AbstractBreast cancer is the most common cancer in women in the UK. In over 90% of cases mortality is due to tumour recurrence and metastasis, which are hard to treat because most tumours become resistant to conventional therapies. During tumour progression, changes to the breast cancer microenvironment and extracellular matrix result in part from altered secretomes of neoplastic cells. There is a need to identify the molecular pathways that control these changes.
Previous research in Professor Adams’ laboratory showed that Protein Disulphide-Isomerase A3 (PDIA3) supports a pro-metastatic phenotype in breast cancer cell lines through effects on secreted proteins. In this study, pharmacological inhibition of PDIA3 in MDA-MB-231 invasive breast cancer cells is shown to decrease cell area, focal adhesions and to alter F-actin organisation. Formation of 3-dimensional spheroids was also decreased. By comparing the activities of conditioned media from control or PDIA3-inhibited MDA-MB-A231 cells, secreted PDIA3-dependent or heparin-binding proteins were shown to be necessary for cell spreading.
Comparative TMT-based quantitative proteomic analysis of the PDIA3-dependent, heparin- binding fractions of conditioned media identified that PDIA3 inhibition significantly decreased (≥ 2-fold) 80 proteins, 48 of which were extracellular proteins, including lysyl-oxidase like 2 (LOXL2) (p=0.003). However, pharmacological inhibition of LOXL2in MDA-MB-231 cells with β-aminopropionitrile did not affect 3-dimensional spheroid formation.
Analysis by Gene Ontology and other computational tools highlighted that 20 of these proteins are components of Extracellular Structure Organisation and 17 proteins are involved in Epithelial-to-Mesenchymal Transition. The extracellular proteins also showed pathway enrichment for Platelet Degranulation. The altered abundance of selected extracellular proteins was validated by immunoblotting.
These results demonstrate a PDIA3-dependence of secreted proteins of A231 cells that have known roles in a pro-metastatic phenotype. Future investigation into the role of PDIA3 in breast cancer microenvironment and metastasis may help direct new drug targets to prevent metastasis.
|Date of Award||28 Nov 2019|
|Supervisor||Jo Adams (Supervisor)|
Identification of Protein Disulphide-Isomerase A3 Dependent Proteins from the Secretome of MDA-MB-231 Breast Cancer Cells
Germon, A. L. (Author). 28 Nov 2019
Student thesis: Master's Thesis › Master of Science (MSc)