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Current Cancer Drug Targets

Author(s): Cíntia Regina Niederauer Ramos, Renato José Silva Oliveira, Marcela Nunes Rosa, Ariane Stéfani Pereira, Renata Barbosa Vahia de Abreu, Andre van Helvoort Lengert, Rui Manuel Reis, Viviane Aline Oliveira Silva, Edenir Inêz Palmero* and Matias Eliseo Melendez*

DOI: 10.2174/1568009623666230418101511

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RAD50 Deficient in a Breast Cancer Model Predicts Sensitivity to PARP Inhibitors

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Abstract

Background: Breast and ovarian tumors with pathogenic variants in BRCA1 or BRCA2 genes are more sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi) treatment than wildtype tumors. Pathogenic variants in non-BRCA1/2 homologous recombination repair genes (HRR) also concede sensitivity to PARPi treatment. RAD50 participates in the Mre11-RAD50-Nbn (MRN) complex of the HRR pathway and plays an important role in DNA repair.

Objective: The objective of this study is to evaluate whether RAD50 protein deficiency modulates the PARPi response in breast cancer cell lines.

Methods: T47D breast cancer cell line was modified using small interfering RNA and CRISPR/Cas9 technology, to knockout the RAD50 gene. PARPi response (niraparib, olaparib and rucaparib alone or in combination with carboplatin), in T47D and T47D-edited clones, was evaluated by cell viability, cell cycle, apoptosis and protein expression analyses.

Results: Treatment with niraparib and carboplatin exerted a synergistic effect on T47D-RAD50 deficient cells and an antagonistic effect on T47D cells parental. Cell cycle analysis demonstrated an increase in the G2/M population in cells treated with niraparib or rucaparib alone or in combination with carboplatin. T47D-RAD50 deficient cells treated with rucaparib and carboplatin exhibited twofold levels in late apoptosis, also showing differences in PARP activation. All T47D RAD50 deficient clones treated with niraparib or rucaparib combined with carboplatin, or rucaparib alone showed increased levels of H2AX phosphorylation.

Conclusions: T47D RAD50 deficient cells treated with PARP inhibitors alone or in combination with carboplatin showed cell cycle arrest in the G2/M phase, leading to death by apoptosis. Thus, RAD50 deficiency may be a good biomarker for predicting PARPi response.

Keywords: PARP inhibitors, synthetic lethality, DNA repair, homologous recombination, breast cancer, target therapy.

Graphical Abstract

[1]
Lynch, H.T.; Snyder, C.; Lynch, J. Hereditary breast cancer: Practical pursuit for clinical translation. Ann. Surg. Oncol., 2012, 19(6), 1723-1731.
[http://dx.doi.org/10.1245/s10434-012-2256-z] [PMID: 22434244]
[2]
Futreal, P.A.; Liu, Q.; Shattuck-Eidens, D.; Cochran, C.; Harshman, K.; Tavtigian, S.; Bennett, L.M.; Haugen-Strano, A.; Swensen, J.; Miki, Y.; Eddington, K.; McClure, M.; Frye, C.; Weaver-Feldhaus, J.; Ding, W.; Gholami, Z.; Söderkvist, P.; Terry, L.; Jhanwar, S.; Berchuck, A.; Iglehart, J.D.; Marks, J.; Ballinger, D.G.; Barrett, J.C.; Skolnick, M.H.; Kamb, A.; Wiseman, R. BRCA1 mutations in primary breast and ovarian carcinomas. Science, 1994, 266(5182), 120-122.
[http://dx.doi.org/10.1126/science.7939630] [PMID: 7939630]
[3]
Miki, Y.; Swensen, J.; Shattuck-Eidens, D.; Futreal, P.A.; Harshman, K.; Tavtigian, S.; Liu, Q.; Cochran, C.; Bennett, L.M.; Ding, W.; Bell, R.; Rosenthal, J.; Hussey, C.; Tran, T.; McClure, M.; Frye, C.; Hattier, T.; Phelps, R.; Haugen-Strano, A.; Katcher, H.; Yakumo, K.; Gholami, Z.; Shaffer, D.; Stone, S.; Bayer, S.; Wray, C.; Bogden, R.; Dayananth, P.; Ward, J.; Tonin, P.; Narod, S.; Bristow, P.K.; Norris, F.H.; Helvering, L.; Morrison, P.; Rosteck, P.; Lai, M.; Barrett, J.C.; Lewis, C.; Neuhausen, S.; Cannon-Albright, L.; Goldgar, D.; Wiseman, R.; Kamb, A.; Skolnick, M.H. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science, 1994, 266(5182), 66-71.
[http://dx.doi.org/10.1126/science.7545954] [PMID: 7545954]
[4]
Wooster, R.; Bignell, G.; Lancaster, J.; Swift, S.; Seal, S.; Mangion, J.; Collins, N.; Gregory, S.; Gumbs, C.; Micklem, G.; Barfoot, R.; Hamoudi, R.; Patel, S.; Rices, C.; Biggs, P.; Hashim, Y.; Smith, A.; Connor, F.; Arason, A.; Gudmundsson, J.; Ficenec, D.; Kelsell, D.; Ford, D.; Tonin, P.; Timothy Bishop, D.; Spurr, N.K.; Ponder, B.A.J.; Eeles, R.; Peto, J.; Devilee, P.; Cornelisse, C.; Lynch, H.; Narod, S.; Lenoir, G.; Egilsson, V.; Bjork Barkadottir, R.; Easton, D.F.; Bentley, D.R.; Futreal, P.A.; Ashworth, A.; Stratton, M.R. Identification of the breast cancer susceptibility gene BRCA2. Nature, 1995, 378(6559), 789-792.
[http://dx.doi.org/10.1038/378789a0] [PMID: 8524414]
[5]
Tavtigian, S.V.; Simard, J.; Rommens, J.; Couch, F.; Shattuck-Eidens, D.; Neuhausen, S.; Merajver, S.; Thorlacius, S.; Offit, K.; Stoppa-Lyonnet, D.; Belanger, C.; Bell, R.; Berry, S.; Bogden, R.; Chen, Q.; Davis, T.; Dumont, M.; Frye, C.; Hattier, T.; Jammulapati, S.; Janecki, T.; Jiang, P.; Kehrer, R.; Leblanc, J.F.; Mitchell, J.T.; McArthur-Morrison, J.; Nguyen, K.; Peng, Y.; Samson, C.; Schroeder, M.; Snyder, S.C.; Steele, L.; Stringfellow, M.; Stroup, C.; Swedlund, B.; Swense, J.; Teng, D.; Thomas, A.; Tran, T.; Tranchant, M.; Weaver-Feldhaus, J.; Wong, A.K.C.; Shizuya, H.; Eyfjord, J.E.; Cannon-Albright, L.; Tranchant, M.; Labrie, F.; Skolnick, M.H.; Weber, B.; Kamb, A.; Goldgar, D.E. The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds. Nat. Genet., 1996, 12(3), 333-337.
[http://dx.doi.org/10.1038/ng0396-333] [PMID: 8589730]
[6]
Kuchenbaecker, K.B.; Hopper, J.L.; Barnes, D.R.; Phillips, K.A.; Mooij, T.M.; Roos-Blom, M.J.; Jervis, S.; van Leeuwen, F.E.; Milne, R.L.; Andrieu, N.; Goldgar, D.E.; Terry, M.B.; Rookus, M.A.; Easton, D.F.; Antoniou, A.C.; McGuffog, L.; Evans, D.G.; Barrowdale, D.; Frost, D.; Adlard, J.; Ong, K.; Izatt, L.; Tischkowitz, M.; Eeles, R.; Davidson, R.; Hodgson, S.; Ellis, S.; Nogues, C.; Lasset, C.; Stoppa-Lyonnet, D.; Fricker, J.P.; Faivre, L.; Berthet, P.; Hooning, M.J.; van der Kolk, L.E.; Kets, C.M.; Adank, M.A.; John, E.M.; Chung, W.K.; Andrulis, I.L.; Southey, M.; Daly, M.B.; Buys, S.S.; Osorio, A.; Engel, C.; Kast, K.; Schmutzler, R.K.; Caldes, T.; Jakubowska, A.; Simard, J.; Friedlander, M.L.; McLachlan, S.A.; Machackova, E.; Foretova, L.; Tan, Y.Y.; Singer, C.F.; Olah, E.; Gerdes, A.M.; Arver, B.; Olsson, H. Risks of breast, ovarian, and contralateral breast cancer for BRCA1 and BRCA2 Mutation Carriers. JAMA, 2017, 317(23), 2402-2416.
[http://dx.doi.org/10.1001/jama.2017.7112] [PMID: 28632866]
[7]
Takaoka, M.; Miki, Y. BRCA1 gene: Function and deficiency. Int. J. Clin. Oncol., 2017. INCOMPLETE.
[PMID: 28884397]
[8]
Kleibl, Z.; Kristensen, V.N. Women at high risk of breast cancer: Molecular characteristics, clinical presentation and management. Breast, 2016, 28, 136-144.
[http://dx.doi.org/10.1016/j.breast.2016.05.006] [PMID: 27318168]
[9]
Farmer, H.; McCabe, N.; Lord, C.J.; Tutt, A.N.J.; Johnson, D.A.; Richardson, T.B.; Santarosa, M.; Dillon, K.J.; Hickson, I.; Knights, C.; Martin, N.M.B.; Jackson, S.P.; Smith, G.C.M.; Ashworth, A. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature, 2005, 434(7035), 917-921.
[http://dx.doi.org/10.1038/nature03445] [PMID: 15829967]
[10]
Bryant, H.E.; Schultz, N.; Thomas, H.D.; Parker, K.M.; Flower, D.; Lopez, E.; Kyle, S.; Meuth, M.; Curtin, N.J.; Helleday, T. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature, 2005, 434(7035), 913-917.
[http://dx.doi.org/10.1038/nature03443] [PMID: 15829966]
[11]
Ashworth, A. A synthetic lethal therapeutic approach: poly(ADP) ribose polymerase inhibitors for the treatment of cancers deficient in DNA double-strand break repair. J. Clin. Oncol., 2008, 26(22), 3785-3790.
[http://dx.doi.org/10.1200/JCO.2008.16.0812] [PMID: 18591545]
[12]
McCabe, N.; Turner, N.C.; Lord, C.J.; Kluzek, K.; Białkowska, A.; Swift, S.; Giavara, S.; O’Connor, M.J.; Tutt, A.N.; Zdzienicka, M.Z.; Smith, G.C.M.; Ashworth, A. Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-ribose) polymerase inhibition. Cancer Res., 2006, 66(16), 8109-8115.
[http://dx.doi.org/10.1158/0008-5472.CAN-06-0140] [PMID: 16912188]
[13]
Mendes-Pereira, A.M.; Martin, S.A.; Brough, R.; McCarthy, A.; Taylor, J.R.; Kim, J.S.; Waldman, T.; Lord, C.J.; Ashworth, A. Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors. EMBO Mol. Med., 2009, 1(6-7), 315-322.
[http://dx.doi.org/10.1002/emmm.200900041] [PMID: 20049735]
[14]
Brandt, S.; Samartzis, E.P.; Zimmermann, A.K.; Fink, D.; Moch, H.; Noske, A.; Dedes, K.J. Lack of MRE11-RAD50-NBS1 (MRN) complex detection occurs frequently in low-grade epithelial ovarian cancer. BMC Cancer, 2017, 17(1), 44.
[http://dx.doi.org/10.1186/s12885-016-3026-2] [PMID: 28073364]
[15]
Williams, G.J.; Lees-Miller, S.P.; Tainer, J.A. Mre11–Rad50–Nbs1 conformations and the control of sensing, signaling, and effector responses at DNA double-strand breaks. DNA Repair, 2010, 9(12), 1299-1306.
[http://dx.doi.org/10.1016/j.dnarep.2010.10.001] [PMID: 21035407]
[16]
Damiola, F.; Pertesi, M.; Oliver, J.; Le Calvez-Kelm, F.; Voegele, C.; Young, E.L.; Robinot, N.; Forey, N.; Durand, G.; Vallée, M.P.; Tao, K.; Roane, T.C.; Williams, G.J.; Hopper, J.L.; Southey, M.C.; Andrulis, I.L.; John, E.M.; Goldgar, D.E.; Lesueur, F.; Tavtigian, S.V. Rare key functional domain missense substitutions in MRE11A, RAD50, and NBNcontribute to breast cancer susceptibility: Results from a Breast Cancer Family Registry case-control mutation-screening study. Breast Cancer Res., 2014, 16(3), R58.
[http://dx.doi.org/10.1186/bcr3669] [PMID: 24894818]
[17]
Zhang, M.; Liu, G.; Xue, F.; Edwards, R.; Sood, A.K.; Zhang, W.; Yang, D. Copy number deletion of RAD50 as predictive marker of BRCAness and PARP inhibitor response in BRCA wild type ovarian cancer. Gynecol. Oncol., 2016, 141(1), 57-64.
[http://dx.doi.org/10.1016/j.ygyno.2016.01.004] [PMID: 27016230]
[18]
Silva-Oliveira, R.J.; Silva, V.A.O.; Martinho, O.; Cruvinel-Carloni, A.; Melendez, M.E.; Rosa, M.N.; de Paula, F.E.; de Souza Viana, L.; Carvalho, A.L.; Reis, R.M. Cytotoxicity of allitinib, an irreversible anti-EGFR agent, in a large panel of human cancer-derived cell lines: KRAS mutation status as a predictive biomarker. Cell. Oncol., 2016, 39(3), 253-263.
[http://dx.doi.org/10.1007/s13402-016-0270-z] [PMID: 26920031]
[19]
Cosmic. Available from: http://cancer.sanger.ac.uk/cosmic/help
[20]
Vriend, L.E.M.; Jasin, M.; Krawczyk, P.M. Assaying break and nick-induced homologous recombination in mammalian cells using the DR-GFP reporter and Cas9 nucleases. Methods Enzymol., 2014, 546, 175-191.
[http://dx.doi.org/10.1016/B978-0-12-801185-0.00009-X] [PMID: 25398341]
[21]
Estep, J.A.; Sternburg, E.L.; Sanchez, G.A.; Karginov, F.V. Immunoblot screening of CRISPR/Cas9-mediated gene knockouts without selection. BMC Mol. Biol., 2016, 17(1), 9.
[http://dx.doi.org/10.1186/s12867-016-0061-0] [PMID: 27038923]
[22]
Silva-Oliveira, R.J.; Melendez, M.; Martinho, O.; Zanon, M.F.; de Souza Viana, L.; Carvalho, A.L.; Reis, R.M. AKT can modulate the in vitro response of HNSCC cells to irreversible EGFR inhibitors. Oncotarget, 2017, 8(32), 53288-53301.
[http://dx.doi.org/10.18632/oncotarget.18395] [PMID: 28881811]
[23]
Srivastava, N.; Gochhait, S.; de Boer, P.; Bamezai, R.N.K. Role of H2AX in DNA damage response and human cancers. Mutat. Res. Rev. Mutat. Res., 2009, 681(2-3), 180-188.
[http://dx.doi.org/10.1016/j.mrrev.2008.08.003] [PMID: 18804552]
[24]
Pellegata, N.S.; Antoniono, R.J.; Redpath, J.L.; Stanbridge, E.J. DNA damage and p53-mediated cell cycle arrest: A reevaluation. Proc. Natl. Acad. Sci., 1996, 93(26), 15209-15214.
[http://dx.doi.org/10.1073/pnas.93.26.15209] [PMID: 8986789]
[25]
Ragamin, A.; Yigit, G.; Bousset, K.; Beleggia, F.; Verheijen, F.W.; Wit, M-C.Y.; Strom, T.M.; Dörk, T.; Wollnik, B.; Mancini, G.M.S. HumanRAD50 deficiency: Confirmation of a distinctive phenotype. Am. J. Med. Genet. A., 2020, 182(6), 1378-1386.
[http://dx.doi.org/10.1002/ajmg.a.61570] [PMID: 32212377]
[26]
Luo, J.; Dai, X.; Hu, H.; Chen, J.; Zhao, L.; Yang, C.; Sun, J.; Zhang, L.; Wang, Q.; Xu, S.; Xu, Y.; Liu, N.; Ying, G.; Wang, P. Fluzoparib increases radiation sensitivity of non-small cell lung cancer (NSCLC) cells without BRCA1/2 mutation, a novel PARP1 inhibitor undergoing clinical trials. J. Cancer Res. Clin. Oncol., 2020, 146(3), 721-737.
[http://dx.doi.org/10.1007/s00432-019-03097-6] [PMID: 31786739]
[27]
D’Amours, D.; Jackson, S.P. The MRE11 complex: At the crossroads of DNA repair and checkpoint signalling. Nat. Rev. Mol. Cell Biol., 2002, 3(5), 317-327.
[http://dx.doi.org/10.1038/nrm805] [PMID: 11988766]
[28]
Podhorecka, M.; Skladanowski, A.; Bozko, P. H2AX phosphorylation: Its role in DNA damage response and cancer therapy. J. Nucleic Acids, 2010, 2010, 1-9.
[http://dx.doi.org/10.4061/2010/920161] [PMID: 20811597]
[29]
Flores-Pérez, A.; Rafaelli, L.E.; Ramírez-Torres, N.; Aréchaga-Ocampo, E.; Frías, S.; Sánchez, S.; Marchat, L.A.; Hidalgo-Miranda, A.; Quintanar-Jurado, V.; Rodríguez-Cuevas, S.; Bautista-Piña, V.; Carlos-Reyes, Á.; López-Camarillo, C. RAD50 targeting impairs DNA damage response and sensitizes human breast cancer cells to cisplatin therapy. Cancer Biol. Ther., 2014, 15(6), 777-788.
[http://dx.doi.org/10.4161/cbt.28551] [PMID: 24642965]
[30]
Liao, M.; Beltman, J.; Giordano, H.; Harding, T.C.; Maloney, L.; Simmons, A.D.; Xiao, J.J. Clinical pharmacokinetics and pharmacodynamics of rucaparib. Clin. Pharmacokinet., 2022, 61(11), 1477-1493.
[http://dx.doi.org/10.1007/s40262-022-01157-8] [PMID: 36107395]
[31]
Akay, M.; Funingana, I.G.; Patel, G.; Mustapha, R.; Gjafa, E.; Ng, T.; Ng, K.; Flynn, M.J. An in-depth review of niraparib in ovarian cancer: Mechanism of action, clinical efficacy and future directions. Oncol. Ther., 2021, 9(2), 347-364.
[http://dx.doi.org/10.1007/s40487-021-00167-z] [PMID: 34363200]
[32]
Sonnenblick, A.; de Azambuja, E.; Azim, H.A., Jr; Piccart, M. An update on PARP inhibitors—moving to the adjuvant setting. Nat. Rev. Clin. Oncol., 2015, 12(1), 27-41.
[http://dx.doi.org/10.1038/nrclinonc.2014.163] [PMID: 25286972]
[33]
Coleman, R.L.; Fleming, G.F.; Brady, M.F.; Swisher, E.M.; Steffensen, K.D.; Friedlander, M.; Okamoto, A.; Moore, K.N.; Efrat Ben-Baruch, N.; Werner, T.L.; Cloven, N.G.; Oaknin, A.; DiSilvestro, P.A.; Morgan, M.A.; Nam, J.H.; Leath, C.A., III; Nicum, S.; Hagemann, A.R.; Littell, R.D.; Cella, D.; Baron-Hay, S.; Garcia-Donas, J.; Mizuno, M.; Bell-McGuinn, K.; Sullivan, D.M.; Bach, B.A.; Bhattacharya, S.; Ratajczak, C.K.; Ansell, P.J.; Dinh, M.H.; Aghajanian, C.; Bookman, M.A. Veliparib with first-line chemotherapy and as maintenance therapy in ovarian cancer. N. Engl. J. Med., 2019, 381(25), 2403-2415.
[http://dx.doi.org/10.1056/NEJMoa1909707] [PMID: 31562800]
[34]
Dal Molin, G.Z.; Omatsu, K.; Sood, A.K.; Coleman, R.L. Rucaparib in ovarian cancer: An update on safety, efficacy and place in therapy. Ther. Adv. Med. Oncol., 2018, 10. [INCOMPLETE].
[http://dx.doi.org/10.1177/1758835918778483] [PMID: 29977351]
[35]
Swisher, E.M.; Lin, K.K.; Oza, A.M.; Scott, C.L.; Giordano, H.; Sun, J.; Konecny, G.E.; Coleman, R.L.; Tinker, A.V.; O’Malley, D.M.; Kristeleit, R.S.; Ma, L.; Bell-McGuinn, K.M.; Brenton, J.D.; Cragun, J.M.; Oaknin, A.; Ray-Coquard, I.; Harrell, M.I.; Mann, E.; Kaufmann, S.H.; Floquet, A.; Leary, A.; Harding, T.C.; Goble, S.; Maloney, L.; Isaacson, J.; Allen, A.R.; Rolfe, L.; Yelensky, R.; Raponi, M.; McNeish, I.A. Rucaparib in relapsed, platinum-sensitive high-grade ovarian carcinoma (ARIEL2 Part 1): An international, multicentre, open-label, phase 2 trial. Lancet Oncol., 2017, 18(1), 75-87.
[http://dx.doi.org/10.1016/S1470-2045(16)30559-9] [PMID: 27908594]