Protein disulfide isomerase A3 activity promotes extracellular accumulation of proteins relevant to basal breast cancer outcomes in human MDA-MB-A231 breast cancer cells

Metastasis and recurrence of breast cancer remain major causes of patient mortality, and there is an ongoing need to identify new therapeutic targets relevant to tumor invasion. Protein disulfide isomerase A3 (PDIA3) is a disulfide oxidoreductase and isomerase of the endoplasmic reticulum that has known extracellular substrates and has been correlated with aggressive breast cancers. We show that either prior PDIA3 inhibition by the disulfide isomerase inhibitor 16F16 or depletion of heparin-binding proteins strongly reduces the activity of conditioned medium (CM) of MDA-MB-231 human breast cancer cells to support promi-gratory cell spreading and F-actin organization by newly adherent MDA-MB-231 cells. Quantitative proteomics to investigate effects of 16F16 inhibition on heparin-binding proteins in the CM of MDA-MB-231 cells identified 80 proteins reproducibly decreased at least twofold (at q < 0.05) after 16F16 treatment. By Gene Ontology analysis, many of these have roles in extracel-lular matrix (ECM) structure and function and cell adhesion; ribosomal proteins that also correlate with extracellular vesicles were also identified. Protein-protein interaction analysis showed that many of the extracellular proteins have known network interac-tions with each other. The predominant types of disulfide-bonded domains in the extracellular proteins contained b-hairpin folds, with the knottin fold the most common. From human breast cancer data sets, the extracellular proteins were found to correlate specifically with the basal subtype of breast cancer and their high expression in tumors correlated with reduced distant metasta-sis-free survival. These data provide new evidence that PDIA3 may be a relevant therapeutic target to alter properties of the ECM-associated microenvironment in basal breast cancer.