Boric Acid Modulates Hippo, TGF-β/SMAD, and Epigenetic Signaling Pathways in MDA-MB-231 Triple-Negative Breast Cancer Cells

Öz

Background: The Hippo signaling pathway plays a critical role in the regulation of cell proliferation, apoptosis, and tumor progression, and its dysregulation has been strongly associated with triple-negative breast cancer (TNBC). Boric acid has been reported to exhibit anti-proliferative and pro-apoptotic effects in various cancer models; however, its effects on Hippo signaling and related molecular pathways in TNBC remain poorly understood. This study aimed to investigate the time-dependent effects of boric acid on Hippo pathway-related gene expression and associated protein markers in MDA-MB-231 breast cancer cells. Methods: MDA-MB-231 cells were treated with boric acid, and cell viability was assessed using the MTT assay. The half-maximal inhibitory concentration (IC₅₀) was determined and used for subsequent analyses. Expression levels of Hippo pathway-related genes (YAP, TAZ, TEAD1–4), TGF-β/SMAD pathway components (SMAD2, SMAD3), the chromatin remodeling factor CHD2, and extracellular matrix remodeling genes (COL1A1, MMP2, TIMP1–3) were evaluated by quantitative real-time PCR after 24, 48, and 72 h of treatment. Immunocytochemical analyses were performed to assess Cleaved Caspase-3, TNF-α, and TGFβR1 expression. Results: Boric acid reduced cell viability in a concentration-dependent manner, with an IC₅₀ value of approximately 240 μg/mL after 48 h of treatment. Gene expression analyses revealed significant downregulation of YAP and TEAD2 at 24 h, CHD2 at 48 h, and TAZ, YAP, SMAD2, and CHD2 at 72 h. In contrast, MMP2 expression was significantly increased at 72 h. Immunocytochemical evaluation demonstrated a time-dependent increase in Cleaved Caspase-3 immunoreactivity, moderate alterations in TNF-α expression, and reduced TGFβR1 staining intensity following boric acid treatment. Conclusions: These findings indicate that boric acid modulates multiple interconnected signaling pathways in TNBC cells, including Hippo, TGF-β/SMAD, apoptotic, inflammatory, and extracellular matrix remodeling pathways. The observed suppression of YAP/TAZ-associated signaling together with CHD2 downregulation suggests that both transcriptional and epigenetic mechanisms contribute to the anti-proliferative effects of boric acid. Boric acid may therefore represent a promising modulatory agent for targeting key regulatory networks in aggressive breast cancer subtypes.

Anahtar Kelimeler

• Boric acid
• Triple-negative breast cancer
• Hippo signaling pathway
• TGF-β/SMAD
• Apoptosis

Etik Beyan

This study did not require ethical approval, as it was based solely on commercially available established cell lines and did not involve human participants, human-derived samples or experimental animals.

Borik Asit, MDA-MB-231 Üçlü Negatif Meme Kanseri Hücrelerinde Hippo, TGF-β/SMAD ve Epigenetik Sinyal Yollarını Modüle Eder

Öz

Amaç: Hippo sinyal yolakları, hücre proliferasyonu, apoptoz ve tümör ilerlemesinin düzenlenmesinde kritik bir rol oynar ve bu yolakların düzensizliği, üçlü negatif meme kanseri (TNBC) ile güçlü bir şekilde ilişkilendirilmiştir. Borik asidin çeşitli kanser modellerinde anti-proliferatif ve pro-apoptotik etkiler gösterdiği bildirilmiştir; ancak, TNBC'de Hippo sinyalizasyonu ve ilgili moleküler yollar üzerindeki etkileri henüz tam olarak anlaşılamamaktadır. Bu çalışma, borik asidin MDA-MB-231 meme kanseri hücrelerinde Hippo yolu ile ilgili gen ekspresyonu ve ilişkili protein belirteçleri üzerindeki zaman bağımlı etkilerini araştırmayı amaçlamıştır. Yöntemler: MDA-MB-231 hücreleri borik asit ile tedavi edildi ve hücre canlılığı MTT testi kullanılarak değerlendirildi. Yarı maksimum inhibitör konsantrasyonu (IC50) belirlendi ve sonraki analizler için kullanıldı. Hippo yolu ile ilgili genlerin (YAP, TAZ, TEAD1–4), TGF-β/SMAD yolu bileşenlerinin (SMAD2, SMAD3), kromatin yeniden şekillendirme faktörü CHD2'nin ve ekstraselüler matris yeniden şekillendirme genlerinin (COL1A1, MMP2, TIMP1–3) ekspresyon seviyeleri, tedaviden sonra 24, 48 ve 72 saatlik sürelerde kantitatif gerçek zamanlı PCR ile değerlendirildi. İmmünositokimyasal analizler, Cleaved Caspase-3, TNF-α ve TGFβR1 ekspresyonunu değerlendirmek için yapıldı. Sonuçlar: Borik asit, hücre canlılığını konsantrasyona bağlı olarak azalttı ve 48 saatlik muamele sonrası yaklaşık 240 μg/mL'lik bir IC50 değeri gösterdi. Gen ekspresyon analizleri, 24 saatte YAP ve TEAD2'nin, 48 saatte CHD2'nin ve 72 saatte TAZ, YAP, SMAD2 ve CHD2'nin önemli ölçüde aşağı regüle olduğunu ortaya koydu. Buna karşılık, MMP2 ifadesi 72 saatte önemli ölçüde arttı. İmmünositokimyasal değerlendirme, borik asit muamelesinin ardından Cleaved Caspase-3 immünoreaktivitesinde zamanla artan bir artış, TNF-α ifadesinde orta dereceli değişiklikler ve TGFβR1 boyama yoğunluğunda azalma gösterdi. Sonuçlar: Bu bulgular, borik asidin TNBC hücrelerinde Hippo, TGF-β/SMAD, apoptoz, inflamatuar ve ekstraselüler matris yeniden şekillendirme yolları da dahil olmak üzere çok sayıda birbirine bağlı sinyal yollarını modüle ettiğini göstermektedir. Gözlemlenen YAP/TAZ ile ilişkili sinyal yolaklarının baskılanması ve CHD2'nin aşağı regülasyonu, hem transkripsiyonel hem de epigenetik mekanizmaların borik asidin anti-proliferatif etkilerine katkıda bulunduğunu düşündürmektedir. Bor asidi bu nedenle, agresif meme kanseri alt tiplerinde anahtar düzenleyici ağları hedeflemek için umut verici bir modülatör ajanı temsil edebilir.

Anahtar Kelimeler

• Borik asit
• Üçlü-negatif meme kanseri
• Hippo sinyal yolakları
• TGF-β/SMAD
• Apoptoz

Kaynakça

1. 1.Jemal A, Siegel R, Sung H, et al. Cancer statistics,2025. CA Cancer J Clin. 2025; 75(1): 10-45.
2. 2.Pratelli G, Carlisi D, Di Liberto D, et al. MCL1Inhibition Overcomes the Aggressiveness Featuresof Triple-Negative Breast Cancer MDA-MB-231Cells. Int J Mol Sci. 2023; 24(13): 11149.
3. 3.Arroyo-Crespo JJ, Armiñán A, Charbonnier D, et al.Characterization of triple-negative breast cancerpreclinical models provides functional evidence ofmetastatic progression. Int J Cancer. 2019; 145(8):2267-81.
4. 4.Cailleau R, Young R, Olive M, et al. Breast tumorcell lines from pleural effusions. J Natl Cancer Inst.1974; 53(3): 661-74.
5. 5.Huang Z, Yu P, Tang J. Characterization of Triple-Negative Breast Cancer MDA-MB-231 Cell SpheroidModel. Onco Targets Ther. 2020; 13: 5395-405.
6. 6.Pan D. The Hippo signaling pathway indevelopment and cancer. Dev Cell. 2010; 19(4): 491-505.
7. 7.Yu FX, Zhao B, Guan KL. Hippo pathway in organsize control, tissue homeostasis, and cancer. Cell.2015; 163(4): 811-28.
8. 8.Zanconato F, Cordenonsi M, Piccolo S. YAP/TAZ atthe roots of cancer. Cancer Cell. 2016; 29(6): 783-803.

9. 9.Harvey, K. F., Zhang, X., & Thomas, D. M. The Hippopathway and human cancer. Nature reviews. Cancer. 2013; 13(4), 246–57.
10. 10.Massague J. TGFbeta in cancer. Cell. 2008;134(2): 215-30.
11. 11.Ikushima H, Miyazono K. TGFbeta signalling: acomplex web in cancer progression. Nat Rev Cancer.2010; 10(6): 415-24.
12. 12.Varelas X. The Hippo pathway effectors TAZ andYAP in development, homeostasis and disease.Development. 2014; 141: 1614-26.
13. 13.Liu H, Sun M, Wu N, et al. TGF-β/Smads signalingpathway, Hippo-YAP/TAZ signaling pathway, andVEGF: their mechanisms and roles in vascularremodeling related diseases. Immun Inflamm Dis.2023; 11:e1060.
14. 14. Zhang J, Wu H, Ren X, et al. Hippo/YAP signaling's multifaceted crosstalk in cancer. Front Cell Dev Biol.2025; 13:1595362.
15. 15.Li J, Chen B, Huang Y, et al. TNF-α modulates cellproliferation via SOX4/TGF-β/Smad signaling inbenign prostatic hyperplasia. Cell Death Dis. 2025;16:472.
16. 16.Pizzorno L. Nothing Boring About Boron. IntegrMed (Encinitas). 2015; 14(4): 35-48.
17. 17.Scorei RI, Popa R Jr. Boron-containingcompounds as preventive and chemotherapeuticagents for cancer. Anticancer Agents Med Chem.2010; 10(4): 346-51.
18. 18.Marfella CG, Imbalzano AN. The CHD family ofchromatin remodelers. Mutat Res. 2007; 618 (1-2):30-40.
19. 19.Sarıbaş GS, Turna Saltoğlu G, Yalçın Azarkan S, etal. Effects of irinotecan (CPT-11), ellagic acid andcombination of both on HeLa cells in 2D and 3Dculture. Cyprus J Med Sci. 2023; 8: 173-83.
20. 20.Akalın H, Bilgiç B, Avcil S, et al. Low expressionlevels of MAOA and TPH1 genes may represent riskfactors in boys with autism spectrum disorder-acase-control study. Int J Dev Neurosci. 2025; 85:e70073.
21. 21.Bustin SA, Benes V, Garson JA, et al. The MIQEguidelines: minimum information for publication ofquantitative real-time PCR experiments. Clin Chem.2009; 55: 611-22.
22. 22.Schou JS, Jansen JA, Aggerbeck B. Humanpharmacokinetics and safety of boric acid. ArchToxicol Suppl. 1984; 7: 232-5.
23. 23.Bayram D, Özgöçmen M, Çelik DA, et al. Doesboric acid inhibit cell proliferation on MCF-7 andMDA-MB-231 cells in monolayer and spheroidcultures by using apoptosis pathways? Biol TraceElem Res. 2024; 202: 2008-21.
24. 24.Elsori D, Pandey P, Alshaikh K, et al. TargetingHippo-YAP/TAZ signaling pathway: an updatedreview demonstrating the therapeutic potential ofkey plant derived anticancer compounds. FrontPharmacol. 2026; 16: 1743102.
25. 25.Li X, Zhuo S, Zhuang T, et al. YAP inhibits ERα andER+ breast cancer growth by disrupting a TEAD-ERαsignaling axis. Nat Commun. 2022; 13: 3075.
26. 26.Labibi B, Bashkurov M, Wrana JL, et al. Modelingthe control of TGF-β/Smad nuclear accumulation bythe Hippo pathway effectors TAZ/YAP. iScience.2020; 23(8): 101416.
27. 27.Dou H, Yu PY, Liu YQ, et al. Recent advances incaspase-3, breast cancer, and traditional Chinesemedicine: a review. J Chemother. 2024; 36(5): 370-88.
28. 28.Ciurescu S, Buciu V, Serban D, et al. Role ofcytokines in breast cancer: a systematic review andmeta-analysis. Biomedicines. 2025; 13(9): 2203.
29. 29.Liu JC, Ferreira CG, Yusufzai T. Human CHD2 is achromatin assembly ATPase regulated by itschromo- and DNA-binding domains. J Biol Chem.2015; 290(1): 25-34.
30. 30.Siggens L, Cordeddu L, Ronnerblad M, et al.Transcription-coupled recruitment of human CHD1and CHD2 influences chromatin accessibility andhistone H3 and H3.3 occupancy at active chromatinregions. Epigenetics Chromatin. 2015; 8: 4.