Ponatinib miRNA İfadelerini Düzenleyerek Meme Kanseri Hücrelerini Hedefler

Öz

Meme kanseri kadınlarda en yaygın gözlenen kanser türüdür. Mevcut tedavilerin düşük seçicilik ya da zamanla oluşan ilaç direnci gibi eksiklerini giderebilecek yeni stratejilerin belirlenmesine ihtiyaç vardır. Çalışmamızda, çoklu hedefli bir tirozin kinaz inhibitörü olan ponatinibin meme kanseri hücreleri üzerindeki anti-kanser etkisini değerlendirmeyi ve ponatinib yanıtında yer alan miRNA'ların biyoinformatik yaklaşım ile sinyal yolaklarındaki potansiyel işlevini tanımlamayı hedefledik. Bu amaçla, MCF-7 hücrelerinde ponatinibin sitotoksik etkileri xCELLigence ile gerçek-zamanlı olarak belirlendi. Ponatinib uygulaması sonrasında apoptoz, proliferasyon hızı, hücre döngüsündeki değişimler akım sitometriyle, miRNA'ların ifadelerindeki düzenlenmeler qRT-PCR ile değerlendirildi. İfadelerinde anlamlı değişim belirlenen miRNA’ların ilişkili olduğu olası mRNA’lar ve sinyal yolakları KEGG yolak analizi ile tanımlandı. Ponatinibin MCF-7 hücreleri üzerinde sitotoksik etkiye sahip olduğu (IC50: 4,59 μM) belirlendi. Ponatinib uygulaması ile MCF-7 hücrelerinde anlamlı olarak apoptozun indüklendiği, proliferasyonun baskılandığı ve hücre döngüsünün G0/G1, S evrelerinde durakladığı belirlendi. Ayrıca, let-7a-5p, miR-29a-3p, miR-7-5p, miR-125b-5p, miR-212-3p ifadelerinde artış (p<0,05), miR-210-3p, miR-19b-3p, miR-140-5p, miR-181b-5p, miR-155-5p, miR-223-3p, miR-141-3p, miR-21-5p ifadelerinde azalma olduğu (p<0,05), miR-19a-3p ifadesinin ise tamamen baskılandığı belirlendi. Biyoinformatik analizler ile, ifadesi değişen miRNA’ların kanserde proteoglikanlar, Hippo, p53, TGF-beta, kanser-ilişkili, PI3K-Akt, prolaktin, hücre döngüsü, östrojen, mTOR sinyal yolakları ile ilişkili olduğu ortaya koyuldu. Ponatinib uygulaması meme kanseri hücrelerinde apoptozu indükleyerek, proliferasyonu baskılayarak ve hücre döngüsünü durdurarak güçlü anti-kanser aktivite sergilemiştir. Ponatinibin belirlenen anti-kanser etkilerinde miRNA’ların rolleri olabileceği gösterilmiştir. Olası miRNA-mRNA etkileşimleri ile meme kanserindeki hedef sinyal yolaklarının tanımlanması ışığında, ponatinibin tek başına veya diğer tedavilerle kombinasyon halinde meme kanseri tedavisi için potansiyel bir strateji olabileceği görüşündeyiz.

Anahtar Kelimeler

• Meme kanseri
• Ponatinib
• miRNA

Ponatinib Targets Breast Cancer Cells by Regulating miRNA Expressions

Öz

Breast cancer is the most common cancer in women. There is a need to identify novel strategies that can overcome the failure of existing treatments, such as low selectivity or drug-resistance. In our study, we aimed to evaluate the anti-cancer effect of ponatinib, a multi-targeted tyrosine kinase inhibitor, on breast cancer cells and to define the potential function of miRNAs involved in the ponatinib response in signaling pathways with bioinformatics analysis. For this purpose, the cytotoxic effects of ponatinib on MCF-7 cells were measured in real-time by xCELLigence. After ponatinib treatment, changes in apoptosis, proliferation, cell-cycle regulation were evaluated by flow cytometry, and the regulations of miRNAs were evaluated by qRT-PCR. mRNAs and signaling pathways interacted with miRNAs that significantly changed were predicted by KEGG pathway analysis. It was determined that ponatinib had a cytotoxic effect (IC50: 4.59 μM) on MCF-7 cells. After ponatinib treatment, it was determined that apoptosis was induced, proliferation was suppressed and the cell-cycle was arrested at the G0/G1 and S phases significantly in MCF-7 cells. Ponatinib up-regulated let-7a-5p, miR-29a-3p, miR-7-5p, miR-125b-5p, miR-212-3p expressions (p<0,05). Following the ponatinib exposure, miR-210-3p, miR-19b-3p, miR-140-5p, miR-181b-5p, miR-155-5p, miR-223-3p, miR-141-3p, miR-21-5p were down-regulated (p<0,05) while the expression of miR-19a-3p was completely suppressed. Bioinformatics analyzes revealed that ponatinib-regulated miRNAs are associated with proteoglycans in cancers, Hippo, p53, TGF-beta, cancer-related, PI3K-Akt, prolactin, cell-cycle, estrogen, mTOR signaling pathways. Ponatinib treatment exhibited potent anti-cancer activity by inducing apoptosis, suppressing proliferation and blocking the cell-cycle progression in breast cancer cells. It has been shown that miRNAs play roles in the anti-cancer efficiency of ponatinib. In light of the identification of target signaling pathways based on the predicted miRNA-mRNA interactions, we believe that ponatinib alone or in combination with other treatments may be a potential strategy for the treatment of breast cancer.

Anahtar Kelimeler

• Breast cancer
• Ponatinib
• miRNA

Kaynakça

1. 1. Arya GC, Kaur K, Jaitak V. Isoxazole derivatives as anticancer agent: A review on synthetic strategies, mechanism of action and SAR studies. Eur J Med Chem 2021;221:113511.
2. 2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69(1):7-34.
3. 3. Lumachi F, Santeufemia DA, Basso SM. Current medical treatment of estrogen receptor-positive breast cancer. World J Biol Chem 2015;6(3):231-9.
4. 4. Eric I, Petek Eric A, Kristek J, Koprivcic I, Babic M. Breast Cancer in Young Women: Pathologic and Immunohistochemical Features. Acta Clin Croat 2018;57(3):497-502.
5. 5. Dickson C, Spencer-Dene B, Dillon C, Fantl V. Tyrosine kinase signalling in breast cancer: fibroblast growth factors and their receptors. Breast Cancer Res 2000;2(3):191-6.
6. 6. Jitariu AA, Raica M, Cimpean AM, Suciu SC. The role of PDGF-B/PDGFR-BETA axis in the normal development and carcinogenesis of the breast. Crit Rev Oncol Hematol 2018;131:46-52.
7. 7. Singh DD, Yadav DK. TNBC: Potential Targeting of Multiple Receptors for a Therapeutic Breakthrough, Nanomedicine, and Immunotherapy. Biomedicines 2021;9(8).
8. 8. Huang WS, Metcalf CA, Sundaramoorthi R, Wang Y, Zou D, Thomas RM, ve ark. Discovery of 3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methylpiperazin-1-y l)methyl]-3-(trifluoromethyl)phenyl}benzamide (AP24534), a potent, orally active pan-inhibitor of breakpoint cluster region-abelson (BCR-ABL) kinase including the T315I gatekeeper mutant. J Med Chem 2010;53(12):4701-19.

9. 9. Zhou T, Commodore L, Huang WS, Wang Y, Thomas M, Keats J, ve ark. Structural mechanism of the Pan-BCR-ABL inhibitor ponatinib (AP24534): lessons for overcoming kinase inhibitor resistance. Chem Biol Drug Des 2011;77(1):1-11.
10. 10. O'Hare T, Shakespeare WC, Zhu X, Eide CA, Rivera VM, Wang F, ve ark. AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance. Cancer Cell 2009;16(5):401-12.
11. 11. Musumeci F, Greco C, Grossi G, Molinari A, Schenone S. Recent Studies on Ponatinib in Cancers Other Than Chronic Myeloid Leukemia. Cancers (Basel) 2018;10(11).
12. 12. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116(2):281-97.
13. 13. Iorio MV, Croce CM. MicroRNAs in cancer: small molecules with a huge impact. J Clin Oncol 2009;27(34):5848-56.
14. 14. Esquela-Kerscher A, Slack FJ. Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer 2006;6(4):259-69.
15. 15. Wong JS, Cheah YK. Potential miRNAs for miRNA-Based Therapeutics in Breast Cancer. Noncoding RNA 2020;6(3).
16. 16. Vlachos IS, Zagganas K, Paraskevopoulou MD, Georgakilas G, Karagkouni D, Vergoulis T, ve ark. DIANA-miRPath v3.0: deciphering microRNA function with experimental support. Nucleic Acids Res 2015;43(W1):W460-6.
17. 17. Okcanoğlu TB, Kayabaşı Ç, Süslüer SY, Gündüz C. The Relationship Between Long Non-Coding RNA Expressions and Ponatinib in Breast Cancer. Cyprus Journal of Medical Sciences 2019;4(2):125-30.
18. 18. Shao W, Li S, Li L, Lin K, Liu X, Wang H, ve ark. Chemical genomics reveals inhibition of breast cancer lung metastasis by Ponatinib via c-Jun. Protein Cell 2019;10(3):161-77.
19. 19. Kim S, You D, Jeong Y, Yoon SY, Kim SA, Lee JE. Inhibition of platelet-derived growth factor receptor synergistically increases the pharmacological effect of tamoxifen in estrogen receptor alpha positive breast cancer. Oncol Lett 2021;21(4):294.
20. 20. Kim S, You D, Jeong Y, Yoon SY, Kim SA, Lee JE. Inhibition of platelet-derived growth factor C and their receptors additionally increases doxorubicin effects in triple-negative breast cancer cells. Eur J Pharmacol 2021;895:173868.
21. 21. Bauer K, Berger D, Zielinski CC, Valent P, Grunt TW. Hitting two oncogenic machineries in cancer cells: cooperative effects of the multi-kinase inhibitor ponatinib and the BET bromodomain blockers JQ1 or dBET1 on human carcinoma cells. Oncotarget 2018;9(41):26491-506.
22. 22. Fridman JS, Lowe SW. Control of apoptosis by p53. Oncogene 2003;22(56):9030-40.
23. 23. Zhao B, Chen YG. Regulation of TGF-beta Signal Transduction. Scientifica (Cairo) 2014;2014:874065.
24. 24. Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2(7):489-501.
25. 25. Dong S, Ma M, Li M, Guo Y, Zuo X, Gu X, ve ark. LncRNA MEG3 regulates breast cancer proliferation and apoptosis through miR-141-3p/RBMS3 axis. Genomics 2021;113(4):1689-704.
26. 26. Muluhngwi P, Krishna A, Vittitow SL, Napier JT, Richardson KM, Ellis M, ve ark. Tamoxifen differentially regulates miR-29b-1 and miR-29a expression depending on endocrine-sensitivity in breast cancer cells. Cancer Lett 2017;388:230-8.
27. 27. Shi Y, Luo X, Li P, Tan J, Wang X, Xiang T, ve ark. miR-7-5p suppresses cell proliferation and induces apoptosis of breast cancer cells mainly by targeting REGgamma. Cancer Lett 2015;358(1):27-36.
28. 28. Lee S, Lee H, Bae H, Choi EH, Kim SJ. Epigenetic silencing of miR-19a-3p by cold atmospheric plasma contributes to proliferation inhibition of the MCF-7 breast cancer cell. Sci Rep 2016;6:30005.
29. 29. Jin J, Sun Z, Yang F, Tang L, Chen W, Guan X. miR-19b-3p inhibits breast cancer cell proliferation and reverses saracatinib-resistance by regulating PI3K/Akt pathway. Arch Biochem Biophys 2018;645:54-60.
30. 30. Yao A, Xiang Y, Si YR, Fan LJ, Li JP, Li H, ve ark. PKM2 promotes glucose metabolism through a let-7a-5p/Stat3/hnRNP-A1 regulatory feedback loop in breast cancer cells. J Cell Biochem 2019;120(4):6542-54.
31. 31. Sun H, Ding C, Zhang H, Gao J. Let7 miRNAs sensitize breast cancer stem cells to radiationinduced repression through inhibition of the cyclin D1/Akt1/Wnt1 signaling pathway. Mol Med Rep 2016;14(4):3285-92.
32. 32. Li Z, Li Y, Wang X, Liang Y, Luo D, Han D, ve ark. LINC01977 Promotes Breast Cancer Progression and Chemoresistance to Doxorubicin by Targeting miR-212-3p/GOLM1 Axis. Front Oncol 2021;11:657094.
33. 33. Li X, Zou ZZ, Wen M, Xie YZ, Peng KJ, Luo T, ve ark. ZLM-7 inhibits the occurrence and angiogenesis of breast cancer through miR-212-3p/Sp1/VEGFA signal axis. Mol Med 2020;26(1):109.
34. 34. Wu Y, Li M, Lin J, Hu C. Hippo/TEAD4 signaling pathway as a potential target for the treatment of breast cancer. Oncol Lett 2021;21(4):313.
35. 35. Zhu Y, Bo H, Chen Z, Li J, He D, Xiao M, ve ark. LINC00968 can inhibit the progression of lung adenocarcinoma through the miR-21-5p/SMAD7 signal axis. Aging (Albany NY) 2020;12(21):21904-22.
36. 36. Tzanakakis G, Giatagana EM, Kuskov A, Berdiaki A, Tsatsakis AM, Neagu M, ve ark. Proteoglycans in the Pathogenesis of Hormone-Dependent Cancers: Mediators and Effectors. Cancers (Basel) 2020;12(9).
37. 37. Subramani R, Nandy SB, Pedroza DA, Lakshmanaswamy R. Role of Growth Hormone in Breast Cancer. Endocrinology 2017;158(6):1543-55.