A proteomic approach to investigate the effects of extracellular matrix stiffness on endothelial cells

Reid, Steven Edward (2015) A proteomic approach to investigate the effects of extracellular matrix stiffness on endothelial cells. PhD thesis, University of Glasgow.

Abstract

One of the most common symptoms that people associate with cancer is the formation of a lump in soft tissue. This is a sign of abnormal tissue stiffness and can be derived from changes in the extracellular matrix (ECM). This physical property is known to be a consequence and promoter of tumour development. The abnormally high tumour stiffness is typically derived from increased crosslinking of the ECM. The aim of this project was to ascertain how the increased tissue stiffness, that is found within and around the tumour affects the endothelial cells that make up the vasculature. To do this I generated hydrogels representing an ECM stiffness of normal tissue and a tumour like ECM stiffness. Using high resolution mass spectrometry combined with stable isotope labelling of amino acids in cell culture (SILAC), I analysed the global proteome of primary human umbilical vein endothelial cells (HUVECs) cultured at each ECM stiffness. I found that many proteins involved in cell adhesion were upregulated, including N-Cadherin and CCN1. I demonstrated that stiffness induced CCN1 mediates both β-catenin activity and N-Cadherin expression. For the first time I show that CCN1, which is known as a secreted protein, can mediate these effects without exiting the cell, demonstrating an intracellular role for CCN1 in HUVECs. N-Cadherin is known to play a key role in trans-endothelial migration of cancer cells through the blood vessels, and I found that CCN1 induces cancer cell adhesion to HUVECs via N-Cadherin. This suggests that CCN1 can increase cancer cell metastasis by aiding the exit of cancer cells from the primary tumour and entry at distant sites. To test this hypothesis in vivo, we used a C57BL/6 Ccn1 loxP/loxP syngeneic melanoma model. To knock out Ccn1, a soluble form of Cre was utilised, which can be up taken into cells and has been shown previously to specifically target cells of the vasculature. Our results show that knocking out Ccn1 in the ECs decreased the number of circulating tumour cells and subsequent metastases, showing that Ccn1 loss in the vasculature can decrease cancer cell intravasation. Our work provides evidence of an unprecedented mechanism through which CCN1 in the stromal cells promotes invasion of cancer cells, which compliments the findings that CCN1 can induce invasion in the cancer cells. We provide further evidence that targeting CCN1 therapeutically in the clinic may decrease the spread of cancer from the perspective of the endothelial cells.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Due to copyright restrictions the full text of this thesis cannot be made available online. Access to the printed version is available once any embargo periods have expired.
• Keywords: Tumour stiffness, Endothelial cells, HUVECs, SILAC, proteomics, CYR61, CCN1, N-Cadherin, Transendothelial migration, intravasation, metastasis, cancer.
• Subjects: Q Science > Q Science (General); R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences > Beatson Institute of Cancer Research
• Supervisor's Name: Zanivan, Dr. Sara
• Date of Award: 2015
• Depositing User: Mr Steven Reid
• Unique ID: glathesis:2015-7098
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 26 Feb 2016 10:25
• Last Modified: 18 Feb 2019 15:56
• URI: https://theses.gla.ac.uk/id/eprint/7098