The main goal of the proposed studies is to define the molecular signaling pathways whereby activated Ras proteins induce a novel form of non-apoptotic cell death in human glioblastoma cells. Gliomas are notoriously resistant to apoptotic death. Therefore, the long-term goal of this research is to uncover molecular targets that can be manipulated in a therapeutic context to activate non-apoptotic death in this type of cancer. These studies were prompted by the observation that ectopic expression of activated H-Ras or K-Ras in glioblastoma cells causes accumulation of cytoplasmic vacuoles that ultimately disrupt cell viability. The vacuoles are distinct from autophagosomes and may arise from late endosome or lysosome compartments. Active Ras produces similar effects in nine different human glioma cell lines, and the phenotype is recapitulated in stable glioblastoma cell lines where Ras is conditionally expressed. The unusual effects of Ras depend on its membrane association, but are independent from its stimulation of well- known effectors like Raf or PI3K. Preliminary studies indicate that activated Rac1, but not RhoA or Cdc42, can mimic the effects of activated Ras, whereas dominant-negative Rac1 blocks the Ras-induced phenotype. These findings lead to the central hypothesis that Ras activates Rac1-dependent effector pathways in glioblastoma to cause lethal disruptions of endosome or lysosome morphogenesis. To test this hypothesis, studies will focus on four specific aims:
(Aim 1) We will define the defective organelles and trafficking events that contribute to non-apoptotic death in human glioblastoma cells. This will include identification of membrane compartments involved in the biogenesis of the vacuoles and delineation of alterations in vesicular trafficking.
(Aim 2) We will evaluate the effects of Ras and Rac1 expression on the growth and viability of glioblastoma xenografts in mice, using established cell lines for conditional expression of the proteins.
(Aim 3) We will identify the specific Ras effector pathway(s) required for activated Ras to elicit the vacuolar phenotype, focusing on Ras-regulated nucleotide exchange factors (e.g., Tiam1, RasGRF) that activate Rac1 signaling. This will involve the use of dominant-negative mutants, RNAi-mediated gene silencing, and affinity isolation and proteomic characterization of Ras binding partners.
(Aim 4) We will define the downstream molecular connections between Rac1 signaling pathway(s) and the endo-lysosomal trafficking machinery in glioblastoma. These studies will focus on Rac1 interactions with Rab GTPases and phosphoinositide 4-phosphate 5'-kinases, which regulate trafficking in early and late endocytic pathways. These studies will contribute to a better understanding of connections between Ras and Rac1 signaling pathways and endo-lysosomal function, and they will provide new knowledge about a novel and poorly understood form of non-apoptotic death with potential clinical significance for treatment of brain tumors.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA115495-04
Application #
7851048
Study Section
Cancer Molecular Pathobiology Study Section (CAMP)
Program Officer
Salnikow, Konstantin
Project Start
2007-08-01
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2012-05-31
Support Year
4
Fiscal Year
2010
Total Cost
$281,770
Indirect Cost
Name
University of Toledo
Department
Biochemistry
Type
Schools of Medicine
DUNS #
807418939
City
Toledo
State
OH
Country
United States
Zip Code
43614
Li, Zehui; Mbah, Nneka E; Maltese, William A (2017) Vacuole-inducing compounds that disrupt endolysosomal trafficking stimulate production of exosomes by glioblastoma cells. Mol Cell Biochem :
Mbah, Nneka E; Overmeyer, Jean H; Maltese, William A (2017) Disruption of endolysosomal trafficking pathways in glioma cells by methuosis-inducing indole-based chalcones. Cell Biol Toxicol 33:263-282
Trabbic, Christopher J; George, Sage M; Alexander, Evan M et al. (2016) Synthesis and biological evaluation of isomeric methoxy substitutions on anti-cancer indolyl-pyridinyl-propenones: Effects on potency and mode of activity. Eur J Med Chem 122:79-91
Maltese, William A; Overmeyer, Jean H (2015) Non-apoptotic cell death associated with perturbations of macropinocytosis. Front Physiol 6:38
Trabbic, Christopher J; Overmeyer, Jean H; Alexander, Evan M et al. (2015) Synthesis and biological evaluation of indolyl-pyridinyl-propenones having either methuosis or microtubule disruption activity. J Med Chem 58:2489-512
Trabbic, Christopher J; Dietsch, Heather M; Alexander, Evan M et al. (2014) Differential Induction of Cytoplasmic Vacuolization and Methuosis by Novel 2-Indolyl-Substituted Pyridinylpropenones. ACS Med Chem Lett 5:73-77
Maltese, William A; Overmeyer, Jean H (2014) Methuosis: nonapoptotic cell death associated with vacuolization of macropinosome and endosome compartments. Am J Pathol 184:1630-42
Robinson, Michael W; Overmeyer, Jean H; Young, Ashley M et al. (2012) Synthesis and evaluation of indole-based chalcones as inducers of methuosis, a novel type of nonapoptotic cell death. J Med Chem 55:1940-56
Overmeyer, Jean H; Maltese, William A (2011) Death pathways triggered by activated Ras in cancer cells. Front Biosci (Landmark Ed) 16:1693-713
Wilson, Eden N; Bristol, Molly L; Di, Xu et al. (2011) A switch between cytoprotective and cytotoxic autophagy in the radiosensitization of breast tumor cells by chloroquine and vitamin D. Horm Cancer 2:272-85

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