Investigation into the Effect of Protein Disulphide Isomerase A3 on the Prom-Metastatic Phenotype of Breast Cancer Cells

Abstract

Breast cancer has the second highest incidence in women and has high mortality when metastasis occurs. Protein disulphide isomerase A3 (PDIA3) is an endoplasmic reticulum-resident protein that effects post-translational disulphide bond formation on substrates with cysteine-rich domains including extracellular matrix (ECM) proteins. PDIA3 protein is increased in invasive ductal carcinomas (IDC) of the breast, but little is known about its functions in breast cancer. The Objective of this M.Res. project was to investigate the role of PDIA3 in pro-metastatic activities of three human breast cancer cell lines in vitro.

In pilot immunohistochemical studies of IDC, PDIA3 was present in luminal and triple-negative IDC. PDIA3 was present in the breast cancer cell lines by immunoblot. Effects of pharmacological inhibition of PDIA3 were examined with regard to cell-substratum attachment, F-actin organization and migration by fluorescence microscopy or 2-dimensional directional cell migration. PDIA3 inhibition, compared to PDIA1 inhibition or solvent-control, resulted in reduced cell attachment and spreading of all the cell lines with time, and decreased migration. However, 5-fluorouracil or cyclophosphamide mediated cell killing was unaffected.

To explore if PDIA3-dependent secreted proteins contribute to the tumour microenvironment, ECM was isolated from control or PDIA3-inhibited breast cancer cells. PDIA3-inhibition did not affect attachment of ‘naïve” cells, but resulted in decreased spreading and altered F-actin organization. To explore possible PDIA3-dependent fibroblast-cancer communications, cells were treated with conditioned media (CM) from wild-type or Pdia3-/- mouse embryo fibroblasts (MEF). CM from Pdia3-/- MEF resulted in reduced spreading and F-actin organisation of all cell lines, compared to cells incubated with CM from WT-MEF.

These results contribute knowledge that PDIA3 functions in support of adhesion and migratory capacity of breast cancer cells, by processes involving secreted proteins of cancer cells or fibroblasts. Targeting of PDIA3 could have potential for inhibition of breast cancer metastasis.

Date of Award	23 Jan 2019
Original language	English
Awarding Institution	University of Bristol
Supervisor	Jon D Lane (Supervisor) & Jo Adams (Supervisor)