TARGETING CIRCULATING BREAST CANCER STEM CELLS VIA MTOR AND WNT INHIBITORS AFTER BONE METASTASIS

ÖZLEM ALTUNDAĞ ERDOĞAN

TARGETING CIRCULATING BREAST CANCER STEM CELLS VIA MTOR AND WNT INHIBITORS AFTER BONE METASTASIS

Tezin Türü: Doktora

Tezin Yürütüldüğü Kurum: Hacettepe Üniversitesi, Sağlık Bilimleri Enstitüsü, Kök Hücre Bilimleri A.B.D., Türkiye

Tezin Onay Tarihi: 2025

Tezin Dili: Türkçe

Öğrenci: ÖZLEM ALTUNDAĞ ERDOĞAN

Danışman: Betül Çelebi Saltık

Özet:

Cancer stem cells (CSCs), a rare subpopulation in breast tissue, are strongly associated with tumor progression, distant metastasis and secondary tumor formation. The aim of this thesis is to determine the changes in apoptosis, EMT and stemness properties of CSCs isolated from TNBC MDA-MB-231, after they are localized in a 3D bone-like structure and targeted with Temsirolimus and Niclosamide. A scaffold was fabricated using a 3D printer with PLA filaments and a PU cylindrical vessel-like structure was passed through the scaffold. The interior of the scaffold was coated with a composite material comprising 3.1 mg/mL collagen-I, 3.6 mg/mL poly-γ-glutamic acid, and 500 µM sodium metasilicate. BM-MSCs were seeded within this material and differentiated osteogenically for 21 days. Osteogenic differentiation was confirmed on days 14 and 21 through Alizarin Red staining and calcium quantification. The impacts of 5 µM Temsirolimus (p-mTOR) and 100 µM Niclosamide (p-STAT3) treatment for 6h were assessed in CSCs. Gene expression of CSCs were evaluated based on CD133 selection (MACS), for both drugs. Stemness (NANOG), EMT (MMP2, CXCR4), aassociated gene expression levels were decreased in the CD133⁺ groups of both drugs (p < 0.05). After Temsirolimus treatment, while Vimentin protein level was decreased, E-cadherin protein level was increased (p<0.05) within 3D model. Besides, apoptotic (Bax) and metastatic (Vimentin, ZEB1) protein levels were also decreased significantly after Niclosamide treatment as a result of Western Blot experiments. Additionally, PCR results indicated a significant downregulation of genes such as CXCR4, ABCG1, MMP2, and NANOG following Temsirolimus and Niclosamide treatment in both CD133+, CD133- cell groups. The increase in Bax protein, a key player in apoptosis induction, along with the decrease in the anti-apoptotic protein Bcl-2, suggests the activation of cell death mechanisms. Notably, its targeted impact on the CD44+/CD24- population suggests that these drugs could enhance the sensitivity of CSCs to treatment, preventing tumor recurrence. Keywords: Breast cancer stem cell, Temsirolimus, Niclosamide, collagen type I, poly-γ glutamic acid, sodium metasilicate.