Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation, and impacts on metastasis free survival in breast cancer patients.
Cell Cycle. 2015-08-03; 14(19): 3066-3078
DOI: 10.1080/15384101.2015.1078020
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1. Cell Cycle. 2015;14(19):3066-78. doi: 10.1080/15384101.2015.1078020.
Cdk5 promotes DNA replication stress checkpoint activation through RPA-32
phosphorylation, and impacts on metastasis free survival in breast cancer
patients.
Chiker S(1)(2)(3), Pennaneach V(1)(2), Loew D(4), Dingli F(4), Biard D(5),
Cordelières FP(1)(6)(7), Gemble S(1)(6), Vacher S(8), Bieche I(8), Hall
J(1)(2)(9), Fernet M(1)(2).
Author information:
(1)a Institut Curie; Centre de Recherche; Centre Universitaire ; Orsay Cedex ,
France.
(2)b Inserm; U612; Centre Universitaire ; Orsay Cedex , France.
(3)c Université Paris-XI; Faculté de Médecine ; Le Kremlin Bicêtre , France.
(4)d Institut Curie; Centre de Recherche; Laboratoire de Spectrométrie de Masse
Protéomique ; Paris , France.
(5)e Commissariat à l’Energie Atomique; DSV; iMETI; SEPIA; Team Cellular
Engineering and Human Syndromes ; Fontenay aux Roses , France.
(6)f CNRS; UMR3348; Centre Universitaire ; Orsay Cedex , France.
(7)g Plateforme IBiSA d’Imagerie Cellulaire et Tissulaire; Institut Curie; Centre
Universitaire ; Orsay , France.
(8)h Pharmacogenetics Unit; Genetics Service ; Department of Tumour Biology ;
Institut Curie ; Paris , France.
(9)i Centre de Recherche en Cancérologie de Lyon -UMR Inserm 1052 – CNRS 5286 ;
Lyon , France.
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.
DOI: 10.1080/15384101.2015.1078020
PMCID: PMC4825598
PMID: 26237679 [Indexed for MEDLINE]