Peroxiredoxin-1 protects estrogen receptor α from oxidative stress-induced suppression and is a protein biomarker of favorable prognosis in breast cancer

Introduction Peroxiredoxin-1 (PRDX1) is a multifunctional protein, acting as a hydrogen peroxide (H2O2) scavenger, molecular chaperone and immune modulator. Although differential PRDX1 expression has been described in many tumors, the potential role of PRDX1 in breast cancer remains highly ambiguous. Using a comprehensive antibody-based proteomics approach, we interrogated PRDX1 protein as a putative biomarker in estrogen receptor (ER)-positive breast cancer. Methods An anti-PRDX1 antibody was validated in breast cancer cell lines using immunoblotting, immunohistochemistry and reverse phase protein array (RPPA) technology. PRDX1 protein expression was evaluated in two independent breast cancer cohorts, represented on a screening RPPA (n = 712) and a validation tissue microarray (n = 498). In vitro assays were performed exploring the functional contribution of PRDX1, with oxidative stress conditions mimicked via treatment with H2O2, peroxynitrite, or adenanthin, a PRDX1/2 inhibitor. Results In ER-positive cases, high PRDX1 protein expression is a biomarker of improved prognosis across both cohorts. In the validation cohort, high PRDX1 expression was an independent predictor of improved relapse-free survival (hazard ratio (HR) = 0.62, 95% confidence interval (CI) = 0.40 to 0.96, P = 0.032), breast cancer-specific survival (HR = 0.44, 95% CI = 0.24 to 0.79, P = 0.006) and overall survival (HR = 0.61, 95% CI = 0.44 to 0.85, P = 0.004). RPPA screening of cancer signaling proteins showed that ERα protein was upregulated in PRDX1 high tumors. Exogenous H2O2 treatment decreased ERα protein levels in ER-positive cells. PRDX1 knockdown further sensitized cells to H2O2- and peroxynitrite-mediated effects, whilst PRDX1 overexpression protected against this response. Inhibition of PRDX1/2 antioxidant activity with adenanthin dramatically reduced ERα levels in breast cancer cells. Conclusions PRDX1 is shown to be an independent predictor of improved outcomes in ER-positive breast cancer. Through its antioxidant function, PRDX1 may prevent oxidative stress-mediated ERα loss, thereby potentially contributing to maintenance of an ER-positive phenotype in mammary tumors. These results for the first time imply a close connection between biological activity of PRDX1 and regulation of estrogen-mediated signaling in breast cancer.

Chemiluminescence was detected by autoradiography using X-ray film (Fujifilm, Japan).

Reverse phase protein array analysis
Proteins were extracted from tissue or cell lines using RPPA lysis buffer (1% Triton X-100, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1.5 mM MgCl 2 , 1 mM EGTA, 100 mM NaF, 10 mM Na pyrophosphate, 1 mM Na 3 VO 4 , 10% glycerol, containing freshly added protease and phosphatase inhibitors (Roche)). Samples were centrifuged at 20,817 x g (14,000 rpm) at 4°C. The supernatants were removed and the protein concentrations determined by the bicinchoninic acid (BCA) method (Pierce, IL, USA) and adjusted by diluting in lysis buffer to 1-1.5mg/ml. Cellular proteins were denatured by 1% SDS (with beta-mercaptoethanol) and diluted in five 2-fold serial dilutions in dilution buffer (lysis buffer containing 1% SDS). Serial diluted lysates were arrayed on nitrocellulose-coated FAST slides (Whatman) using the Aushon 2470 Arrayer (Aushon Biosystems). A total of 5808 array spots were arranged on each slide, including the spots corresponding to positive and negative controls prepared from mixed cell lysates or dilution buffer, respectively.
Each slide was probed with a validated primary antibody plus a biotin-conjugated secondary antibody. Only antibodies which generated a Pearson correlation coefficient (r) between RPPA and Western blotting densitometry data of greater than 0.7 in respect to cell culture models were used in RPPA-based analysis of tissue extracts. In more detail, antibodies which provided a discrete signal via Western blotting were further assessed by direct comparison to RPPA using cell lines with a dynamic range of protein expression, using protein lysates from recombinant cell lines with overexpression or knockdown of the protein-of-interest. The signal obtained was amplified using a Dako Cytomation-catalyzed system (Dako) and visualized by a DAB colorimetric reaction. The slides were scanned, analyzed, and quantified using a customized-software Microvigene (VigeneTech Inc.) to generate spot intensity measurements.
Each dilution curve was fitted with a logistic model ("Supercurve Fitting" developed by the

Department of Bioinformatics and Computational Biology at the MD Anderson Cancer
Center, "http://bioinformatics.mdanderson.org/OOMPA"). This fits a single curve using all the samples (i.e. dilution series) on a slide with the signal intensity as the response variable and the dilution steps as the independent variable. The fitted curve is plotted with the signal intensitiesboth observed and fitted -on the y-axis and the log2-concentration of proteins on the x-axis. The protein concentrations of each set of slides were then normalized by median polish, which was corrected across samples by the linear expression values using the median expression levels of all antibody experiments to calculate a loading correction factor for each sample.

Immunohistochemistry
To construct cell pellet arrays, cells were trypsinized and fixed for 10 hours in 10% formalin (Sigma) following which they were centrifuged, washed with phosphate buffered saline (PBS) (Thermo Scientific) and resuspended in 1% agarose (Pronadisa). The tumor cellcontaining agarose plugs were processed through gradient alcohols before being cleared in xylene and washed in molten paraffin. These cell pellets were embedded in paraffin and arrayed in quadruplicate 1.0mm cores using a manual tissue arrayer (MTA-1, Beecher Inc, WI). IHC was carried out on 5μm sections. Tissue microarrays (TMA) were constructed as previously described [1].
Cell pellet arrays or tissue microarrays were deparaffinized in xylene and rehydrated in descending gradient alcohols. Heat-mediated antigen retrieval was performed using 10mM sodium citrate buffer (pH 6) using a PT module (LabVision, UK) for 15 minutes at 95°C. The were carried out at room temperature. The sections were counterstained in haematoxylin, dehydrated in alcohol and xylene and mounted using an automated coverslipper (Leica, Germany). As a negative control, the anti-PRDX1 antibody was substituted with an antirabbit immunoglobulin G isotype control and with a PBST only negative control.

TCGA (cohort 3) data download and analysis approach
PRDX1 protein expression as determined by RPPA analysis and clinical follow-up data were downloaded directly from the TCGA data portal (https://tcga-data.nci.nih.gov/tcga/). After merging the data based on patient ID number, the cohort was separated into ER-negative and -positive subgroups using expression levels of ERα protein as determined by RPPA. A threshold of < -1.8 and > -1.4 of the log2 transformed signal was used to stratify the ERnegative and -positive subgroups, respectively. Pearson's correlation test was used to check for proteins correlating with PRDX1 protein or mRNA expression levels. The two-sided t-test was used to identify proteins co-regulated between the lower and upper quartile of PRDX1 protein expression cases, using a fold change threshold of 0.67 < FC < 1.5. A p-value of less than 0.05 was considered significant.

Peroxynitrite treatment
Peroxynitrite treatment was for 16 hours in duration. Peroxynitrite is not stable in FBS conditions, so all peroxynitrite treatments were carried out in phenol-red free media with 5% charcoal dextran FBS. Degraded peroxynitrite was used as a control, along with complete media (DMEM + 10% FBS). Experiments were performed using a FACS Aria III cell sorter (BD Bioscience) and data processing was performed using BD FACSDiva™ Software (BD Bioscience).

Apoptosis Assay
A total of 3×10 3 cells were seeded per 384-well and the cells were allowed to adhere overnight. Caspase 3/7 activity was measured after 1 hour incubations, using the Apotox-Glo™ Triplex assay kit and the GloMax®-Multi Microplate Multimode Reader (kits and equipment from Promega). Treatments were carried out using triplicate wells in each biological replicate.