title: Activated forms of H-RAS and K-RAS differentially regulate membrane association of PI3K, PDK-1, and AKT and the effect of therapeutic kinase inhibitors on cell survival.
abstract: The abilities of mutated active RAS proteins to modulate cell survival following exposure to ionizing radiation and small molecule kinase inhibitors were examined. Homologous recombination in HCT116 cells to delete the single allele of K-RAS D13 resulted in a cell line that exhibited an approximately 75% reduction in basal extracellular signal-regulated kinase 1/2, AKT, and c-jun-NH2-kinase 1/2 activity. Transfection of cells lacking K-RAS D13 with H-RAS V12 restored extracellular signal-regulated kinase 1/2 and AKT activity to basal levels but did not restore c-jun-NH2-kinase 1/2 phosphorylation. In cells expressing H-RAS V12, radiation caused prolonged intense activation of AKT. Inhibition of H-RAS V12 function, blockade of phosphatidylinositol 3-kinase (PI3K) function using small interfering RNA/small-molecule inhibitors, or expression of dominant-negative AKT abolished radiation-induced AKT activation, and radiosensitized these cells. Inhibition of PI3K function did not significantly radiosensitize parental HCT116 cells. Inhibitors of the AKT PH domain including perifosine, SH-(5, 23-25) and ml-(14-16) reduced the plating efficiency of H-RAS V12 cells in a dose-dependent fashion. Inhibition of AKT function using perifosine enhanced radiosensitivity in H-RAS V12 cells, whereas the SH and ml series of AKT PH domain inhibitors failed to promote radiation toxicity. In HCT116 H-RAS V12 cells, PI3K, PDK-1, and AKT were membrane associated, whereas in parental cells expressing K-RAS D13, only PDK-1 was membrane bound. In H-RAS V12 cells, membrane associated PDK-1 was phosphorylated at Y373/376, which was abolished by the Src family kinase inhibitor PP2. Inhibition of PDK-1 function using the PH domain inhibitor OSU-03012 or using PP2 reduced the plating efficiency of H-RAS V12 cells and profoundly increased radiosensitivity. OSU-03012 and PP2 did not radiosensitize and had modest inhibitory effects on plating efficiency in parental cells. A small interfering RNA generated against PDK1 also radiosensitized HCT116 cells expressing H-RAS V12. Collectively, our data argue that molecular inhibition of AKT and PDK-1 signaling enhances the radiosensitivity of HCT116 cells expressing H-RAS V12 but not K-RAS D13. Small-molecule inhibitory agents that blocked stimulated and/or basal PDK-1 and AKT function profoundly reduced HCT116 cell survival but had variable effects at enhancing tumor cell radiosensitivity.
authors.affiliation: Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, 401 College Street, Richmond, VA 23298-0058, USA.
authors.forename: Rubén W
authors.initials: RW
authors.lastname: Carón
authors.forename: Adly
authors.initials: A
authors.lastname: Yacoub
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Zhu
authors.forename: Clint
authors.initials: C
authors.lastname: Mitchell
authors.forename: Young
authors.initials: Y
authors.lastname: Hong
authors.forename: William
authors.initials: W
authors.lastname: Hawkins
authors.forename: Takehiko
authors.initials: T
authors.lastname: Sasazuki
authors.forename: Senji
authors.initials: S
authors.lastname: Shirasawa
authors.forename: Alan P
authors.initials: AP
authors.lastname: Kozikowski
authors.forename: Philip A
authors.initials: PA
authors.lastname: Dennis
authors.forename: Michael P
authors.initials: MP
authors.lastname: Hagan
authors.forename: Steven
authors.initials: S
authors.lastname: Grant
authors.forename: Paul
authors.initials: P
authors.lastname: Dent
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D002277:Carcinoma / Q000201:enzymology* / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology*; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017353:Gene Deletion; D006801:Humans; D009154:Mutation / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000032:analysis / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D011836:Radiation Tolerance / Q000502:physiology*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D064413:3-Phosphoinositide-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017853:Phosphotransferases (Alcohol Group Acceptor); D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D018631:ras Proteins; C494918:AKT1 protein, human; C578562:PDPK1 protein, human