A Drosophila Model for EGFR-Ras and PI3K-Dependent Human Glioma

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.

[1]  T. Orr-Weaver,et al.  Regulation of cell cycles in Drosophila development: intrinsic and extrinsic cues. , 2003, Annual review of genetics.

[2]  R. Henkelman,et al.  Identification of human brain tumour initiating cells , 2004, Nature.

[3]  M. Freeman,et al.  Pointed and Tramtrack69 establish an EGFR-dependent transcriptional switch to regulate mitosis , 2002, Nature Cell Biology.

[4]  Roger A Hoskins,et al.  The Carnegie Protein Trap Library: A Versatile Tool for Drosophila Developmental Studies , 2007, Genetics.

[5]  R. Bodmer,et al.  Activated FOXO-mediated insulin resistance is blocked by reduction of TOR activity. , 2006, Cell metabolism.

[6]  K. Wallace,et al.  The pan‐neural bHLH proteins DEADPAN and ASENSE regulate mitotic activity and cdk inhibitor dacapo expression in the Drosophila larval optic lobes , 2000, Genesis.

[7]  M. Freeman,et al.  Glial cell biology in Drosophila and vertebrates , 2006, Trends in Neurosciences.

[8]  C. Klämbt,et al.  Organization and Function of the Blood–Brain Barrier in Drosophila , 2008, The Journal of Neuroscience.

[9]  Li Zhang,et al.  Prognostic Associations of Activated Mitogen-Activated Protein Kinase and Akt Pathways in Glioblastoma , 2006, Clinical Cancer Research.

[10]  B. Dickson,et al.  The Drosophila Tuberous Sclerosis Complex Gene Homologs Restrict Cell Growth and Cell Proliferation , 2001, Cell.

[11]  B. Edgar,et al.  Genomic binding and transcriptional regulation by the Drosophila Myc and Mnt transcription factors. , 2005, Cold Spring Harbor symposia on quantitative biology.

[12]  B. Dickson,et al.  A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila , 2007, Nature.

[13]  D. Prober,et al.  Interactions between Ras1, dMyc, and dPI3K signaling in the developing Drosophila wing. , 2002, Genes & development.

[14]  P. Sicinski,et al.  Cell Cycle Progression without Cyclin D-CDK4 and Cyclin D-CDK6 Complexes , 2005, Cell cycle.

[15]  A. Teleman,et al.  Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila. , 2008, Cell metabolism.

[16]  T. P. Neufeld,et al.  Inhibition of cellular growth and proliferation by dTOR overexpression in Drosophila , 2002, Genesis.

[17]  Webster K. Cavenee,et al.  Feedback Circuit among INK4 Tumor Suppressors Constrains Human Glioblastoma Development , 2008, Cancer cell.

[18]  T. Igaki,et al.  Loss of Cell Polarity Drives Tumor Growth and Invasion through JNK Activation in Drosophila , 2006, Current Biology.

[19]  P. Pandolfi,et al.  mTOR promotes survival and astrocytic characteristics induced by Pten/AKT signaling in glioblastoma. , 2005, Neoplasia.

[20]  D. Busam,et al.  An Integrated Genomic Analysis of Human Glioblastoma Multiforme , 2008, Science.

[21]  E. Hafen,et al.  The Drosophila phosphoinositide 3‐kinase Dp110 promotes cell growth. , 1996, The EMBO journal.

[22]  L. Chin,et al.  Malignant astrocytic glioma: genetics, biology, and paths to treatment. , 2007, Genes & development.

[23]  N. Perrimon,et al.  Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. , 1993, Development.

[24]  Nancy M Bonini,et al.  Drosophila as a model for human neurodegenerative disease. , 2005, Annual review of genetics.

[25]  R. DePinho,et al.  Epidermal growth factor receptor and Ink4a/Arf: convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis. , 2002, Cancer cell.

[26]  C. Strambi,et al.  Decreasing Glutamate Buffering Capacity Triggers Oxidative Stress and Neuropil Degeneration in the Drosophila Brain , 2004, Current Biology.

[27]  Ethan Bier,et al.  Using Drosophila melanogaster to uncover human disease gene function and potential drug target proteins , 2002, Expert opinion on therapeutic targets.

[28]  G M Rubin,et al.  Ectopic expression of activated Ras1 induces hyperplastic growth and increased cell death in Drosophila imaginal tissues. , 1998, Development.

[29]  P. Salvaterra,et al.  Functional Analysis and Tissue‐Specific Expression of Drosophila Na+,K+‐ATPase Subunits , 1998, Journal of neurochemistry.

[30]  I. Hariharan,et al.  A Cyclin-Dependent Kinase Inhibitor, Dacapo, Is Necessary for Timely Exit from the Cell Cycle during Drosophila Embryogenesis , 1996, Cell.

[31]  Yan Geng,et al.  Requirement for CDK4 kinase function in breast cancer. , 2006, Cancer cell.

[32]  B. Calvi,et al.  Developmental and cell cycle regulation of the Drosophila histone locus body. , 2007, Molecular biology of the cell.

[33]  Joseph Gera,et al.  mTORC2 activity is elevated in gliomas and promotes growth and cell motility via overexpression of rictor. , 2007, Cancer research.

[34]  Gerald C. Chu,et al.  P53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation , 2008, Nature.

[35]  Eric C. Holland,et al.  Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice , 2000, Nature Genetics.

[36]  I. Rebay,et al.  Signal integration during development: Insights from the Drosophila eye , 2004, Developmental dynamics : an official publication of the American Association of Anatomists.

[37]  Junhyong Kim,et al.  Unwrapping Glial Biology Gcm Target Genes Regulating Glial Development, Diversification, and Function , 2003, Neuron.

[38]  S. Shibata,et al.  Expression of the Ets-1 proto-oncogene correlates with malignant potential in human astrocytic tumors. , 1999, Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc.

[39]  D. Bohmann,et al.  JNK‐ and Fos‐regulated Mmp1 expression cooperates with Ras to induce invasive tumors in Drosophila , 2006, The EMBO journal.

[40]  I. Hariharan,et al.  The Drosophila F Box Protein Archipelago Regulates dMyc Protein Levels In Vivo , 2004, Current Biology.

[41]  J. Cerón,et al.  Segregation of postembryonic neuronal and glial lineages inferred from a mosaic analysis of the Drosophila larval brain , 2007, Mechanisms of Development.

[42]  Marion Müller-Borg,et al.  Direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveals a link between the PAR/aPKC complex and phosphoinositide signaling , 2005, Development.

[43]  M. Stern,et al.  Phosphatidylinositol 3-Kinase and Akt Nonautonomously Promote Perineurial Glial Growth in Drosophila Peripheral Nerves , 2007, The Journal of Neuroscience.

[44]  I. Hariharan,et al.  Regulation of imaginal disc growth by tumor-suppressor genes in Drosophila. , 2006, Annual review of genetics.

[45]  David M Sabatini,et al.  Defining the role of mTOR in cancer. , 2007, Cancer cell.

[46]  T. P. Neufeld,et al.  Coordination of Growth and Cell Division in the Drosophila Wing , 1998, Cell.

[47]  C. Rickert,et al.  The homeobox gene repo is required for the differentiation and maintenance of glia function in the embryonic nervous system of Drosophila melanogaster. , 1995, Development.

[48]  Stella Pelengaris,et al.  c-MYC: more than just a matter of life and death , 2002, Nature Reviews Cancer.

[49]  K. Mechtler,et al.  Asymmetric Segregation of the Tumor Suppressor Brat Regulates Self-Renewal in Drosophila Neural Stem Cells , 2006, Cell.

[50]  N E Baker,et al.  Several levels of EGF receptor signaling during photoreceptor specification in wild-type, Ellipse, and null mutant Drosophila. , 1999, Developmental biology.

[51]  Eric C. Holland,et al.  Mouse Models of Brain Tumors and Their Applications in Preclinical Trials , 2006, Clinical Cancer Research.

[52]  Joshua M. Korn,et al.  Comprehensive genomic characterization defines human glioblastoma genes and core pathways , 2008, Nature.

[53]  D. Prober,et al.  Ras1 Promotes Cellular Growth in the Drosophila Wing , 2000, Cell.

[54]  John Tyler Bonner,et al.  Morphogenesis , 1965, Cell.

[55]  R. DePinho,et al.  Malignant glioma: genetics and biology of a grave matter. , 2001, Genes & development.

[56]  T. Schüpbach,et al.  Ectopic activation of torpedo/Egfr, a Drosophila receptor tyrosine kinase, dorsalizes both the eggshell and the embryo. , 1997, Development.

[57]  V. Hartenstein,et al.  Morphogenesis and proliferation of the larval brain glia in Drosophila. , 2005, Developmental biology.

[58]  Christian Klämbt,et al.  The Ets transcription factors encoded by the Drosophila gene pointed direct glial cell differentiation in the embryonic CNS , 1994, Cell.

[59]  A. Shearn,et al.  Drosophila brain tumor metastases express both neuronal and glial cell type markers. , 2007, Developmental biology.

[60]  Liqun Luo,et al.  Mosaic Analysis with a Repressible Cell Marker for Studies of Gene Function in Neuronal Morphogenesis , 1999, Neuron.

[61]  Hongye Liu,et al.  Olig2-Regulated Lineage-Restricted Pathway Controls Replication Competence in Neural Stem Cells and Malignant Glioma , 2007, Neuron.

[62]  E. Hafen,et al.  Ras controls growth, survival and differentiation in the Drosophila eye by different thresholds of MAP kinase activity. , 2001, Development.

[63]  S. Cohen,et al.  Re-evaluating AKT regulation: role of TOR complex 2 in tissue growth. , 2007, Genes & development.

[64]  E. Caussinus,et al.  Induction of tumor growth by altered stem-cell asymmetric division in Drosophila melanogaster , 2005, Nature Genetics.

[65]  Bassem A. Hassan,et al.  atonal Regulates Neurite Arborization but Does Not Act as a Proneural Gene in the Drosophila Brain , 2000, Neuron.

[66]  I. Mellinghoff,et al.  AKT Activity Determines Sensitivity to Mammalian Target of Rapamycin (mTOR) Inhibitors by Regulating Cyclin D1 and c-myc Expression* , 2004, Journal of Biological Chemistry.

[67]  I. Salecker,et al.  glial cells missing and gcm2 Cell Autonomously Regulate Both Glial and Neuronal Development in the Visual System of Drosophila , 2005, Neuron.

[68]  E. Petricoin,et al.  Drosophila screening model for metastasis: Semaphorin 5c is required for l(2)gl cancer phenotype , 2003, Proceedings of the National Academy of Sciences of the United States of America.