TGF-ß Signaling, Via Smad3, Mediates Notch Pathway-Induced Stemness and Epithelial to Mesenchymal Transition in Colon Cancer Cells

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Alexander G. Clark (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Late stage colorectal cancer (CRC) remains a challenging disease to treat due to several factors including stemness and epithelial to mesenchymal transition (EMT). Dysfunctional signaling pathways in CRC contribute to these phenomena, including the Notch and Transforming Growth Factor-Beta (TGF-[beta]) pathways. These pathways integrate external signals by cross-talking with one another to fine-tune cellular responses. We previously found that cells expressing constitutively active Notch1 also had increased expression of Smad3, an important member of the TGF-[beta] signaling pathway. Therefore, we hypothesized that the TGF-[beta] pathway, via Smad3, mediates the Notch-induced stemness and EMT observed in these cells. To test our hypothesis, we used the human colorectal carcinoma cell line HCT-116 and derivative cell lines GFP-v (a control cell line transfected with a retrovirus containing the green fluorescent protein), and ICN1 (a cell line transfected with a retrovirus containing both the green fluorescent protein and constitutively active intracytoplasmic Notch1). These cells were treated with different combinations of TGF-[beta]1 (a TGF-[beta] receptor ligand), DAPT (a [gamma]-secretase inhibitor), or SIS3 (a novel Smad3 inhibitor). Western blot procedures and statistical analysis were performed to determine the cross-talk between the Notch and TGF-[beta] pathways and to assess the effects of Smad3 stimulation and inhibition on Notch and its downstream targets. The role of Smad3 on colosphere formation was also determined using the aforementioned cell lines. Smad3 inhibition induced a decrease in Notch1 and Notch3 receptor expression. Additionally, SIS3 effectively inhibited key stemness and EMT markers such as CD44, Slug, Snail, and Hes1. Colosphere forming ability was also considerably reduced in cells treated with SIS3. These results indicate a key role of TGF-[beta] signaling in Notch1-induced tumorigenesis, and they also suggest a potential use for Smad3 inhibitors in combination with Notch1 inhibitors that are already in use for CRC treatments.

Additional Information

Publication

Thesis

Language: English

Date: 2023

Subjects

Notch signaling

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