Effects of CRISPR-Cas9 silencing of VPS28 gene on cisplatin response in chemotherapy-resistant ovarian cancer cells

Abstract

Aim: Today, ovarian cancer is still common in women and is the leading cause of deaths from gynecologic cancer. Although cisplatin, a chemotherapeutic agent, is frequently used in the treatment, resistance to cisplatin develops in patients over time and genetic factors are known to be responsible for the development of resistance. In this study, it was aimed to determine the possible role of the ESCRT-I complex component VPS28 gene, which is active in multivesicular body biogenesis, in ovarian cancer cisplatin resistance. Method: After silencing the VPS28 gene in cisplatin-resistant A2780 ovarian cancer cells using the CRISPR-Cas9 method, the effects on proliferation, cisplatin response, apoptosis and cell cycle were investigated. Results: Functional analyses revealed that VPS28 gene silencing in chemotherapy-resistant ovarian cancer increased cisplatin sensitivity, induced apoptosis, and caused mitotic catastrophe by arresting the cell cycle in the S phase. Conclusion: The VPS28 gene has been shown to be involved in acquired cisplatin resistance in A2780 ovarian cancer cells, and its silencing has a synergistic effect with the cisplatin used in treatment.

Keywords

• Ovarian cancer
• cisplatin resistance
• VPS28
• CRISPR-Cas9

Kemoterapi dirençli over kanseri hücrelerinde VPS28 geninin CRISPR-Cas9 yöntemi ile susturulmasının cisplatin yanıtı üzerine etkileri

Abstract

Amaç: Günümüzde yumurtalık (over) kanseri kadınlarda halen yaygın olarak görülmekte ve jinekolojik kansere bağlı ölüm nedenlerinin başında gelmektedir. Tedavisinde sıklıkla kemoterapötik bir ajan olan cisplatin kullanılmakla birlikte, zamanla hastalarda cisplatine karşı direnç gelişmekte ve direnç gelişiminde genetik faktörlerin sorumlu olduğu bilinmektedir. Bu çalışmada multiveziküler cisim biyogenezinde etkin ESCRT-I kompleks bileşeni VPS28 geninin, over kanseri cisplatin direncindeki olası rolünün belirlenmesi amaçlanmıştır. Yöntem: Cisplatin dirençli A2780 over kanseri hücre serisinde VPS28 geni CRISPR-Cas9 yöntemiyle susturulduktan sonra proliferasyon, cisplatin yanıtı, apoptoz ve hücre döngüsü üzerine etkileri araştırılmıştır. Bulgular: Yapılan fonksiyonel analizler sonucunda, cisplatin dirençli over kanseri hücrelerinde VPS28 geninin susturulması ile cisplatin duyarlılığının arttığı, apoptozun indüklendiği ve hücre döngüsü S evresinde mitotik katastrofa neden olduğu belirlenmiştir. Sonuç: VPS28 geninin A2780 over kanseri hücrelerindeki kazanılmış cisplatin direncinde etkili olduğu ve susturulmasının, tedavisinde kullanılan cisplatin ile birlikte sinerjistik etki yarattığı gösterilmiştir.

Keywords

• Over kanseri
• cisplatin direnci
• VPS28
• CRISPR-Cas9

Supporting Institution

Bu çalışma, Türkiye Bilimsel ve Teknolojik Araştırma Kurumu (TÜBİTAK) tarafından 123Z193 Numaralı proje ile desteklenmiştir.

Ethical Statement

İn vitro çalışmalar yapıldığı için etik kurul onayına ihtiyaç duyulmamıştır.

Thanks

Projeye verdiği destekten ötürü TÜBİTAK’a teşekkürlerimizi sunarız. Ayrıca laboratuvar çalışmalarında bize yardımcı olan ve her türlü imkanı sağlayan Harran Üniversitesi Tıp Fakültesi Tıbbi Biyoloji Anabilim Dalı Öğretim Üyesi Arş. Gör. Dr. Neslihan Pınar Özateş’e, Harran Üniversitesi Tıp Fakültesi Fizyoloji Anabilim Dalı Öğretim Üyesi Dr. Öğr. Üyesi Seyhan Taşkın’a, Mehmet Akif Ersoy Üniversitesi Fen Edebiyat Fakültesi, Moleküler Biyoloji ve Genetik Bölümü Öğretim Üyesi Dr. Muzaffer Dükel’e teşekkür ederiz.

References

1. Solati A, Thvimi S, Khatami S, Shabaninejad Z, Malekzadegan Y, Alizadeh M, et.al. Non-coding RNAs in gynecologic cancer. Clinica Chimica Acta. 2023;551:117618.
2. Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians. 2024;74(3):229-63.
3. Lheureux S, Braunstein M, Oza AM. Epithelial ovarian cancer: evolution of management in the era of precision medicine. CA: a cancer journal for clinicians. 2019;69(4):280-304.
4. Wang L, Zhao X, Fu J, Xu W, Yuan J. The role of tumour metabolism in cisplatin resistance. Frontiers in molecular biosciences. 2021;8:691795.
5. Köberle B, Tomicic MT, Usanova S, Kaina B. Cisplatin resistance: preclinical findings and clinical implications. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer. 2010;1806(2):172-82.
6. Konstantinopoulos PA, Matulonis UA. Clinical and translational advances in ovarian cancer therapy. Nature Cancer. 2023;4(9):1239-57.
7. Zamwar UM, Anjankar AP. A etiology, epidemiology, histopathology, classification, detailed evaluation, and treatment of ovarian cancer. Cureus. 2022;14(10).
8. Makovec T. Cisplatin and beyond: molecular mechanisms of action and drug resistance development in cancer chemotherapy. Radiology and oncology. 2019;53(2):148-58.

9. Tchounwou PB, Dasari S, Noubissi FK, Ray P, Kumar S. Advances in our understanding of the molecular mechanisms of action of cisplatin in cancer therapy. Journal of experimental pharmacology. 2021;303-28.
10. Ranasinghe R, Mathai ML, Zulli A. Cisplatin for cancer therapy and overcoming chemoresistance. Heliyon. 2022;8(9).
11. Dasari S, Njiki S, Mbemi A, Yedjou CG, Tchounwou PB. Pharmacological effects of cisplatin combination with natural products in cancer chemotherapy. International journal of molecular sciences. 2022 ;23(3):1532.
12. Peng X, Yang L, Ma Y, Li Y, Li H. Focus on the morphogenesis, fate and the role in tumor progression of multivesicular bodies. Cell Communication and Signaling. 2020;18:1-5.
13. Liu CC, Liu YY, Zhou JF, Chen X, Chen H, Hu JH, et al. Cellular ESCRT components are recruited to regulate the endocytic trafficking and RNA replication compartment assembly during classical swine fever virus infection. PLoS Pathogens. 2022;18(2):e1010294.
14. Booth A, Marklew CJ, Ciani B, Beales PA. In vitro membrane remodeling by ESCRT is regulated by negative feedback from membrane tension. Iscience. 2019;15:173-84.
15. Minaidou A, Nicolaou P, Christodoulou K. Deregulation of LRSAM1 expression impairs the levels of TSG101, UBE2N, VPS28, MDM2 and EGFR. Plos one. 2019;14(2):e0211814.
16. Shi W, Hu D, Xing Y, Zhuo R, Lao Q, Liu H, et al. Deciphering the oncogenic role of VPS28 modulated by miR-491-5p in breast cancer cells using in silico and functional analysis. Frontiers in Molecular Biosciences. 2021;8:634183.
17. Hu X, Wang Y. Protocol to identify receptors of secreted proteins through CRISPR-Cas9 whole-genome screening technology. STAR protocols. 2024; 20;5(4):103315.
18. O’Shea AS. Clinical staging of ovarian cancer. Ovarian Cancer: Methods and Protocols. 2022:3-10.
19. Garg V, Oza AM. Treatment of ovarian cancer beyond PARP inhibition: current and future options. Drugs. 2023;83(15):1365-85.
20. Ashrafizadeh M, Zarrabi A, Hushmandi K, Hashemi F, Moghadam ER, et al. Lung cancer cells and their sensitivity/resistance to cisplatin chemotherapy: Role of microRNAs and upstream mediators. Cellular signalling. 2021;78:109871.
21. Davodabadi F, Sajjadi SF, Sarhadi M, Mirghasemi S, Hezaveh MN, Khosravi S, et al. Cancer chemotherapy resistance: Mechanisms and recent breakthrough in targeted drug delivery. European Journal of Pharmacology. 2023;958:176013.
22. Eslami M, Memarsadeghi O, Davarpanah A, Arti A, Nayernia K, Behnam B. Overcoming chemotherapy resistance in metastatic cancer: A comprehensive review. Biomedicines. 2024 ;12(1):183.
23. Kryczka J, Kryczka J, Czarnecka-Chrebelska KH, Brzeziańska-Lasota E. Molecular mechanisms of chemoresistance induced by cisplatin in NSCLC cancer therapy. International Journal of Molecular Sciences. 2021;22(16):8885.
24. Xue HY, Ji LJ, Gao AM, Liu P, He JD, Lu XJ. CRISPR-Cas9 for medical genetic screens: applications and future perspectives. Journal of medical genetics. 2016;53(2):91-7.
25. Ouyang Q, Liu Y, Tan J, Li J, Yang D, Zeng F, et al. Loss of ZNF587B and SULF1 contributed to cisplatin resistance in ovarian cancer cell lines based on Genome-scale CRISPR/Cas9 screening. American journal of cancer research. 2019;9(5):988.
26. Xu D, Liang SQ, Yang H, Bruggmann R, Berezowska S, Yang Z, et al. CRISPR screening identifies WEE1 as a combination target for standard chemotherapy in malignant pleural mesothelioma. Molecular cancer therapeutics. 2020;19(2):661-72.
27. Xu M, Ji J, Jin D, Wu Y, Wu T, Lin R, et al. The biogenesis and secretion of exosomes and multivesicular bodies (MVBs): Intercellular shuttles and implications in human diseases. Genes & Diseases. 2023;10(5):1894-907.
28. Hornung T, O’Neill HA, Logie S, Fowler KM, Duncan JE, Rosenow M, et al. ADAPT identifies an ESCRT complex composition that discriminates VCaP from LNCaP prostate cancer cell exosomes. Nucleic acids research. 2020;48(8):4013-27.
29. Fader CM, Colombo MI. Autophagy and multivesicular bodies: two closely related partners. Cell Death & Differentiation. 2009;16(1):70-8.
30. Mattissek C, Teis D. The role of the endosomal sorting complexes required for transport (ESCRT) in tumorigenesis. Molecular membrane biology. 2014;31(4):111-9.
31. Hurley JH. The ESCRT complexes. Critical reviews in biochemistry and molecular biology. 2010;45(6):463-87.
32. Schmidt O, Teis D. The ESCRT machinery. Current Biology. 2012;22(4):R116-20.
33. Kolmus K, Erdenebat P, Szymańska E, Stewig B, Goryca K, Derezińska-Wołek E, et al. Concurrent depletion of Vps37 proteins evokes ESCRT-I destabilization and profound cellular stress responses. Journal of cell science. 2021;134(1):jcs250951.
34. Zhao J, Cui X, Zhan Q, Zhang K, Su D, Yang S, et al. CRISPR-Cas9 library screening combined with an exosome-targeted delivery system addresses tumorigenesis/TMZ resistance in the mesenchymal subtype of glioblastoma. Theranostics. 2024;14(7):2835.
35. Tufan AB, Lazarow K, Kolesnichenko M, Sporbert A, von Kries JP, Scheidereit C. TSG101 associates with PARP1 and is essential for PARylation and DNA damage‐induced NF‐κB activation. The EMBO Journal. 2022;41(21):e110372