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Replication Protein a Availability During DNA Replication Stress Is A Published OnlineFirst August 2, 2018; DOI: 10.1158/0008-5472.CAN-18-0618 Cancer Molecular Cell Biology Research Replication Protein A Availability during DNA Replication Stress Is a Major Determinant of Cisplatin Resistance in Ovarian Cancer Cells Francois¸ Belanger 1, Emile Fortier1,2, Maxime Dube1,2, Jean-Francois¸ Lemay1, Remi Buisson3, Jean-Yves Masson3, Abdelhamid Elsherbiny1,2, Santiago Costantino1,4, Euridice Carmona5, Anne-Marie Mes-Masson2,5,6, Hugo Wurtele1,2,6, and Elliot Drobetsky1,2,6 Abstract Intrinsic and acquired resistance to cisplatin remains a pri- via NER, specifically during S phase. Such defects in NER were mary hurdle to treatment of high-grade serous ovarian cancer attributable to RPA exhaustion arising from aberrant activation (HGSOC). Cisplatin selectively kills tumor cells by inducing of DNA replication origins during replication stress. Reduced DNA crosslinks that block replicative DNA polymerases. Single- RPA availability promoted Mre11-dependent degradation of stranded DNA (ssDNA) generated at resulting stalled replica- nascent DNA at stalled RF in cell lines exhibiting elevated tion forks (RF) is bound and protected by heterotrimeric sensitivity to cisplatin. Strikingly, defective S-phase NER, RF replication protein A (RPA), which then serves as a platform instability, and cisplatin sensitivity could all be rescued by for recruitment and activation of replication stress response ectopic overexpression of RPA. Taken together, our findings factors. Cells deficient in this response are characterized by indicate that RPA exhaustion represents a major determinant of extensive ssDNA formation and excessive RPA recruitment that cisplatin sensitivity in HGSOC cell lines. exhausts the available pool of RPA, which (i) inhibits RPA- dependent processes such as nucleotide excision repair (NER) Significance: The influence of replication protein A exhaus- and (ii) causes catastrophic failure of blocked RF. Here, we tion on cisplatin sensitivity harbors important implications investigated the influence of RPA availability on chemosensi- toward improving therapy of various cancers that initially tivity using a panel of human HGSOC cell lines. Our data respond to platinum-based agents but later relapse due to revealed a striking correlation among these cell lines between intrinsic or acquired drug resistance. Cancer Res; 78(19); 5561–73. cisplatin sensitivity and the inability to efficiently repair DNA Ó2018 AACR. Introduction followed by combination chemotherapy with a platinum-based drug such as cisplatin (CDDP). This regimen yields a high initial High-grade serous ovarian cancer (HGSOC) is the most lethal response rate, often resulting in clinical remission. However, most gynecologic malignancy, with a 5-year survival rate of approxi- patients eventually relapse, at which point their cancer manifests mately 50% (1). Frontline treatment consists of debulking surgery strong resistance to CDDP (2). The therapeutic efficacy of CDDP is attributable to the capacity of its platinum atom to form covalent 1Centre de Recherche de l'Hopital^ Maisonneuve-Rosemont, Montreal, Canada. bonds involving the N7 position of purines in DNA generating 2Programme de Biologie Moleculaire, Universite de Montreal, Montreal, Canada. 1,2- and 1,3-intrastrand crosslinks (98%) in addition to a much 3CHU de Quebec Research Center, Oncology Axis, and Laval University Cancer lower yield (2%) of interstrand crosslinks (3). These adducts Research Center, Quebec City, Canada. 4Departement d'Ophtalmologie, Uni- strongly block the progression of DNA polymerases, inducing a 5 versite de Montreal, Montreal, Canada. Centre de Recherche du Centre Hospi- state of "replication stress" that selectively promotes the elimi- talier de l'Universite de Montreal, and Institut du Cancer de Montreal, Montreal, nation of rapidly proliferating cancer cells (4). Canada. 6Departement de Medecine, Universite de Montreal, Montreal, Canada. The mechanism of clinical resistance to CDDP is complex and Note: Supplementary data for this article are available at Cancer Research remains incompletely understood. The cytotoxicity of CDDP can Online (http://cancerres.aacrjournals.org/). be reduced by processes that counter its ability to react with DNA, F. Belanger and E. Fortier contributed equally to this article. including increased efflux/reduced uptake and inactivation by Current address for R. Buisson: Massachusetts General Hospital Cancer Center, sulfur-containing molecules (5). In addition, CDDP-induced Harvard Medical School, Boston, MA. intrastrand crosslinks are removed by nucleotide excision repair fi Corresponding Authors: Elliot Drobetsky, Centre de Recherche de l'Hopital^ (NER; ref. 6), and cells de cient in this pathway are sensitive to the Maisonneuve-Rosemont, 5415 boul de l'Assomption, Montreal H1T 2M4, drug (7, 8). Indeed, based on various lines of evidence, increased Canada. Phone: 514-252-3400; Fax: 514-252-3430; E-mail: NER capacity has been proposed as an important mechanism of [email protected]; and Hugo Wurtele, Centre de Recherche de CDDP resistance in ovarian cancer (5). ^ l'Hopital Maisonneuve-Rosemont, 5415 Boulevard de l'Assomption, Montreal In addition to mechanisms outlined above that remove DNA H1T 2M4, Canada. Fax: 514-252-3430; E-mail: [email protected] lesions or inhibit their formation, modulation of the cellular doi: 10.1158/0008-5472.CAN-18-0618 response to replication stress, i.e., pathways that contribute to Ó2018 American Association for Cancer Research. the stabilization/resolution of stalled replication forks (RF), is www.aacrjournals.org 5561 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst August 2, 2018; DOI: 10.1158/0008-5472.CAN-18-0618 Belanger et al. emerging as a key determinant of CDDP resistance in cancer (9). FBS, L-glutamine, and antibiotics. All cell lines were used within For example, germline mutations in BRCA1 or BRCA2 greatly 20 passages after thawing and routinely tested for mycoplasma increase susceptibility to ovarian and breast tumors (10). contamination by DAPI staining/fluorescence microscopy. Although BRCA1/2 were initially characterized for their key roles in DNA double-strand break (DSB) repair by homologous recom- Ectopic RPA expression and siRNA treatment bination (HR), recent data reveal that these proteins also stabilize The RPA expression plasmid (pAC-GFP-RPA; ref. 16), a generous stalled RF by inhibiting Mre11-dependent nucleolytic degrada- gift of Dr. J. Lukas (University of Copenhagen, Copenhagen, tion of nascent DNA (11, 12). Moreover, sensitivity to replication- Denmark), was transfected using Lipofectamine 2000 (Thermo- blocking drugs such as CDDP was recently shown to correlate Fisher). Stable clones were sorted by FACS in 96-well plates and strongly with the inability of BRCA1/2-defective cells to protect selected with 500 mg/mL Geneticin (ThermoFisher). siRNA smart- nascent DNA from nuclease activity (12). pools for knockdown of RPA1 and BRCA1 were purchased from Following exposure to replication stress–inducing drugs, Dharmacon and transfected using RNAiMax (ThermoFisher). uncoupling of DNA polymerases from the MCM helicase complex Pools of nontargeting (NT) duplexes were used as controls. at blocked RF generates regions of single-stranded DNA (ssDNA), which are rapidly coated by heterotrimeric replication protein A Cell irradiation and drug treatment (RPA; ref. 13). RPA-ssDNA acts as a recruitment/activation plat- Cell monolayers were irradiated with 254-nm UV (hereafter form for DNA damage response (DDR) factors that mitigate UV) at a fluence rate of 0.4 J/m2/s, or with ionizing radiation (dose À replication stress, notably the apical kinase ataxia telangiectasia rate of 5 Â 10 2 Gy/s) as previously described (24). The ATR and Rad3-related (ATR; ref. 14). Activated ATR phosphorylates inhibitor VE-821 (Cedarlane), hydroxyurea (HU; Bioshop multiple substrates, which cooperate to (i) prevent further origin Canada), cyclin-dependent kinase inhibitor roscovitine (Abcam), activation, (ii) stabilize stalled RF, and (iii) block cell-cycle or MRE11 inhibitor mirin (Sigma-Aldrich) were added to cell progression. Abrogation of ATR activity during replication stress monolayers in fresh media. leads to unrestricted firing of DNA replication origins, excessive accumulation of ssDNA, and greatly increased levels of chroma- NER assay tin-bound RPA (15). This eventually results in global exhaustion Removal of 6-4 pyrimidine-pyrimidone photoproducts of cellular RPA and extensive DSB formation at persistently stalled (6-4PP) as a function of cell cycle was quantified as described RF, a process recently termed "replication catastrophe" (16). (19). For repair determinations in the presence of roscovitine, However, the precise mechanisms leading to RF instability in cells were treated with 20 mmol/L of the drug (or mock treated) for response to RPA exhaustion, and their potential influence on 2 or 72 hours before the assay and during post-UV incubations. cancer chemoresistance, remain incompletely characterized. Under conditions of limited RPA availability, important RPA- dependent processes aside from DNA replication can be compro- Clonogenic survival mised, which might contribute to increased RF stalling/failure. Cells were seeded on 100 mm dishes and treated with CDDP Indeed, we and others have shown, in various mutant yeast strains (Cedarlane) or mitomycin C (MMC; Abcam) for 2 hours in and mammalian
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