Protein Disulphide Isomerase A1 Is Involved in the Regulation of Breast Cancer Cell Adhesion and Transmigration via Lung Microvascular Endothelial Cells

Abstract

Cancer cell cross-talk with the host endothelium plays a crucial role in metastasis, but the underlying mechanisms are still not fully understood. We studied the involvement of protein disulphide isomerase A1 (PDIA1) in human breast cancer cell (MCF-7 and MDA-MB-231) adhesion and transendothelial migration. For comparison, the role of PDIA1 in proliferation, migration, cell cycle and apoptosis was also assessed. Pharmacological inhibitor, bepristat 2a and PDIA1 silencing were used to inhibit PDIA1. Inhibition of PDIA1 by bepristat 2a markedly decreased the adhesion of breast cancer cells to collagen type I, fibronectin and human lung microvascular endothelial cells. Transendothelial migration of breast cancer cells across the endothelial monolayer was also inhibited by bepristat 2a, an effect not associated with changes in ICAM-1 expression or changes in cellular bioenergetics. The silencing of PDIA1 produced less pronounced anti-adhesive effects. However, inhibiting extracellular free thiols by non-penetrating blocker p-chloromercuribenzene sulphonate substantially inhibited adhesion. Using a proteomic approach, we identified that β1 and α2 integrins were the most abundant among all integrins in breast cancer cells as well as in lung microvascular endothelial cells, suggesting that integrins could represent a target for PDIA1. In conclusion, extracellular PDIA1 plays a major role in regulating the adhesion of cancer cells and their transendothelial migration, in addition to regulating cell cycle and caspase 3/7 activation by intracellular PDIA1. PDIA1-dependent regulation of cancer-endothelial cell interactions involves disulphide exchange and most likely integrin activation but is not mediated by the regulation of ICAM-1 expression or changes in cellular bioenergetics in breast cancer or endothelial cells.

Keywords: adhesion; disulphide exchange; protein disulphide isomerase A1; transendothelial migration.

Figure 1

Effect of PDIA1 inhibition on clonogenic capacity of breast cancer cells. The clonogenicity of MCF-7 (A) and MDA-MB-231 (B) cells treated with bepristat 2a as well as MCF-7 (C) and MDA-MB-231 (D) cells transduced with lentiviral vectors carrying short hairpin RNA (shRNA) against PDIA1 or negative sequence after selection with puromycin in regarding to wild type cell line. Data represent the means ± SD with points for individual measurements, the representative photos are also presented. Statistical analysis was calculated using parametric one-way ANOVA followed by Dunnett’s multiple comparisons test (* p = 0.05, ** p = 0.01, *** p = 0.001, **** p = 0.0001).

Figure 2

Effects of PDIA1 inhibition on wound-healing and migration of breast cancer cells and endothelial cells. ECIS Wound-Healing Assay of hLMVEC, MCF-7 and MDA-MB-231 cells and breast cancer sublines with silencing of PDIA1 (shN, shPDIA1-1 and shPDIA1-3). Real time tracings in hLMVECs (A), MCF-7 (C) and MDA-MB-231 (E) cell lines after addition of bepristat 2a at various concentrations (1, 10, 30 or 50 µM) and after PDIA1 silencing in MCF-7 (G) and MDA-MB-231 (I) cell lines. Area under the curve boxplots represent AUC quantitation of changes in migration rate of bepristat 2a-treated hLMVECs (B), MCF-7 (D) and MDA-MB-231 (F) cell lines versus non-treated controls as well as MCF-7 (H) or MDA-MB-231 (J) sublines transduced against PDIA1 (shPDIA1-1, shPDIA1-3) or wild type cells regarding to negative sequence (shN). The line graphs and AUC boxplots represent mean ± SD of three independent experiments. Statistical analysis was calculated using parametric one-way ANOVA followed by Dunnett’s multiple comparisons test (* p = 0.05, ** p = 0.01, *** p = 0.001).

Figure 3

Effect of PDIA1 inhibition on adhesion of breast cancer cells to the endothelium, collagen type I and fibronectin and their transendothelial migration across hLMVEC monolayer. Effect of bepristat 2a on adhesion of MCF-7 (A,E) and MDA-MB-231 (B,F) cells to hLMVEC, collagen type I and fibronectin, respectively, and MCF-7 (C,G) and MDA-MB-231 (D,H) cells transduced with lentiviral vectors carrying short hairpin RNA (shRNA) against PDIA1 or negative sequence after selection with puromycin in regarding to wild type cell line. Transendothelial migration of MCF-7 (I) and MDA-MB-231 (J) cells treated with bepristat 2a and MCF-7 (K) and MDA-MB-231 (L) sublines with silenced PDIA1 expression across hLMVEC monolayers. Data represent mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test or nonparametric Kruskal–Wallis followed by Dunn’s multiple comparisons test (* p = 0.05, ** p = 0.01, *** p = 0.001, **** p = 0.0001).

Figure 4

Effects of exogenous PDIA1 and PDIA3 on adhesive interaction between breast cancer cells and different substrates. Effect of exogenous proteins PDIA1 and PDIA3 on adhesion of MCF-7 (A–F) and MDA-MB-231 (G–L) cells to collagen type I, fibronectin and lung microvascular hLMVEC cells, respectively. Data represent mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test (* p = 0.05, ** p = 0.01, *** p = 0.001).

Figure 5

Effects of thiol blocker, pCMBS on adhesion of cancer cells to fibronectin and collagen type I. Effect of inhibiting ecto-sulfhydryls by p-chloromercuribenzene sulphonate (pCMBS) on adhesion of MCF-7 (A,B) and MDA-MB-231 (C,D) cells to collagen type I and fibronectin. Data represent mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test (* p = 0.05, ** p = 0.01, *** p = 0.001).

Figure 6

The scheme of lentiviral vector used for silencing of PDIA1 expression and sequences of short hairpin RNAs (shRNAs) cloned to the plasmid.