CDK2 Depletion Impairs Tumour Growth in the 3D Luminal Breast Cancer Model via CRISPR-CAS9 Gene Editing

Abstract

Objective: Cyclin-Dependent Kinase 2 (CDK2) is a key regulator of the G1/S transition and genomic stability. While recent studies have linked CDK2 activity to therapy resistance in hormone receptor-positive breast cancer, its functional role under tumourrelevant conditions remains unclear. This study aims to elucidate the context-specific requirement of CDK2 in luminal breast cancer models. Material and Methods: CDK2 was knocked out in T47D cells using CRISPR-Cas9. Phenotypic effects were evaluated in both 2D monolayer and 3D spheroid cultures. Quantitative PCR validated gene knockout. Colony formation and GFP-based competition assays assessed 2D proliferation, while 3D spheroid size was quantified using ImageJ. Results: CDK2 depletion resulted in a 40% reduction in 2D colony formation and a 50% decrease in spheroid size. The fitness disadvantage was more pronounced in 3D cultures, suggesting increased dependency on CDK2 in complex microenvironments. Conclusion: CDK2 plays a critical role in sustaining tumour growth and structural organization, particularly in 3D environments that mimic the tumour microenvironment. These findings highlight CDK2 as a potential therapeutic target in luminal breast cancer, especially in the context of anti-oestrogen resistance.

Keywords

• CDK2
• Luminal breast cancer
• 3D culture
• CRISPR-Cas9

Project Number

na

Ethical Statement

We confirm that our study does not involve research conducted on either animals or humans. Therefore, ethical approval was not required. Please do not hesitate to contact me should you need any further information.

CDK2 Geninin CRISPR-CAS9 ile Hedeflenmesi 3B Luminal Meme Kanseri Modellerinde Tümöral Büyümeyi Baskılar

Abstract

Amaç: Siklin-Bağımlı Kinaz 2 (CDK2), G1/S geçişi ve genomik stabilitenin önemli bir düzenleyicisidir. Son çalışmalar, hormon reseptörü pozitif meme kanserinde CDK2 aktivitesini tedavi direnciyle ilişkilendirse de, tümörle ilişkili koşullarda fonksiyonel rolü net değildir. Bu çalışmanın amacı, luminal meme kanseri modellerinde CDK2’ye özgü bağlama-bağımlı gerekliliği ortaya koymaktır. Gereç ve Yöntemler: CDK2, CRISPR-Cas9 sistemi kullanılarak T47D hücrelerinde devre dışı bırakıldı. Fenotipik etkiler hem 2D monolayer hem de 3D sferoid kültür modellerinde değerlendirildi. Gen silinmesi kantitatif PCR ile doğrulandı. 2D proliferasyon kolon oluşumu ve GFP-temelli rekabet analizleriyle incelendi. 3D sferoid boyutları ImageJ ile ölçüldü. Bulgular: CDK2 silinmesi, 2D kolon oluşumunda yaklaşık %40, 3D sferoid boyutunda ise yaklaşık %50 oranında azalmaya neden oldu. Özellikle 3D ortamda CDK2’ye olan bağımlılık daha belirgin hale geldi. Sonuç: CDK2, özellikle tümör mikroçevresini taklit eden 3D yapılarda tümöral büyüme ve yapısal bütünlüğün korunmasında kritik rol oynamaktadır. Bulgular, CDK2’nin luminal meme kanserinde, özellikle anti-östrojen direnci bağlamında potansiyel bir terapötik hedef olabileceğini göstermektedir.

Keywords

• CDK2
• Luminal meme kanseri
• 3B hücre kültürü
• CRISPR-Cas9

Project Number

na

Ethical Statement

We confirm that our study does not involve research conducted on either animals or humans. Therefore, ethical approval was not required. Please do not hesitate to contact me should you need any further information.

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