title: Hsp90 inhibitor-mediated disruption of chaperone association of ATR with hsp90 sensitizes cancer cells to DNA damage.
abstract: Following DNA damage that results in stalled replication fork, activation of ATR-CHK1 signaling induces the DNA damage response (DDR) in transformed cells. In the present studies on human cervical and breast cancer cells, we determined the effects of hsp90 inhibition on the levels and accumulation of DNA damage/repair-associated proteins following exposure to γ-ionizing radiation (IR; 4 Gy). We show that hsp90 inhibition with 17-allylamino-demehoxygeldanamycin or the novel, nongeldanamycin analogue AUY922 (resorcinylic isoxazole amide; Novartis Pharma) dose-dependently reduced the levels of ATR and CHK1 without affecting ATM levels. AUY922-mediated depletion of ATR and CHK1 was associated with an increase in their polyubiquitylation and decreased binding to hsp90. Cotreatment with bortezomib partially restored AUY922-mediated depletion of ATR and CHK1 levels. Additionally, treatment with AUY922 reduced the accumulation of ATR, p53BP1, and CHK1 but not γ-H2AX to the sites of DNA damage. Following exposure to IR, AUY922 treatment abrogated IR-induced phospho (p)-ATR and p-CHK1 levels, but significantly enhanced γ-H2AX levels. AUY922 treatment also increased IR-induced accumulation of the cells in G(2)-M phase of the cell cycle, inhibited the repair of IR-induced DNA damage, and augmented IR-mediated loss of clonogenic survival. Short hairpin RNA-mediated depletion of ATR also inhibited IR-induced p-ATR and p-CHK1, but increased γ-H2AX levels, sensitizing cancer cells to IR-induced apoptosis and loss of clonogenic survival. These findings indicate that ATR is a bona fide hsp90 client protein and post-IR administration of AUY922, by inhibiting ATR-CHK1-mediated DDR, sensitizes cancer cells to IR.
authors.affiliation: Medical College of Georgia, Augusta, Georgia, USA.
authors.forename: Kyungsoo
authors.initials: K
authors.lastname: Ha
authors.forename: Warren
authors.initials: W
authors.lastname: Fiskus
authors.forename: Rekha
authors.initials: R
authors.lastname: Rao
authors.forename: Ramesh
authors.initials: R
authors.lastname: Balusu
authors.forename: Sreedhar
authors.initials: S
authors.lastname: Venkannagari
authors.forename: Narasimha Rao
authors.initials: NR
authors.lastname: Nalabothula
authors.forename: Kapil N
authors.initials: KN
authors.lastname: Bhalla
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000187:drug effects*; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D004789:Enzyme Activation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000528:radiation effects; D045325:HCT116 Cells; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects; D011494:Protein Kinases / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011836:Radiation Tolerance / Q000235:genetics; D011839:Radiation, Ionizing; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: D018797:Cell Cycle Proteins; C468783:H2AX protein, human; D018841:HSP90 Heat-Shock Proteins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D011838:Radiation-Sensitizing Agents; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex