title: Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation, and impacts on metastasis free survival in breast cancer patients.
abstract: Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing.
authors.affiliation: a Institut Curie; Centre de Recherche; Centre Universitaire ; Orsay Cedex , France.|b Inserm; U612; Centre Universitaire ; Orsay Cedex , France.|c Université Paris-XI; Faculté de Médecine ; Le Kremlin Bicêtre , France.
authors.forename: Sara
authors.initials: S
authors.lastname: Chiker
authors.affiliation: a Institut Curie; Centre de Recherche; Centre Universitaire ; Orsay Cedex , France.|b Inserm; U612; Centre Universitaire ; Orsay Cedex , France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Pennaneach
authors.affiliation: d Institut Curie; Centre de Recherche; Laboratoire de Spectrométrie de Masse Protéomique ; Paris , France.
authors.forename: Damarys
authors.initials: D
authors.lastname: Loew
authors.affiliation: d Institut Curie; Centre de Recherche; Laboratoire de Spectrométrie de Masse Protéomique ; Paris , France.
authors.forename: Florent
authors.initials: F
authors.lastname: Dingli
authors.affiliation: e Commissariat à l'Energie Atomique; DSV; iMETI; SEPIA; Team Cellular Engineering and Human Syndromes ; Fontenay aux Roses , France.
authors.forename: Denis
authors.initials: D
authors.lastname: Biard
authors.affiliation: a Institut Curie; Centre de Recherche; Centre Universitaire ; Orsay Cedex , France.|f CNRS; UMR3348; Centre Universitaire ; Orsay Cedex , France.|g Plateforme IBiSA d'Imagerie Cellulaire et Tissulaire; Institut Curie; Centre Universitaire ; Orsay , France.
authors.forename: Fabrice P
authors.initials: FP
authors.lastname: Cordelières
authors.affiliation: a Institut Curie; Centre de Recherche; Centre Universitaire ; Orsay Cedex , France.|f CNRS; UMR3348; Centre Universitaire ; Orsay Cedex , France.
authors.forename: Simon
authors.initials: S
authors.lastname: Gemble
authors.affiliation: h Pharmacogenetics Unit; Genetics Service ; Department of Tumour Biology ; Institut Curie ; Paris , France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Vacher
authors.affiliation: h Pharmacogenetics Unit; Genetics Service ; Department of Tumour Biology ; Institut Curie ; Paris , France.
authors.forename: Ivan
authors.initials: I
authors.lastname: Bieche
authors.affiliation: a Institut Curie; Centre de Recherche; Centre Universitaire ; Orsay Cedex , France.|b Inserm; U612; Centre Universitaire ; Orsay Cedex , France.|i Centre de Recherche en Cancérologie de Lyon -UMR Inserm 1052 - CNRS 5286 ; Lyon , France.
authors.forename: Janet
authors.initials: J
authors.lastname: Hall
authors.affiliation: a Institut Curie; Centre de Recherche; Centre Universitaire ; Orsay Cedex , France.|b Inserm; U612; Centre Universitaire ; Orsay Cedex , France.
authors.forename: Marie
authors.initials: M
authors.lastname: Fernet
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D051360:Cyclin-Dependent Kinase 5 / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D005260:Female; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics; D011839:Radiation, Ionizing
chemical_list: C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human; D006918:Hydroxyurea
keywords: Cdk5; PARP-1; hydroxyurea; ionizing radiation; replication stress