brains (E) are glia (‘G’), which do not stain for HRP. 2 µm optical sections. (F–I) dCyclinE (F,G) and dCyclinB (H,I) expression (red) shown alone (left panels) and with Repo (blue nuclei) and CD8GFP (green, membranes/cytoplasm) glial markers (right in each panel). ‘G’ denotes representative glia, ‘NB’, representative neuroblasts. In wild-type (F, H), glia rarely expressed dCyclinE or dCyclinB, while neuroblasts (‘NB’) showed predominant expression. dCyclinE expression is primarily nuclear in dEGFRλ;dp110CAAX glia (G), visible as purple in overlay (right panel), although not all mutant glia express dCyclinE. Inset in (G) shows dCyclinE expression in polyploid glia (‘ppG’). (I) dCyclinB expression in dEGFRλ;dp110CAAX glia is primarily cytoplasmic, visible as yellow in overlay (right panel). ppG in repo>dEGFRλ;dp110CAAX brains do not express dCyclinB, as seen by the absence of yellow or purple in overlay. (F,G) 6.5 µm and (H,I) 5 µm optical projections. (J–M) 2 µm optical sections of larval brain hemispheres from late 3rd instar larvae approximately 130 hr AED, displayed at the same scale. Frontal sections, midway through brains. repo>dEGFRλ;dp110CAAX brains (K) show a dramatic increase in number of glial nuclei (red) relative to wild-type (J). Glial cell nuclei are decreased by co-overexpression of Dap (L) or Rbf1 (M) with dEGFRλ;dp110CAAX. Glial cell bodies and membranes (green, repo>CD8GFP reporter) appear relatively normal in (L) and (M) compared to (J). HRP counter-stain (blue) reveals neuropil at high intensity and neuronal cell bodies at low intensity. Genotypes: (A), (D), (F), (H), and (J) repo-Gal4 UAS-CD8GFP/+, (B), (C), (E), (G), (I), and (K) UAS-dEGFRλ UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/+, (L) UAS-dEGFRλ UAS-dp110CAAX/UAS-dap; repo-Gal4 UAS-CD8GFP/+, (M) UAS-dEGFRλ;UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/UAS-Rbf1.
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pgen-1000374-g002: Coactivation of EGFR and PI3K cell-autonomously promotes cell cycle entry.(A–F) Close-ups of late 3rd instar larval brain hemispheres. Anterior up; midline to left. 20 µm scale bars. (A–C) Glial cell nuclei are labeled with Repo (red). Glial cell bodies and membranes are labeled with CD8GFP (green) driven by repo-Gal4. In wild-type (A), surface glia (SG) that cover the brain form a single-cell layer of fairly flat cells whereas astrocyte-like cortex glia are often evenly spaced and form a honeycomb-like network of fine projections throughout the brain. Superficial cortex glia show stellate morphology (arrowheads), and cortex glia deeper in the brain sometimes show a radial morphology (asterisk). In repo>dEGFRλ;dp110CAAX brains (B,C) surface glia (SG) and superficial cortex glia (brighter GFP, arrowheads) form thick multicellular aggregations throughout the brain and often adopt a spindle-shaped morphology (arrowheads). In repo>dEGFRλ;dp110CAAX brains, deeper cortex glia (C) can still form fine projections, but more often lack these and create loose aggregates of abnormally shaped cells (asterisks). (D,E) BrdU labeling (red) of S-phase cells. The prominent replicative cells in normal brains (D) are neuroblasts (‘NB’) and optic lobe neural precursors (‘NP’), identified by their stereotyped position and/or staining for HRP (faint blue). The prominent replicative cells in repo>dEGFRλ;dp110CAAX brains (E) are glia (‘G’), which do not stain for HRP. 2 µm optical sections. (F–I) dCyclinE (F,G) and dCyclinB (H,I) expression (red) shown alone (left panels) and with Repo (blue nuclei) and CD8GFP (green, membranes/cytoplasm) glial markers (right in each panel). ‘G’ denotes representative glia, ‘NB’, representative neuroblasts. In wild-type (F, H), glia rarely expressed dCyclinE or dCyclinB, while neuroblasts (‘NB’) showed predominant expression. dCyclinE expression is primarily nuclear in dEGFRλ;dp110CAAX glia (G), visible as purple in overlay (right panel), although not all mutant glia express dCyclinE. Inset in (G) shows dCyclinE expression in polyploid glia (‘ppG’). (I) dCyclinB expression in dEGFRλ;dp110CAAX glia is primarily cytoplasmic, visible as yellow in overlay (right panel). ppG in repo>dEGFRλ;dp110CAAX brains do not express dCyclinB, as seen by the absence of yellow or purple in overlay. (F,G) 6.5 µm and (H,I) 5 µm optical projections. (J–M) 2 µm optical sections of larval brain hemispheres from late 3rd instar larvae approximately 130 hr AED, displayed at the same scale. Frontal sections, midway through brains. repo>dEGFRλ;dp110CAAX brains (K) show a dramatic increase in number of glial nuclei (red) relative to wild-type (J). Glial cell nuclei are decreased by co-overexpression of Dap (L) or Rbf1 (M) with dEGFRλ;dp110CAAX. Glial cell bodies and membranes (green, repo>CD8GFP reporter) appear relatively normal in (L) and (M) compared to (J). HRP counter-stain (blue) reveals neuropil at high intensity and neuronal cell bodies at low intensity. Genotypes: (A), (D), (F), (H), and (J) repo-Gal4 UAS-CD8GFP/+, (B), (C), (E), (G), (I), and (K) UAS-dEGFRλ UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/+, (L) UAS-dEGFRλ UAS-dp110CAAX/UAS-dap; repo-Gal4 UAS-CD8GFP/+, (M) UAS-dEGFRλ;UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/UAS-Rbf1.
Mentions: In repo>dEGFRλ;dp110CAAX brains, excess glia emerged in early larval stages and accumulated over 5–7 days. dEGFRλ;dp110CAAX glia severely disrupt the normal cellular architecture of the larval brain (Figure 1A and 1B and Figure 2A–C), lose normal stellate glial morphologies (Figure 2A–C), and generate multilayered aggregations of abnormal glia throughout the brain (Figure 2A–C); in these ways dEGFRλ;dp110CAAX glia are neoplastic [18]. Like neoplastic epithelial cells, dEGFRλ;dp110CAAX glia ectopically expressed an active form of the matrix metalloprotease dMMP1 (Figure S3), which can confer an invasive potential [19],[20], implying that abnormal dEGFRλ;dp110CAAX glia may be invasive within the brain. Unlike neoplastic epithelia, neoplastic neural cells, such as dEGFRλ;dp110CAAX glia, typically retain expression of genes that regulate neural cell fate, such as Repo [21],[22].

Bottom Line: This network acts synergistically to coordinately stimulate cell cycle entry and progression, protein translation, and inappropriate cellular growth and migration.In particular, we found that the fly orthologs of CyclinE, Cdc25, and Myc are key rate-limiting genes required for glial neoplasia.Moreover, orthologs of Sin1, Rictor, and Cdk4 are genes required only for abnormal neoplastic glial proliferation but not for glial development.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, California, United States of America. rread@salk.edu

ABSTRACT
Gliomas, the most common malignant tumors of the nervous system, frequently harbor mutations that activate the epidermal growth factor receptor (EGFR) and phosphatidylinositol-3 kinase (PI3K) signaling pathways. To investigate the genetic basis of this disease, we developed a glioma model in Drosophila. We found that constitutive coactivation of EGFR-Ras and PI3K pathways in Drosophila glia and glial precursors gives rise to neoplastic, invasive glial cells that create transplantable tumor-like growths, mimicking human glioma. Our model represents a robust organotypic and cell-type-specific Drosophila cancer model in which malignant cells are created by mutations in signature genes and pathways thought to be driving forces in a homologous human cancer. Genetic analyses demonstrated that EGFR and PI3K initiate malignant neoplastic transformation via a combinatorial genetic network composed primarily of other pathways commonly mutated or activated in human glioma, including the Tor, Myc, G1 Cyclins-Cdks, and Rb-E2F pathways. This network acts synergistically to coordinately stimulate cell cycle entry and progression, protein translation, and inappropriate cellular growth and migration. In particular, we found that the fly orthologs of CyclinE, Cdc25, and Myc are key rate-limiting genes required for glial neoplasia. Moreover, orthologs of Sin1, Rictor, and Cdk4 are genes required only for abnormal neoplastic glial proliferation but not for glial development. These and other genes within this network may represent important therapeutic targets in human glioma.

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Related in: MedlinePlus