title: Suppression of RAF/MEK or PI3K synergizes cytotoxicity of receptor tyrosine kinase inhibitors in glioma tumor-initiating cells.
abstract: BACKGROUND
  The majority of glioblastomas have aberrant receptor tyrosine kinase (RTK)/RAS/phosphoinositide 3 kinase (PI3K) signaling pathways and malignant glioma cells are thought to be addicted to these signaling pathways for their survival and proliferation. However, recent studies suggest that monotherapies or inappropriate combination therapies using the molecular targeted drugs have limited efficacy possibly because of tumor heterogeneities, signaling redundancy and crosstalk in intracellular signaling network, indicating necessity of rationale and methods for efficient personalized combination treatments. Here, we evaluated the growth of colonies obtained from glioma tumor-initiating cells (GICs) derived from glioma sphere culture (GSC) in agarose and examined the effects of combination treatments on GICs using targeted drugs that affect the signaling pathways to which most glioma cells are addicted.
  
  
  METHODS
  Human GICs were cultured in agarose and treated with inhibitors of RTKs, non-receptor kinases or transcription factors. The colony number and volume were analyzed using a colony counter, and Chou-Talalay combination indices were evaluated. Autophagy and apoptosis were also analyzed. Phosphorylation of proteins was evaluated by reverse phase protein array and immunoblotting.
  
  
  RESULTS
  Increases of colony number and volume in agarose correlated with the Gompertz function. GICs showed diverse drug sensitivity, but inhibitions of RTK and RAF/MEK or PI3K by combinations such as EGFR inhibitor and MEK inhibitor, sorafenib and U0126, erlotinib and BKM120, and EGFR inhibitor and sorafenib showed synergy in different subtypes of GICs. Combination of erlotinib and sorafenib, synergistic in GSC11, induced apoptosis and autophagic cell death associated with suppressed Akt and ERK signaling pathways and decreased nuclear PKM2 and β-catenin in vitro, and tended to improve survival of nude mice bearing GSC11 brain tumor. Reverse phase protein array analysis of the synergistic treatment indicated involvement of not only MEK and PI3K signaling pathways but also others associated with glucose metabolism, fatty acid metabolism, gene transcription, histone methylation, iron transport, stress response, cell cycle, and apoptosis.
  
  
  CONCLUSIONS
  Inhibiting RTK and RAF/MEK or PI3K could induce synergistic cytotoxicity but personalization is necessary. Examining colonies in agarose initiated by GICs from each patient may be useful for drug sensitivity testing in personalized cancer therapy.
authors: Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. tshingu@mdanderson.org.|Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, 1881 East Road, Houston, TX, 77054, USA. tshingu@mdanderson.org.
authors: Takashi
authors: T
authors: Shingu
authors: Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. lindsay.holmes@bcm.edu.|Baylor College of Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, USA. lindsay.holmes@bcm.edu.
authors: Lindsay
authors: L
authors: Holmes
authors: Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. vkhenry@mdanderson.org.
authors: Verlene
authors: V
authors: Henry
authors: Department of Bioinformatics and Computational Biology, The University of Texas M. D. Anderson Cancer Center, 1881 East Road, Houston, TX, 77054, USA. QWang5@mdanderson.org.|Department of Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. QWang5@mdanderson.org.
authors: Qianghu
authors: Q
authors: Wang
authors: Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. klatha@mdanderson.org.
authors: Khatri
authors: K
authors: Latha
authors: Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. Anupama.E.Gururaj@uth.tmc.edu.
authors: Anupama E
authors: AE
authors: Gururaj
authors: Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. lagibson@mdanderson.org.|Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, 1881 East Road, Houston, TX, 77054, USA. lagibson@mdanderson.org.
authors: Laura A
authors: LA
authors: Gibson
authors: Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. tifslater@hotmail.com.
authors: Tiffany
authors: T
authors: Doucette
authors: Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. flang@mdanderson.org.
authors: Frederick F
authors: FF
authors: Lang
authors: Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. grao@mdanderson.org.
authors: Ganesh
authors: G
authors: Rao
authors: Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, 1881 East Road, Houston, TX, 77054, USA. LYuan3@mdanderson.org.
authors: Liang
authors: L
authors: Yuan
authors: Department of Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. EPSulman@mdanderson.org.
authors: Erik P
authors: EP
authors: Sulman
authors: Department of Chemistry, Virginia Commonwealth University, 901 West Franklin Street, Richmond, VA, 23284-9005, USA. npfarrell@vcu.edu.
authors: Nicholas P
authors: NP
authors: Farrell
authors: Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, 1881 East Road, Houston, TX, 77054, USA. wpriebe@mdanderson.org.
authors: Waldemar
authors: W
authors: Priebe
authors: Department of Biostatistics, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. khess@mdanderson.org.
authors: Kenneth R
authors: KR
authors: Hess
authors: Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, 1881 East Road, Houston, TX, 77054, USA. yalanwang@mdanderson.org.
authors: Yaoqi A
authors: YA
authors: Wang
authors: Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, 1881 East Road, Houston, TX, 77054, USA. JHu3@mdanderson.org.
authors: Jian
authors: J
authors: Hu
authors: Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA. obogler@mdanderson.org.|The University of Texas M. D. Anderson Cancer Center, 7007 Bertner Ave., Houston, TX, 77030, USA. obogler@mdanderson.org.
authors: Oliver
authors: O
authors: Bögler
mesh_terms: D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D014408:Biomarkers, Tumor / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004357:Drug Synergism; D005910:Glioma / Q000188:drug therapy* / Q000473:pathology*; D006801:Humans; D020128:Inhibitory Concentration 50; D008297:Male; D008819:Mice, Nude; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use*; D015398:Signal Transduction / Q000187:drug effects; D048490:raf Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D020929:Mitogen-Activated Protein Kinase Kinases; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D048490:raf Kinases