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Cyclin-Dependent Kinase 2 Functions in Normal DNA Repair and Is a Therapeutic Target in BRCA1-Deficient Cancers

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Cyclin-Dependent Kinase 2 Functions in Normal DNA Repair and Is a Therapeutic Target in BRCA1-Deficient Cancers Research Article Cyclin-Dependent Kinase 2 Functions in Normal DNA Repair and Is a Therapeutic Target in BRCA1-Deficient Cancers Andrew J. Deans,1 Kum Kum Khanna,2 Carolyn J. McNees,3 Ciro Mercurio,4 Jo¨rg Heierhorst,3,5 and GrantA. McArthur 1,5 1Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; 2Queensland Institute of Medical Research, Herston, Queensland, Australia; 3St. Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia; 4Nerviano Medical Sciences, BU-Oncology and BU-Preclinical Science, Milan, Italy; and 5Department of Medicine, St Vincent’s Hospital, The University of Melbourne, Parkville, Victoria, Australia Abstract CDKs are a family of serine/threonine kinases that control the progression of the cell cycle, including the onset of DNA synthesis Abnormal regulation of progression from G1 to S phase of the cell cycle by altered activity of cyclin-dependent kinases and mitosis (2). However, recent knockout and small interfering (CDKs) is a hallmark of cancer.However, inhibition of CDKs, RNA (siRNA) studies suggested that CDK2 is not essential for particularly CDK2, has not shown selective activity against somatic cell division and that other kinases can substitute for its most cancer cells because the kinase seems to be redundant in function in the normal cell cycle. As CDK2 is considered a potential control of cell cycle progression.Here, we show a novel role in therapeutic target in cancer and multiple CDK2 inhibitors have shown efficacy in preclinical models and clinical trials, this was an the DNA damage response and application of CDK inhibitors À À in checkpoint-deficient cells.CDK2 / mouse fibroblasts and unexpected outcome. Given that CDK2 has several non-cell cycle– associated substrates in the DNA damage response pathway small interfering RNA–mediated or small-molecule–mediated BRCA1 BRCA2 CDK2 inhibition in MCF7 or U2OS cells lead to delayed [e.g., familial breast cancer genes (3) and (4), the damage signaling through Chk1, p53, and Rad51.This tumor suppressor p53 (5), and the NHEJ factor Ku70 (6)], we exa- mined the rate of DNA repair in cells where CDK2 was abrogated. coincided with reduced DNA repair using the single-cell in vitro comet assay and defects observed in both homologous Using several systems, we found that inhibition of CDK2 recombination and nonhomologous end-joining in cell-based using small-molecule inhibitors leads to a decrease in DNA repair assays.Furthermore, tumor cells lacking cancer predisposi- activity in mammalian cells. Induction of DNA damage following specific ablation of CDK2 activity using short hairpin RNA (shRNA) tion genes BRCA1 or ATM are 2- to 4-fold more sensitive to À À or in CDK2 / mouse embryonic fibroblasts (MEF) leads to a CDK inhibitors.These data suggest that inhibitors of CDK2 can be applied to selectively enhance responses of cancer cells similar reduction in either the repair of an exogenous double-strand to DNA-damaging agents, such as cytotoxic chemotherapy and break in end-joining or HR assays or endogenous DNA damage after radiotherapy.Moreover, inhibitors of CDKs may be useful ionizing radiation. We show that after CDK inhibition, multiple therapeutics in cancers with defects in DNA repair, such as components of the DNA damage response are altered in their mutations in the familial breast cancer gene BRCA1. (Cancer response to damage caused by ionizing radiation, including Res 2006; 66(16): 8219-26) phosphorylation of H2A.X, p53, and Chk1, and increased expression of p21Cip1. Based on this finding, we tested the efficacy of CDK inhibition on cells that are deficient in other components of DNA Introduction repair, BRCA1 or ATM. Interestingly, CDK inhibition showed up to The DNA damage response in mammalian cells is an evolu- 4-fold increased toxicity in cells deficient in these DNA repair tionarily conserved pathway that protects cells from the lethal or components, which are often mutated in familial breast cancer tumor-promoting consequences of genotoxic insult. Mutation in families. These findings show that CDK2 activity plays a key role in DNA repair genes, such as BRCA1, BRCA2, and ATM, accounts for the normal DNA damage response and that CDK inhibition may be a significant proportion of the breast cancers attributed to a a chemotherapeutic option for specifically targeting cancer cells familial predisposition, highlighting the importance of this process with defects in DNA repair. in tumor protection. Cell cycle regulation is thought to be important in regulating the DNA repair process. For example, Materials and Methods nonhomologous end-joining (NHEJ) and homologous recombina- À/À tion (HR) are differently regulated in cycling versus quiescent cells, Cell culture and flow cytometry. Immortalized CDK2 MEFs and and the overall rate of DNA repair is faster in cells that are rapidly matched wild-type controls were kindly provided by Mariano Barbacid’s laboratory (Spanish National Cancer Institute, Madrid, Spain). MCF7, U2OS, cycling compared with those that are quiescent (1). We propose and MEF cells were cultured at 37jC in DMEM supplemented with 10% fetal a candidate and therapeutic target in this temporal regulation, bovine serum (FBS). FTY-pEB and FTY-pEB-(Flag)ATM cells (7) were cyclin-dependent kinase 2 (CDK2). cultured in RPMI1640 supplemented with 10% FBS. Bromodeoxyuridine (BrdUrd) flow cytometry was done as described (8) on an LSR flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Note: Supplementary data for this article are available at Cancer Research Online Drug treatments. Roscovitine (LC Laboratories, Woburn, MA) and  (http://cancerres.aacrjournals.org/). PHA533533 (9) were dissolved in DMSO at 1,000 final concentration. IC70 Requests for reprints: Grant A. McArthur, Peter MacCallum Cancer Centre, doses were determined after 72-hour drug exposure using the sulforhod- Locked Bag 1, A’Beckett Street, Melbourne, Victoria 8006, Australia. Phone: 61-3-9656- amine B assay as described (10). 1195; Fax: 61-3-9656-1411; E-mail: [email protected]. I2006 American Association for Cancer Research. DNA damage response assays. Subconfluent MCF7 cells were treated doi:10.1158/0008-5472.CAN-05-3945 with CDK2 inhibitors and then immediately exposed to cesium-137 in a www.aacrjournals.org 8219 Cancer Res 2006; 66: (16). August 15, 2006 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Cancer Research Gammacell 400 at 1.5 min/Gy. Protein samples were then collected at the investigate the role of CDK inhibition in the mammary epithelial indicated time points. normal cell cycle, two small-molecule inhibitors of CDKs were Clonogenic survival assays. MCF7 or FTY cells were plated at 300 to used. The R-anantomer of roscovitine (also known as CYC202 or 10,000 cells per 6-cm plate. Sixteen hours later, cells were treated with drugs Seliciclib) is currently in phase II trials for the treatment of non– at the specified doses and incubated in normal medium containing drug small cell lung cancers and B-cell malignancies and has similar for 12 days, fixed in 90% methanol, and stained with 0.1% crystal violet. Colonies of >50 cells were counted in the surviving fraction. activity against CDK2 and CDK1, whereas PHA533533 is a 3- Single-cell comet assay. The single-cell comet assay was done as aminopyrazole that is in preclinical development and is a more described previously (11). The Olive tail moment was determined by potent and selective inhibitor of CDK2 (9). The structure and analysis of at least 50 nuclei from each treatment group using MetaMorph chemical inhibitory doses of the drugs against a panel of purified software (Universal Imaging Corp., Downington, PA). kinases are shown in Fig. 1. DNA end-joining assay. The plasmid pZeoSV (Invitrogen, Carlsbad, CA) In cultured MCF7 mammary adenocarcinoma cells, doses of was completely digested with PvuII and purified using JetStar DNA 3 Amol/L PHA533533 and 20 Amol/L roscovitine lead to complete purification beads (Genomed, Lohne, Germany). MCF7 cells (1.5  105 per well) were plated in six-well plates. The following day, 0.75 Ag digested plasmid was transfected per well using Fugene 6 reagent (Roche, Mannheim, Germany). Six hours later, CDK2 inhibitors or DMSO vehicle was added to cells. Following 12, 24, or 40 hours of incubation, DNA was extracted with proteinase K digestion overnight at 55jC followed by phenol/chloroform extraction and isopropanol precipitation. DNA (10 ng) was used as a PCR template. Quantitative real-time PCR was done using the SYBR Green detection method (Applied Biosystems, Warrington, United Kingdom) and primers specific for the rejoined cut site [5¶-CGAGAATTCGAACGCGTGA-3¶ (forward) and 5¶-TGTGAAATTTGTGATGCTATTGCTT-3¶ (reverse)] or an uncut region of the plasmid [pZeo, 5¶-ATAATACGACTCACTATAGGAG- GGCC-3¶ (forward) and pZeo, 5¶-GAACTCGACCGCTCCGG-3¶ (reverse)]. HR assay. A U2OS clone containing a single complete copy of the integrated HR reporter hprt-DR-GFP was generated as described previously (12, 13) U2OS-DR-GFP cells (1.5  105 per well) were seeded in six-well plates and, the following morning, transfected with 1 Ag of pCBA-I-SceI(12) plasmid using Fugene 6 reagent. Six hours later, medium was replaced with fresh medium containing drugs at the specified concentration. Cells were harvested after 2 days for flow cytometric analysis on a FACSCalibur cytometer (Becton Dickinson). siRNA-mediated knockdown. Knockdown of BRCA1 using pSuper vector-mediated
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