Elucidating the role of the TGF beta superfamily in metastatic spread of colorectal cancer

Sonthi, Natthamon (2025) Elucidating the role of the TGF beta superfamily in metastatic spread of colorectal cancer. PhD thesis, University of Glasgow.

Abstract

Colorectal cancer (CRC) is an aggressive disease and the leading cause of cancer death, characterised by high heterogeneity and various risk factors related to its etiology (1-3). During carcinogenesis, adenomatous polyps, which represent most premalignant lesions (85-90% of sporadic CRC), can develop into CRC (4-6). About 20-25% of CRC patients are diagnosed with metastatic disease, which is associated with poorer survival rates. CRC can spread to various tissues, including lymph nodes, liver, lungs, peritoneum, bones, and the central nervous system (7). The liver is the most common site for detecting metastatic CRC and is involved in 25% to 50% of cases. However, a rare form of CRC with bone metastasis occurs in 3% to 7% of patients; these individuals often have worse survival outcomes, with shorter survival times and limited treatment options (8-10). TGF-β and BMP signalling pathways are crucial mechanisms in tissue homeostasis, promoting cell proliferation and differentiation during crypt formation in the intestine (11-15). Dysregulation of these pathways in intestinal cells can impair their tumour-suppressing functions and facilitate tumour development (16-18). In particular, mutations in TGFBR2, BMPR1A, and SMAD4 have been identified as contributing to CRC carcinogenesis (2, 3, 19-23). Patients with consensus molecular subtype (CMS) 4, categorised based on gene expression signature, are associated with prominent activation of TGF-β, along with stromal infiltration, epithelial to mesenchymal
transition (EMT), and angiogenesis, leading to poorer survival outcomes (19).

In silico analysis of gene expression levels of TGF-β and BMP signalling components across CRC cell lines using the DepMap database, along with examination of key proteins and their phosphorylation statuses through Western blots, revealed that ligand activation occurs at various levels, with notable activation of upstream receptors such as TGFBR2, ACVR1B, and BMPR1A. Increased expression of signal transduction genes, including SMAD1, SMAD2, SMAD3, and SMAD5, was also observed. Interestingly, although phosphorylation of SMAD1/5/8 was detected in SW620 cell lines (a metastatic CRC cell line), SMAD4, a central element in signal transduction, was found to be downregulated. Apart from the differential expression of TGF-β and BMP signalling in CRC, the investigation of gene expression in these pathways within disease-free bone marrow (BM) cells using the Stemformatics database was also investigated to provide some insight into whether this morphogenic pathway is involved in metastatic spread to the bone. Here we demonstrated high levels of TGF-β and BMP signalling pathways in the bone marrow associated cells, indicating maintenance of homeostasis and serving as a baseline reference for further research.

Assessment of epithelial SMAD4 expression in colorectal polyps using immunohistochemistry (IHC) and digital weighted histoscoring with QuPath revealed that low SMAD4 levels in adenomatous polyps correlated with higher grades of dysplasia, different histological subtypes, the presence of metachronous polyps, and served as a prognostic marker. This marker indicated an increased risk of developing metachronous polyps, particularly in the tubulovillous polyp subtype. Additionally, transcriptomic analysis of tubulovillous polyps showed that upregulation of genes involved in protein deubiquitination occurs in polyps with
low SMAD4 levels, along with a likely enrichment in tyrosine metabolism, PPAR signalling, arginine and proline metabolism, leukocyte transendothelial migration, and basal cell carcinoma. In CRC, lower epithelial SMAD4 expression was strongly associated with higher tumour stages and increased tumour stroma. Moreover, lower SMAD4 expression in CRC tumours had prognostic significance, predicting decreased cancer-specific survival in CRC patients, especially in right-sided tumours. A combination of tumour SMAD4 levels and stroma percentages suggested that the worst survival outcome was in patients with low SMAD4
expression in the tumour and high stroma content. Transcriptomic analysis also identified downregulation of SOD3 and enrichment of aminopeptidase activity in this group.

Differential expression patterns at gene and protein levels, along with phosphorylation activity, were observed across the complex crosstalk of MAPK/ERK, WNT/β-catenin, TGF-β/BMP, and PI3K signalling pathways in CRC cell lines. However, the mechanisms regulating SMAD4 activity in CRC remain unclear.Testing the TGF-β and BMP signalling inhibitor (LDN-212854) in combination with the standard chemotherapy (Fluoropyrimidine; 5-FU) showed synergistic effects on CRC cell viability, cell cycle arrest, cell proliferation, and cell recovery in
an in vitro 2D study. Conditioned media from BM and hepatic cells influence changes in CRC behaviour. The 3D bioprinted SW620 spheroids in 2% alginate and 8% gelatin hydrogel supported physiological interactions, spheroid survival and growth, and were used for drug screening, demonstrating efficacy of the LDN-212854/5-FU combination in a more mechanophysical 3D system.

Further development of this in vitro 3D model to become multicell by incorporating metastatic CRC culture with BM and hepatic niches would enhancefuture CRC research and drug discovery.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by scholarship from Chulabhorn Royal Academy, Thailand, and the Scholar Team from the Office of Educational Affairs at the Royal Thai Embassy.
• Subjects: Q Science > QR Microbiology; R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Funder's Name: Office of Educational Affairs at the Royal Thai Embassy
• Supervisor's Name: Wheadon, Professor Helen, Edwards, Professor Joanne and Jørgensen, Dr. Heather
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85616
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 24 Nov 2025 14:51
• Last Modified: 24 Nov 2025 14:56
• Thesis DOI: 10.5525/gla.thesis.85616
• URI: https://theses.gla.ac.uk/id/eprint/85616