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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-Ras and PI3K creates invasive neoplastic glia.FLP/FRT clones of repo-Gal4-expressing glia within whole adult brains, all displayed at the same scale. CD8GFP (green) labels the cell bodies and membranes of glial clones derived by FLP/FRT mitotic recombination (see text). Repo (red) labels all glial cell nuclei, in both clones and surrounding normal tissue. Red flare is background flourescence in (C–F). Frontal sections; anterior up; midline to left. 20 µm scale bars. (A–G) hs-FLP clones from approximately 2 week-old adults, heat-shocked as late pupae or young adults. (A–B) GFP-positive wild-type control clones composed of 1–3 cells with normal stellate morphology. (C) a dRas85DV12-expressing clone in the same brain region as (D,E). dRas85DV12 clones typically display the most proliferation among single mutant clones, containing approximately 2–5× more cells than controls. (D) a dPTEN−/− clone with abnormal morphology and punctate membrane-bound CD8GFP. (E) a dRas85DV12; PTEN−/− double mutant clone, composed of hundreds of cells that have overtaken the optic lobe and invaded the central brain (arrow), extending through the entire depth of the brain (Video S3). Asterisks demark optically opaque tracheal branches. (F,G) dEGFRλ;dPTEN−/− and dEGFRElp;dPTEN−/− clones, which typically have less cellular density than dRas85DV12; PTEN−/− clones. 5–10 µm optical projections. (H–L) ey-FLP clones derived from a population of ey-FLP and repo-Gal4-expressing mitotic cells. (H–J,L) 8.5 µm confocal optical projections of whole optic lobes from brains of similarly aged adults. Both dRas85DV12 (I) and dPTEN−/− (J) clones contain 2–5-fold more cells than wild-type controls (H). dRas85DV12; PTEN−/− double mutant clones (L) form large glial tumors. White-bordered cut-out shows the high density of glial cell nuclei (red) in dRas85DV12; PTEN−/− double mutant clones (L). (K) shows an earlier stage dRas85DV12; PTEN−/− clone in the larval brain, where dRas85DV12; PTEN−/− clones begin as clusters of tens of cells, and grow to become large clones of hundreds of cells by early adulthood, as in (L). Genotypes: (A) and (B) hs-flp1/+;FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/+, (C) hs-flp1/+; FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/UAS-dRas85DV12, (D) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/+, (E) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dRas85DV12, (F) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dEGFRλ, (G) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dEGFRElp, (H) ey-flp/+; FRT40A tubGal80/FRT40A; repo-Gal4 UAS-CD8GFP/+, (I) ey-flp/+; FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/UAS-dRas85DV12, (J) ey-flp/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/+, (K and L) ey-flp/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dRas85DV12.
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pgen-1000374-g004: Coactivation of EGFR-Ras and PI3K creates invasive neoplastic glia.FLP/FRT clones of repo-Gal4-expressing glia within whole adult brains, all displayed at the same scale. CD8GFP (green) labels the cell bodies and membranes of glial clones derived by FLP/FRT mitotic recombination (see text). Repo (red) labels all glial cell nuclei, in both clones and surrounding normal tissue. Red flare is background flourescence in (C–F). Frontal sections; anterior up; midline to left. 20 µm scale bars. (A–G) hs-FLP clones from approximately 2 week-old adults, heat-shocked as late pupae or young adults. (A–B) GFP-positive wild-type control clones composed of 1–3 cells with normal stellate morphology. (C) a dRas85DV12-expressing clone in the same brain region as (D,E). dRas85DV12 clones typically display the most proliferation among single mutant clones, containing approximately 2–5× more cells than controls. (D) a dPTEN−/− clone with abnormal morphology and punctate membrane-bound CD8GFP. (E) a dRas85DV12; PTEN−/− double mutant clone, composed of hundreds of cells that have overtaken the optic lobe and invaded the central brain (arrow), extending through the entire depth of the brain (Video S3). Asterisks demark optically opaque tracheal branches. (F,G) dEGFRλ;dPTEN−/− and dEGFRElp;dPTEN−/− clones, which typically have less cellular density than dRas85DV12; PTEN−/− clones. 5–10 µm optical projections. (H–L) ey-FLP clones derived from a population of ey-FLP and repo-Gal4-expressing mitotic cells. (H–J,L) 8.5 µm confocal optical projections of whole optic lobes from brains of similarly aged adults. Both dRas85DV12 (I) and dPTEN−/− (J) clones contain 2–5-fold more cells than wild-type controls (H). dRas85DV12; PTEN−/− double mutant clones (L) form large glial tumors. White-bordered cut-out shows the high density of glial cell nuclei (red) in dRas85DV12; PTEN−/− double mutant clones (L). (K) shows an earlier stage dRas85DV12; PTEN−/− clone in the larval brain, where dRas85DV12; PTEN−/− clones begin as clusters of tens of cells, and grow to become large clones of hundreds of cells by early adulthood, as in (L). Genotypes: (A) and (B) hs-flp1/+;FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/+, (C) hs-flp1/+; FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/UAS-dRas85DV12, (D) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/+, (E) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dRas85DV12, (F) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dEGFRλ, (G) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dEGFRElp, (H) ey-flp/+; FRT40A tubGal80/FRT40A; repo-Gal4 UAS-CD8GFP/+, (I) ey-flp/+; FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/UAS-dRas85DV12, (J) ey-flp/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/+, (K and L) ey-flp/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dRas85DV12.

Mentions: In wild-type controls, we observed clones in 68% of brains examined (N = 149). Of the brains with clones, 75% had 1–3 clones, and 83% of these clones consisted of 1–3 cells of the same glial subtype (Figure 4A and 4B). Glial clones overexpressing dRas85DV12, dEGFRλ, or dEGFRElp alone typically contained 2-fold more cells than wild-type (dRas85DV12 shown in Figure 4C). To examine PI3K signaling, we used a dPTEN allele, which became homozygous in FLP-FRT clones. dPTEN−/− glia did not overgrow, but did show aberrant cytoplasmic projections (Figure 4D), perhaps reflecting dPTEN function in the cytoskeleton [38].


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-Ras and PI3K creates invasive neoplastic glia.FLP/FRT clones of repo-Gal4-expressing glia within whole adult brains, all displayed at the same scale. CD8GFP (green) labels the cell bodies and membranes of glial clones derived by FLP/FRT mitotic recombination (see text). Repo (red) labels all glial cell nuclei, in both clones and surrounding normal tissue. Red flare is background flourescence in (C–F). Frontal sections; anterior up; midline to left. 20 µm scale bars. (A–G) hs-FLP clones from approximately 2 week-old adults, heat-shocked as late pupae or young adults. (A–B) GFP-positive wild-type control clones composed of 1–3 cells with normal stellate morphology. (C) a dRas85DV12-expressing clone in the same brain region as (D,E). dRas85DV12 clones typically display the most proliferation among single mutant clones, containing approximately 2–5× more cells than controls. (D) a dPTEN−/− clone with abnormal morphology and punctate membrane-bound CD8GFP. (E) a dRas85DV12; PTEN−/− double mutant clone, composed of hundreds of cells that have overtaken the optic lobe and invaded the central brain (arrow), extending through the entire depth of the brain (Video S3). Asterisks demark optically opaque tracheal branches. (F,G) dEGFRλ;dPTEN−/− and dEGFRElp;dPTEN−/− clones, which typically have less cellular density than dRas85DV12; PTEN−/− clones. 5–10 µm optical projections. (H–L) ey-FLP clones derived from a population of ey-FLP and repo-Gal4-expressing mitotic cells. (H–J,L) 8.5 µm confocal optical projections of whole optic lobes from brains of similarly aged adults. Both dRas85DV12 (I) and dPTEN−/− (J) clones contain 2–5-fold more cells than wild-type controls (H). dRas85DV12; PTEN−/− double mutant clones (L) form large glial tumors. White-bordered cut-out shows the high density of glial cell nuclei (red) in dRas85DV12; PTEN−/− double mutant clones (L). (K) shows an earlier stage dRas85DV12; PTEN−/− clone in the larval brain, where dRas85DV12; PTEN−/− clones begin as clusters of tens of cells, and grow to become large clones of hundreds of cells by early adulthood, as in (L). Genotypes: (A) and (B) hs-flp1/+;FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/+, (C) hs-flp1/+; FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/UAS-dRas85DV12, (D) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/+, (E) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dRas85DV12, (F) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dEGFRλ, (G) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dEGFRElp, (H) ey-flp/+; FRT40A tubGal80/FRT40A; repo-Gal4 UAS-CD8GFP/+, (I) ey-flp/+; FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/UAS-dRas85DV12, (J) ey-flp/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/+, (K and L) ey-flp/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dRas85DV12.
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pgen-1000374-g004: Coactivation of EGFR-Ras and PI3K creates invasive neoplastic glia.FLP/FRT clones of repo-Gal4-expressing glia within whole adult brains, all displayed at the same scale. CD8GFP (green) labels the cell bodies and membranes of glial clones derived by FLP/FRT mitotic recombination (see text). Repo (red) labels all glial cell nuclei, in both clones and surrounding normal tissue. Red flare is background flourescence in (C–F). Frontal sections; anterior up; midline to left. 20 µm scale bars. (A–G) hs-FLP clones from approximately 2 week-old adults, heat-shocked as late pupae or young adults. (A–B) GFP-positive wild-type control clones composed of 1–3 cells with normal stellate morphology. (C) a dRas85DV12-expressing clone in the same brain region as (D,E). dRas85DV12 clones typically display the most proliferation among single mutant clones, containing approximately 2–5× more cells than controls. (D) a dPTEN−/− clone with abnormal morphology and punctate membrane-bound CD8GFP. (E) a dRas85DV12; PTEN−/− double mutant clone, composed of hundreds of cells that have overtaken the optic lobe and invaded the central brain (arrow), extending through the entire depth of the brain (Video S3). Asterisks demark optically opaque tracheal branches. (F,G) dEGFRλ;dPTEN−/− and dEGFRElp;dPTEN−/− clones, which typically have less cellular density than dRas85DV12; PTEN−/− clones. 5–10 µm optical projections. (H–L) ey-FLP clones derived from a population of ey-FLP and repo-Gal4-expressing mitotic cells. (H–J,L) 8.5 µm confocal optical projections of whole optic lobes from brains of similarly aged adults. Both dRas85DV12 (I) and dPTEN−/− (J) clones contain 2–5-fold more cells than wild-type controls (H). dRas85DV12; PTEN−/− double mutant clones (L) form large glial tumors. White-bordered cut-out shows the high density of glial cell nuclei (red) in dRas85DV12; PTEN−/− double mutant clones (L). (K) shows an earlier stage dRas85DV12; PTEN−/− clone in the larval brain, where dRas85DV12; PTEN−/− clones begin as clusters of tens of cells, and grow to become large clones of hundreds of cells by early adulthood, as in (L). Genotypes: (A) and (B) hs-flp1/+;FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/+, (C) hs-flp1/+; FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/UAS-dRas85DV12, (D) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/+, (E) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dRas85DV12, (F) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dEGFRλ, (G) hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dEGFRElp, (H) ey-flp/+; FRT40A tubGal80/FRT40A; repo-Gal4 UAS-CD8GFP/+, (I) ey-flp/+; FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/UAS-dRas85DV12, (J) ey-flp/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/+, (K and L) ey-flp/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dRas85DV12.
Mentions: In wild-type controls, we observed clones in 68% of brains examined (N = 149). Of the brains with clones, 75% had 1–3 clones, and 83% of these clones consisted of 1–3 cells of the same glial subtype (Figure 4A and 4B). Glial clones overexpressing dRas85DV12, dEGFRλ, or dEGFRElp alone typically contained 2-fold more cells than wild-type (dRas85DV12 shown in Figure 4C). To examine PI3K signaling, we used a dPTEN allele, which became homozygous in FLP-FRT clones. dPTEN−/− glia did not overgrow, but did show aberrant cytoplasmic projections (Figure 4D), perhaps reflecting dPTEN function in the cytoskeleton [38].

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