The thioredoxin system in breast cancer cell invasion and migration

Abstract

Metastasis is the most life threatening aspect of breast cancer. It is a multi-step process involving invasion and migration of primary tumor cells with a subsequent colonization of these cells at a secondary location. The aim of the present study was to investigate the role of thioredoxin (Trx1) in the invasion and migration of breast cancer cells and to assess the strength of the association between high levels of Trx1 and thioredoxin reductase (TrxR1) expression with breast cancer patient survival. Our results indicate that the expression of both Trx1 and TrxR1 are statistically significantly increased in breast cancer patient cells compared with paired normal breast tissue from the same patient. Over-expression of Trx1 in MDA-MB-231 breast cancer cell lines enhanced cell invasion in in vitro assays while expression of a redox inactive mutant form of Trx1 (designated 1SS) or the antisense mRNA inhibited cell invasion. Addition of exogenous Trx1 also enhanced cell invasion, while addition of a specific monoclonal antibody that inhibits Trx1 redox function decreased cell invasion. Over-expression of intracellular Trx1 did not increase cell migration but expression of intracellular 1SS inhibited migration. Addition of exogenous Trx1 enhanced cell migration while 1SS had no effect. Treatment with auranofin inhibited TrxR activity, cell migration and clonogenic activity of MDA-MB-231 cells, while increasing reactive oxygen species (ROS) levels. Analysis of 25 independent cohorts with 5910 patients showed that Trx1 and TrxR1 were both associated with a poor patient prognosis in terms of overall survival, distant metastasis free survival and disease free survival. Therefore, targeting the Trx system with auranofin or other specific inhibitors may provide improved breast cancer patient outcomes through inhibition of cancer invasion and migration.

Keywords: Auranofin; Breast cancer; Cell invasion; Cell migration; Patient survival; Thioredoxin.

Graphical abstract

Fig. 1

Expression of Trx1 and TrxR1 in patient breast cancer samples. The normalized gene expression levels (transcripts per million (TPM)) encoding Trx1 and TrxR1 were analyzed according to the RNASeqV2 protocol using data obtained from the TCGA portal. Analysis was restricted to patients with matched tumor and normal samples (n=114). Both Trx1 and TrxR1 expression was shown to be statistically significantly different in tumor samples compared to normal tissue, using a two-sample paired t test, *p<0.001, **p<0.0001.

Fig. 2

Invasion of MDA-MB-231 transfectants in the Boyden chamber invasion assay. DCC – Clones transfected with pcDNA3.1 vector only; DSC – Sense Trx1 clones; DAS – Antisense Trx1 clones; D1S – 1SS redox inactive Trx1 clones (n=3). Results from three independent experiments performed in triplicate are presented as mean±SEM. A one-way ANOVA followed by Tukey's post test was employed and a significant difference in invasion values compared to the control cells is indicated by *p<0.05.

Fig. 3

Effect of exogenously added recombinant human Trx1 on MDA-MB-231 cell invasion. Trx1 concentration is as indicated. SFM is serum free media (negative control) and FBS (fetal bovine serum) in the bottom well was used as a positive control. Results from three independent experiments performed in triplicate are presented as mean±SEM. A two-way ANOVA followed by Tukey’s post test was employed and a significant difference in invasion values compared to the FBS treated control cells and to other concentrations of Trx1 is indicated by *p<0.05.

Fig. 4

Effect of adding anti-Trx1 human monoclonal antibodies on the invasion of MDA-MB-231 cells. Antibodies were added to either the top or bottom well of the Boyden chamber. Results are from three independent experiments performed in triplicate presented as mean±SEM. The invasion value is the cells/field in the test assay less the cells/field in the negative control. A one-way ANOVA followed by Tukey's post test was employed and a significant difference in invasion values compared to the control cells (Nil antibody added) is indicated by *p<0.05.

Fig. 5

Effect of Trx1 on the migration of MDA-MB-231 cells. (A) ROS levels in stable transfected MDA-MB-231 cells analyzed using H₂DCF-DA and represented as a fold change over pIRES-EGFP transfected cells. A one-way ANOVA followed by Sidak’s post test was employed and showed that cells over-expressing the 1SS redox inactive Trx1 protein exhibited significantly higher levels of ROS compared to the control cells, *p<0.05. Results are presented as mean±SEM of three independent experiments performed in triplicate. (B) Representative images of the transfected cells used in the monolayer scratch assays over a period of 48 h. The images were taken at 4× using a Tucsen TCA 3MP camera and the scale bar is 50 µm. (C) Migration of stable transfected cells in response to over-expression of wild-type Trx1 and the 1SS redox inactive Trx1 protein. A repeated-measures two-way ANOVA followed by Sidak’s post test was employed and showed a statistical difference at 48 hour time point between 1SS over-expressing cells and control cells, *p<0.0001. Results are presented as mean±SEM of at least three independent experiments performed in quadruplicate.

Fig. 6

Differential effect of wild-type Trx1 and the 1SS redox inactive Trx1 form on MDA-MB-231 breast cancer cell migration. Two different concentrations of wild-type Trx1 or the 1SS redox inactive Trx1 form were used to treat MDA-MB-231 cells. Untreated cells were used as the control. Scratch closure was expressed as the percentage scratch closure calculated at the 24 or 48 h time points using 0 hour measurements as 0%. A two-way ANOVA employing Tukey's post test showed a significant difference between untreated cells and both the 0.313 μg/mL and 0.626 μg/mL Trx1 treated cells at 24 and 48 h incubation time, *p<0.05. There was also a difference between the Trx1 and 1SS redox inactive Trx1 treated cells at 24 and 48 h incubation time, ^∆p<0.05. Trx: Wild-type Trx1, 1SS: 1SS redox inactive Trx1 form. Results are presented as mean±SEM from three independent experiments conducted in quadruplicate.

Fig. 7

Effect of auranofin induced inhibition of the TrxR system. (A) Proliferation of MDA-MB-231 cells in response to auranofin treatment. The IC₅₀ for auranofin-induced inhibition of MDA-MB-231 cell proliferation was determined to be 5.1 µM. At least three independent experiments were performed in triplicate. (B) TrxR1 specific activity in MDA-MB-231 cells upon auranofin treatment. Auranofin significantly inhibited the TrxR1 activity at 0.31 µM and higher concentrations, *p<0.0001. A one-way ANOVA followed by Sidak's post test was employed and results are presented as mean±SEM of at least three independent experiments performed in triplicate. (C) ROS levels in auranofin treated MDA-MB-231 cells analyzed using H₂DCF-DA. A one-way ANOVA followed by Sidak’s post test was employed. Cells treated with 2 µM auranofin had significantly higher levels of ROS compared to the control cells, *p<0.05. (D) Migration of MDA-MB-231 cells in response to auranofin treatment. Cells treated with 2µM auranofin showed a significant decrease in the distance migrated as compared to the control cells, at both 24 and 48 h time points, *p<0.0001. Data was analyzed using a repeated measures two-way ANOVA followed by Sidak’s post test and results are presented as mean±SEM of three independent experiments.

Fig. 8

Effect of auranofin pre-treatment on the clonogenic capacity of MDA-MB-231 cells. (A) Representative images taken after 5 days of colony formation. (B) Number of colonies obtained after 5 days. Cells that were pre-treated with 2 µM auranofin had significantly less number of colonies compared to the untreated cells, *p<0.05. A one-way ANOVA followed by Sidak's post test was employed and results are presented as mean±SEM of two independent experiments performed in duplicate. (C) Survival data is presented as percentage of the untreated control cells.

Fig. 9

Breast cancer patient survival analysis. Survival analysis for Trx1 and TrxR1 using RNA sequencing data (TCGA cohort) as well as microarray data from 25 independent cohorts, as indicated in the figure with associated number of patients. OS, overall survival; DMFS, distant metastasis-free survival; DFS, disease free survival. A forest plot with the corresponding hazard ratios is shown on the right with the associated confidence intervals indicated. Asterisks mark the results that are statistically significant. A hazard ratio (HR) >1 is indicative of a poor outcome.