Synergistic effect of RAD50 downregulation on combination of rucaparib and doxorubicin

Ramazan Gundogdu; Mehmet Kadir Erdoğan; Aydın Sever; Yusuf Toy

doi:10.19161/etd.1160550

TR EN

RAD50'nin downregülasyonunun rucaparib ve doksorubisin kombinasyonuna sinerjik etkisi

Öz

Amaç: MRN (MRE11-RAD50-NBS1) protein kompleksi bir DNA hasar sensörü olarak işlev görür ve homolog rekombinasyon onarım mekanizması ile DNA çift sarmal kopmalarının onarımının koordine edilmesinde önemli roller oynar. Fonksiyonel olmayan MRN aktivitesinin kanser hücrelerini DNA'ya zarar veren ajanlara veya PARP inhibitörlerine karşı duyarlı hale getirdiği gösterilmiş olsa da, RAD50'nin rucaparib ve doksorubisin tedavileri üzerindeki fonksiyonel önemi henüz araştırılmamıştır. Bu araştırmanın amacı, RAD50 defektif kanser hücrelerinin rucaparib ve doksorubisin kombinasyonuna yanıtının araştırılmasıdır. Gereç ve Yöntem: Bu çalışmada RAD50 ekspresyon seviyelerinin terapötik potansiyelini değerlendirmek için insan kemiği osteosarkoma epitel hücreleri (U2OS) kullanıldı. RAD50 mRNA aktivitesinin ifadesini susturmak için RNA interferans teknolojisi uygulandı. İlgili genlerin mRNA ekspresyon seviyelerini araştırmak için qRT-PCR tekniği kullanıldı. İlgili protein ekspresyon seviyelerini değerlendirmek için Western blot analizi yapıldı. RAD50 kaybının rucaparib ve doksorubisin kombinasyon tedavisi üzerindeki etkisini incelemek için klonojenik sağkalım analizi gerçekleştirildi. Bulgular: Azalan RAD50 ifadesinin, MRE11 ve NBS1 protein seviyelerinde önemli bir düşüşe neden olduğu gözlenirken, p53 ve p21’in mRNA ve protein seviyelerini etkilemediği görüldü. Ayrıca, RAD50 kaybı olan hücrelerin akut doksorubisin tedavisi ile DNA hasar yanıt aktivasyonunu kaybettiği belirlendi. Son olarak, RAD50 susturulmasının PARP inhibitörü olan rucaparib ile birleştirildiğinde doksorubisinin sitotoksisitesini arttırdığı gözlendi. Sonuç: Tüm bu sonuçlar birlikte ele alındığında, klinik öncesi bulgularımız, PARP inhibitörlerinin kanser tedavisinde kullanılmasında RAD50 ekspresyon seviyelerinin prediktif bir biyobelirteç olarak araştırılabileceğini göstermektedir.

Anahtar Kelimeler

Synergistic effect of RAD50 downregulation on combination of rucaparib and doxorubicin

Abstract

Aim: The MRN (MRE11-RAD50-NBS1) protein complex functions as a DNA damage sensor and plays essential roles to coordinate the repair of DNA double-strand breaks. Although dysfunctional MRN activity has been shown to sensitize cancer cells to certain DNA-damaging agents or PARP inhibitors, the functional significance of RAD50 upon rucaparib and doxorubicin treatments has yet to be studied. The aim of this research was to investigate the response of RAD50-defective cancer cells toward the combination of rucaparib and doxorubicin. Materials and Methods: Human bone osteosarcoma epithelial cells (U2OS) were used in this study to assess the therapeutic potential of RAD50 expression levels. The RNA interference technology was applied to silence the expression of the RAD50 mRNA activity. The qRT-PCR technique was used to investigate the mRNA expression levels of the relevant genes. Western blotting analysis was conducted to assess the relevant protein expression levels. Clonogenic survival assay was performed to dissect the effect of RAD50-loss on the rucaparib and doxorubicin combination treatment. Results: RAD50 knockdown resulted in a significant decrease in MRE11 and NBS1 protein levels, whereas it did not affect p53 and p21 expressions at mRNA and protein levels. Furthermore, the cells with RAD50-loss had impaired DNA damage response activation against acute doxorubicin treatment. We finally showed that RAD50 depletion increased the cytotoxicity of doxorubicin when combined with the PARP inhibitor rucaparib. Conclusion: Taken together, our preclinical findings suggest that RAD50 expression levels can be explored as a predictive biomarker in the evaluation for precision cancer treatments involving PARP inhibitors.

Keywords

References

Lindahl T, Barnes DE. Repair of endogenous DNA damage. Cold Spring Harb Symp Quant Biol. 2000;65:127–33.
Ciccia A, Elledge SJSJ, Adamo A, Collis SJ, Adelman CA, Silva N, et al. The DNA Damage Response: Making It Safe to Play with Knives. Mol Cell. 2010 Oct;40(2):179–204.
Vilenchik MM, Knudson AG. Endogenous DNA double-strand breaks: Production, fidelity of repair, and induction of cancer. Proc Natl Acad Sci. 2003 Oct;100(22):12871–6.
Jackson, S. P. 2002. "Sensing and repairing DNA double-strand breaks". Carcinogenesis, 23(5), 687–696.
Goodarzi AA, Jeggo PA. The Repair and Signaling Responses to DNA Double-Strand Breaks. In: Advances in genetics. 2013. p. 1–45.
Hanahan, D., Weinberg, R. A. 2011. "Hallmarks of cancer: the next generation.". Cell, 144(5), 646–674.
Khanna KK, Jackson SP. DNA double-strand breaks: signaling, repair and the cancer connection. Nat Genet. 2001 Mar;27(3):247–54.
Wei Dai, Y. Y. 2014. "Genomic Instability and Cancer". Journal of Carcinogenesis & Mutagenesis, 05(02), 1– 13.

Trenner, A., Sartori, A. A. 2019. "Harnessing DNA Double-Strand Break Repair for Cancer Treatment". Frontiers in Oncology, 9(1388), 1–10.
Rupnik A, Lowndes NF, Grenon M. MRN and the race to the break. Vol. 119, Chromosoma. 2010. p. 115–35.
Krejci L, Altmannova V, Spirek M, Zhao X. Homologous recombination and its regulation. Nucleic Acids Res. 2012 Jul;40(13):5795–818.
Chapman JR, Taylor MRG, Boulton SJ. Playing the End Game: DNA Double-Strand Break Repair Pathway Choice. Mol Cell. 2012 Aug;47(4):497–510.
Bain, A. L., Mastrocola, A. S., Tibbetts, R. S., Khanna, K. K. 2014. "DNA Damage Response: From Tumourigenesis to Therapy". eLS (ss. 329–346). Chichester, UK: John Wiley & Sons, Ltd.
Morales JC, Li L, Fattah FJ, Dong Y, Bey EA, Patel M, et al. Review of poly (ADP-ribose) polymerase (PARP) mechanisms of action and rationale for targeting in cancer and other diseases. Crit Rev Eukaryot Gene Expr [Internet]. 2014 [cited 2022 Feb 22];24(1):15–28. Available from: https://pubmed.ncbi.nlm.nih.gov/24579667/
D’Andrea, A. D. 2018. "Mechanisms of PARP inhibitor sensitivity and resistance". DNA Repair, 71, 172–176.
Lord, C. J., Ashworth, A. 2013. "Mechanisms of resistance to therapies targeting BRCA-mutant cancers". Nature Medicine, 19(11), 1381–1388.
Lord CJ, Tutt ANJ, Ashworth A. Synthetic Lethality and Cancer Therapy: Lessons Learned from the Development of PARP Inhibitors. Annu Rev Med. 2015 Jan;66(1):455–70.
Lord, C. J., Ashworth, A. 2017. "PARP inhibitors: Synthetic lethality in the clinic". Science, 355(6330), 1152– 1158.
Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature. 2005 Apr;434(7035):913–7.
Farmer H, McCabe N, Lord CJ, Tutt ANJ, Johnson DA, Richardson TB, et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature. 2005 Apr;434(7035):917–21.
Shirley M. Rucaparib: A Review in Ovarian Cancer. Target Oncol. 2019;14(2):237–46.
Sachdev E, Tabatabai R, Roy V, Rimel BJ, Mita MM. PARP Inhibition in Cancer: An Update on Clinical Development. Target Oncol [Internet]. 2019;14(6):657–79. Available from: https://doi.org/10.1007/s11523- 019-00680-2
Ashworth A, Lord CJ. Synthetic lethal therapies for cancer: what’s next after PARP inhibitors? Nat Rev Clin Oncol [Internet]. 2018;15(9):564–76. Available from: http://dx.doi.org/10.1038/s41571-018-0055-6
Li H, Liu ZY, Wu N, Chen YC, Cheng Q, Wang J. PARP inhibitor resistance: the underlying mechanisms and clinical implications. Vol. 19, Molecular cancer. NLM (Medline); 2020. p. 107.
Noordermeer, S. M., van Attikum, H. 2019. "PARP Inhibitor Resistance: A Tug-of-War in BRCA-Mutated Cells". Trends in Cell Biology, 29(10), 820–834.
Pilié, P. G., Tang, C., Mills, G. B., Yap, T. A. 2019. "State-of-the-art strategies for targeting the DNA damage response in cancer". Nature Reviews Clinical Oncology, 16(2), 81–104
Cleary, J. M., Aguirre, A. J., Shapiro, G. I., D’Andrea, A. D. 2020. "Biomarker-Guided Development of DNA Repair Inhibitors". Molecular Cell, 18(78), 1070–1085
Hoppe MM, Sundar R, Tan DSP, Jeyasekharan AD. Biomarkers for Homologous Recombination Deficiency in Cancer. JNCI J Natl Cancer Inst. 2018 Jul;110(7):704–13.
Pilié PG, Gay CM, Byers LA, O’Connor MJ, Yap TA. PARP inhibitors: extending benefit beyond BRCA-mutant cancers. Clin Cancer Res. 2019;25(13):3759–71.
Gomez V, Gundogdu R, Gomez M, Hoa L, Panchal N, O’Driscoll M, et al. Regulation of DNA damage responses and cell cycle progression by hMOB2. Cell Signal. 2015 Feb;27(2):326–39.
Franken NAPP, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006 Dec;1(5):2315–9.
Moudry P, Watanabe K, Wolanin KM, Bartkova J, Wassing IE, Watanabe S, et al. TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity. 2016 Feb;212(3):281–8.
Zhong H, Bryson A, Eckersdorff M, Ferguson DO. Rad50 depletion impacts upon ATR-dependent DNA damage responses. Hum Mol Genet. 2005;14(18):2685–93.
Menendez D, Inga A, Resnick MA. The expanding universe of p53 targets. Nat Rev Cancer. 2009;9(10):724– 37.
Georgakilas, A. G., Martin, O. A., Bonner, W. M. 2017. "p21: A Two-Faced Genome Guardian". Trends in Molecular Medicine, 23(4), 310–319.
Kulaberoglu, Y., Gundogdu, R. and Hergovich A. The Role of p53/p21/p16 in DNA-Damage Signaling and DNA Repair. In: Genome Stability. 2016. p. 243–53.
Ahn J, Urist M, Prives C. The Chk2 protein kinase. DNA Repair (Amst). 2004;3(8–9):1039–47.
Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER, Hurov KE, Luo J, et al. ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage. Science (80- ). 2007 May;316(5828):1160–6.
Shepherd GM. Hypersensitivity Reactions to Chemotherapeutic Drugs. Clin Rev Allergy Immunol. 2003 Jun;24(3):253–62.
Desai A, Yan Y, Gerson SL. Advances in therapeutic targeting of the DNA damage response in cancer. Vols. 66–67, DNA Repair. 2018. p. 24–9.
Curtin NJ. DNA repair dysregulation from cancer driver to therapeutic target. Nat Rev Cancer. 2012;12(12):801–17.
Goldstein M, Kastan MB. The DNA Damage Response: Implications for Tumor Responses to Radiation and Chemotherapy. Annu Rev Med. 2015 Jan;66(1):129–43.
Curtin, N. J. 2013. "Inhibiting the DNA damage response as a therapeutic manoeuvre in cancer". British Journal of Pharmacology, 169(8), 1745–1765.
Stover, E. H., Konstantinopoulos, P. A., Matulonis, U. A., Swisher, E. M. 2016. "Biomarkers of response and resistance to DNA repair targeted therapies". Clinical Cancer Research, 22(23), 5651–5660.
Huang A, Garraway LA, Ashworth A, Weber B. Synthetic lethality as an engine for cancer drug target discovery. Nat Rev Drug Discov. 2020;19(1):23–38.
Stover, E. H., Konstantinopoulos, P. A., Matulonis, U. A., Swisher, E. M. 2016. "Biomarkers of response and resistance to DNA repair targeted therapies". Clinical Cancer Research, 22(23), 5651–5660.
Giovannini S, Weller MC, Repmann S, Moch H, Jiricny J. Synthetic lethality between BRCA1 deficiency and poly(ADP-ribose) polymerase inhibition is modulated by processing of endogenous oxidative DNA damage. Nucleic Acids Res. 2019 Sep;47(17):9132–43.
Syed, A., Tainer, J. A. 2018. "The MRE11-RAD50-NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair". Annual Review of Biochemistry, 87, 263– 294.
Varon, R., Vissinga, C., Platzer, M., Cerosaletti, K. M., Chrzanowska, K. H., Saar, K., … Reis, A. 1998. "Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome". Cell, 93(3), 467–476.
Stewart, G. S., Maser, R. S., Stankovic, T., Bressan, D. A., Kaplan, M. I., Jaspers, N. G. J., Taylor, A. M. R. 1999. "The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxiatelangiectasia-like disorder". Cell, 99(6), 577–587.
Hopfner K-P, Karcher A, Craig L, Woo TT, Carney JP, Tainer JA. Structural Biochemistry and Interaction Architecture of the DNA Double-Strand Break Repair Mre11 Nuclease and Rad50-ATPase a template for DNA resynthesis and rejoining (Roth and Wilson The Mre11/Rad50 (MR) complex plays a key role in DSB repair. Homologs of Mre11 and Rad50 are found Mre11 and Rad50 catalytic domains and examined the. Vol. 105, Cell. Gellert; 2001.
Paull TT, Gellert M. The 3 to 5 Exonuclease Activity of Mre11 Facilitates Repair of DNA Double-Strand Breaks Other genetic clues to the identity of DNA repair fac-tors have come from yeast, which primarily utilizes homologous recombination to repair its chromosomal. Vol. 1, Molecular Cell. 1998.
Koppensteiner R, Samartzis EP, Noske A, Von Teichman A, Dedes I, Gwerder M, et al. Effect of MRE11 loss on PARP-inhibitor sensitivity in endometrial cancer In Vitro. PLoS One. 2014;9(6).
Zhang M, Liu G, Xue F, Edwards R, Sood AK, Zhang W, et al. 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.
Flores-Pérez A, Rafaelli LE, Ramírez-Torres N, Aréchaga-Ocampo E, Frías S, Sánchez S, et al. RAD50 targeting impairs DNA damage response and sensitizes human breast cancer cells to cisplatin therapy. Cancer Biol Ther. 2014;15(6):777–88.
Abuzeid WM, Jiang X, Shi G, Wang H, Paulson D, Araki K, et al. Molecular disruption of RAD50 sensitizes human tumor cells to cisplatin-based chemotherapy. J Clin Invest. 2009;119(7):1974–85.
Alblihy A, Alabdullah ML, Toss MS, Algethami M, Mongan NP, Rakha EA, et al. RAD50 deficiency is a predictor of platinum sensitivity in sporadic epithelial ovarian cancers. 2020;1–10.
Yi LL, Kerrigan JE, Lin CP, Azarova AM, Tsai YC, Ban Y, et al. Topoisomerase IIβ-mediated DNA doublestrand breaks: Implications in doxorubicin cardiotoxicity and prevention by dexrazoxane. Cancer Res. 2007;67(18):8839–46.
Wang Y, Gudikote J, Giri U, Yan J, Deng W, Ye R, et al. RAD50 expression is associated with poor clinical outcomes after radiotherapy for resected non-small cell lung cancer. Clin Cancer Res. 2018;24(2):341–50.
Chang L, Huang J, Wang K, Li J, Yan R, Zhu L, et al. Targeting Rad50 sensitizes human nasopharyngeal carcinoma cells to radiotherapy. BMC Cancer. 2016;16(1):1–12.

Details

Primary Language

English

Subjects

Health Care Administration

Journal Section

Research Article

Authors

Ramazan Gundogdu ^*
0000-0001-5230-2121
Türkiye

Mehmet Kadir Erdoğan
0000-0002-1579-5737
Türkiye

Aydın Sever
0000-0002-6727-1556
Türkiye

Yusuf Toy
0000-0003-1901-9994
Türkiye

Publication Date

June 12, 2023

Submission Date

August 11, 2022

Acceptance Date

December 7, 2022

Published in Issue

Year 2023 Volume: 62 Number: 2

DOI

https://doi.org/10.19161/etd.1160550

IZ

https://izlik.org/JA97ZF29YT

Cite

RIS / Bibtex

Vancouver

1.Ramazan Gundogdu, Mehmet Kadir Erdoğan, Aydın Sever, Yusuf Toy. Synergistic effect of RAD50 downregulation on combination of rucaparib and doxorubicin. EJM. 2023 Jun. 1;62(2):289-300. doi:10.19161/etd.1160550