Edited by: Shengtao Zhou, Sichuan University, China
Reviewed by: Carlos Martinez-Perez, Medical Research Council Institute of Genetics and Molecular Medicine (MRC), United Kingdom; Vidya Sethunath, Dana–Farber Cancer Institute, United States
*Correspondence: Constantin Tamvakopoulos,
This article was submitted to Women’s Cancer, a section of the journal Frontiers in Oncology
†Present address: Orestis Argyros, Cell/Gene Therapy, Medicinal Science and Technology, GlaxoSmithKline, London, United Kingdom
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Breast cancer (BC) is a highly heterogeneous disease encompassing multiple subtypes with different molecular and histopathological features, disease prognosis, and therapeutic responses. Among these, the Triple Negative BC form (TNBC) is an aggressive subtype with poor prognosis and therapeutic outcome. With respect to HER2 overexpressing BC, although advanced targeted therapies have improved the survival of patients, disease relapse and metastasis remains a challenge for therapeutic efficacy. In this study the aim was to identify key membrane-associated proteins which are overexpressed in these aggressive BC subtypes and can serve as potential biomarkers or drug targets. We leveraged on the development of a membrane enrichment protocol in combination with the global profiling GeLC-MS/MS technique, and compared the proteomic profiles of a HER2 overexpressing (HCC-1954) and a TNBC (MDA-MB-231) cell line with that of a benign control breast cell line (MCF-10A). An average of 2300 proteins were identified from each cell line, of which approximately 600 were membrane-associated proteins. Our global proteomic methodology in tandem with invigoration by Western blot and Immunofluorescence analysis, readily detected several previously-established BC receptors like HER2 and EPHA2, but importantly STEAP4 and CD97 emerged as novel potential candidate markers. This is the first time that the mitochondrial iron reductase STEAP4 protein up-regulation is linked to BC (HER2+ subtype), while for CD97, its role in BC has been previously described, but never before by a global proteomic technology in TNBC. STEAP4 was selected for further detailed evaluation by the employment of Immunohistochemical analysis of BC xenografts and clinical tissue microarray studies. Results showed that STEAP4 expression was evident only in malignant breast tissues whereas all the benign breast cases had no detectable levels. A functional role of STEAP4 intervention was established in HER2 overexpressing BC by pharmacological studies, where blockage of the STEAP4 pathway with an iron chelator (Deferiprone) in combination with the HER2 inhibitor Lapatinib led to a significant reduction in cell growth
Breast cancer (BC) is one of the most frequently diagnosed malignancies and the leading cause of cancer-related death in women worldwide, with more than one million estimated new cases and nearly five thousand related deaths each year (
We decided to focus our analysis in membrane protein targets due to their central role in all physiological functions, such as cell signalling, cell-cell interactions and cell homeostasis and over all implication in the development and progression of a variety of human cancers (
MS-based proteomics is currently the backbone of cancer biomarker discovery as the technology affords the high throughput study of proteins from complex biological samples aiming to the investigation of their ontology, classification, expression levels and properties (
In this study, we leveraged on a label-free GeLC-MS/MS technique to compare the membrane proteomes of two representative and regularly used epithelial BC cell lines, such as the HCC-1954 (HER2+) and the MDA-MB-231 (TNBC) to a breast benign control cell line, MCF-10A. Several hits of potential interest were generated with CD97 and the iron reductase six-transmembrane epithelial antigen of prostate 4 (STEAP4) distinguished as the most novel and promising of these hits. STEAP4 protein was selected for further downstream analysis by employing Immunohistochemical (IHC) analysis on tumors derived from xenografted mice inoculated with the BC cell lines and on two independent BC tissue microarrays (TMAs). We also performed proof-of-principle functional studies on STEAP4 to assess its involvement in cancer pathophysiology. The STEAP4 pathway was pharmacologically targeted in BC cells, with an iron chelating drug (Deferiprone) with simultaneous blockage of the HER2 pathway with the tyrosine kinase inhibitor Lapatinib, which demonstrated an additive therapeutic potential involvement of STEAP4 in cancer.
Overall, our data suggest that STEAP4 may constitute a novel BC biomarker or a promising new target for HER2+ BC therapy.
The BC cell lines HCC-1954 (CRL-2338), SKBR3 (HTB-30), BT474 (HTB-20), and MDA-MB-231 (HTB-26), and the breast benign epithelial cell line MCF-10A (CRL-10317) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The HCC-1954, the SKBR3 and the BT474 cells were cultured in RPMI 1640 and the MDA-MB-231 in Dulbecco’s modified Eagle’s (DMEM), both medium were supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin). The MCF-10A was cultured in DMEM supplemented with 10% FBS, epidermal growth factor (20 ng/mL), cholera toxin (100 ng/mL and hydrocortisone (0.5 μg/mL). All cells were incubated in a humidified atmosphere at 37°C, 5% CO2 and regularly screened for mycoplasma using the MycoAlert™ Mycoplasma Detection Kit (Lonza, USA). All experiments were performed on cell cultures passaged no more than six times from frozen stock vials of passage 20 for HCC-1954 and SKBR3, 15 for BT474, 25 for MDA-MB-231, and 2 for MCF-10A.
Enriched membrane fractions from the BC cell lines HCC-1954 and MDA-MB-231 and the breast benign control cell line MCF-10A were prepared and separated from cytosolic fractions using a commercially available kit (Mem-PER Plus Membrane Protein Extraction Kit) according to the manufacturer’s instructions. Briefly, 5 × 106 cells were harvested and the cell suspension was centrifuged at 300 × g for 5 min. Then the cell pellet was washed with 3 mL Cell Wash Solution, centrifuged at 300 × g for 5 min and the supernatant was discarded. The cells were resuspended in 1.5 mL Cell Wash Solution, transferred to small tube and centrifuged again at 300 × g for 5 min. The cell pellet was then mixed with 0.75 mL of Permeabilization Buffer, vortexed briefly to obtain a homogeneous cell suspension and incubated 10 min at 4°C with constant mixing. The permeabilized cells were centrifuged for 15 min at 16,000 × g and the supernatant containing the cytosolic proteins was carefully collected to a new tube and stored at -80°C for future use. Subsequently, the protein pellet was resuspended in 0.5 mL of Solubilization Buffer, incubated at 4°C for 30 min with constant mixing and centrifuged at 16,000 × g for 15 min at 4°C. Finally, the supernatant containing solubilized membrane and membrane-associated proteins was transferred to a new tube and stored at -80°C for further analysis. Meanwhile, whole cell lysate fractions were isolated using the Ripa Buffer cell lysis protocol, as described before. The protein concentrations of the resulting membrane cell lysate fractions were measured using the Bradford assay.
One-dimensional SDS-PAGE and in-gel trypsin digestion were performed as previously reported (
All LC-MS/MS experiments were performed on a Dionex Ultimate 3000 UHPLC system coupled with the high resolution nano-ESI Orbitrap-Elite mass spectrometer (Thermo Finnigan, Bremen, Germany), as previously described (
Quantification analysis was performed using a label-free approach based on the peak area intensities of identified proteins (
The peak area intensity measuring and comparing is the most widely used label-free quantification method which is based on the precursor signal intensity as determined by the extracted ion chromatogram (XIC). Precursor signal intensity is strongly correlated with peptide abundance in a specific sample (
Area intensity normalization for each identified protein based on total ion count is a necessary step to eliminate bias in signal intensity (
Cells were lysed in ice-cold RIPA lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM EGTA, 5 mM Na2EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 8 mM sodium fluoride, 1 mM sodium orthovanadate) containing protease and phosphatase inhibitor cocktail (Roche, UK). Protein concentration was determined using the Pierce BCA assay kit (Pierce, Rockford, IL, USA) according to the manufacturer’s specifications. Equal amounts of protein extracted from the HCC-1954, the MDA-MB-231, the SKBR3, the BT474, and the MCF-10A cell lysates (10 μg of protein/cell line), along with NuPAGE Sample Reducing agent and NuPAGE LDS Sample Buffer were then separated by 10% SDS-PAGE gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat dried milk in TBS-T buffer (20 mM Tris, pH 7.6, 137mM NaCl, 0.1% Tween 20) for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 0C and then with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2h at room temperature. The primary antibodies used were: mouse monoclonal anti-ErbB2 (1:1000 dilution; cat. no. ab8054, Abcam, UK), rabbit monoclonal anti-EPHA2 (1:3000 dilution; cat. no. 6997S, Cell Signaling Technology, USA), rabbit monoclonal anti-CD97 (1:1000 dilution; cat. no. ab108368, Abcam, UK) and rabbit polyclonal anti-STEAP4 (1:2500 dilution; cat. no. 11944-1-AP, Proteintech, USA). For normalization of protein concentration, the rabbit monoclonal anti-β-actin (1:10000 dilution; cat. no. ab190476, Abcam, UK) was used as loading control. The secondary antibodies used were: horseradish peroxidase (HRP)-conjugated goat anti-rabbit or horse anti-mouse IgG (1:2000 dilution; cat. no. 7074S and 7076S, Cell Signaling Technology, USA). Reactive protein bands were visualized by incubation of the membranes with Enhanced Chemiluminescence substrate (GE Healthcare, UK) and exposure to x-ray films. All Western blot analyses were repeated at least three times.
HCC-1954, MDA-MB-231, SKBR3, BT474 and MCF-10A cells were grown onto coverslips in 24-well plate at a density of 1 × 104 cells at 37°C. The next day, the culture media was discarded, cells were rinsed with PBS three times and then fixed with 100% methanol at -20°C for 10 min. The cells were further washed twice with ice-cold PBS (5 min per wash) to remove fixative agent. Subsequently, 200 μL of blocking buffer (5% goat serum, 0.1% Triton in PBS) was added to the cells and incubated for 1 h at room temperature to block nonspecific binding with antibody. After blocking, the cells were incubated with the following primary antibodies overnight at 4°C: rabbit polyclonal anti-STEAP4 antibody (1:500 dilution; cat. no. 11944-1-AP, Proteintech, USA), mouse monoclonal anti-ErbB2 (1:100 dilution; cat. no. ab8054, Abcam, UK), rabbit monoclonal anti-EphA2 (1:200 dilution; cat. no. 6997S, Cell Signaling Technology, USA) and rabbit monoclonal anti-CD97 (1:200 dilution; cat. no. ab108368, Abcam, UK). Cells were then washed twice with ice-cold PBS, and incubated with the secondary antibody conjugated to Alexa Fluor 568 donkey anti-rabbit IgG (1:500 dilution; cat. no. ab175470, Abcam, UK) for 2 h in the dark at room temperature. The coverslips containing the cells were then washed twice with ice-cold PBS and mounted on glass slides with ProLong Gold anti-fade reagent containing DAPI (1.5 μg/mL) (Invitrogen Inc., Eugene, OR, USA). Dapi was used to stain the cell nucleus. Immunofluorescent stained cells were dried overnight in the dark at 4°C and the analyses were made using the fluorescence microscope (Leica Microsystems) with a 400 × objective. To allow direct comparisons, all images were captured using the same parameters. The fluorescence intensity was quantified using ImageJ software.
Formalin fixed, paraffin-embedded tissues derived from xenografted mice inoculated with the HCC-1954 and the MDA-MB-231 cell lines were obtained for IHC analysis of STEAP4 expression as previously described (
Two human BC tissue microarray slides (catalog # BRC961; Pantomics, Rockville, MD and catalog # BR251c; US Biomax, Rockville, MD) containing duplicate cores from a total of 36 different cases of BC with 12 cases of normal and benign tumor tissues and 6 quadrupole cases of breast invasive ductal carcinoma with matched adjacent normal breast, respectively, were used for expression studies of STEAP4 in clinical samples. For the TMAs, all human tissues were collected under HIPPA approved protocols, as described in the the providers of the tissue microarrays, US Biomax, Inc. (
For the pharmacological studies, Lapatinib (cat. no. HY-50898) was purchased from MedChem Express and Deferiprone (3-hydroxy-1,2-dimethyl-4(1H)-pyridone) from Sigma-Aldrich.
HCC-1954, SKBR3, BT474 and MCF-10A cells were seeded at a density of 5 × 103 cells per well on 96-well plates. After 24 h incubation (37°C, 5% CO2), the cell medium was removed, and the cells were treated with various concentrations of Lapatinib (10 nM-30 μM) and deferiprone (10 nM-30 μM), either alone or in combination for 72 h. The medium was then removed and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (0.3 mg/mL in PBS) was added to cells for 3 h, after which the MTT solution was removed and the formazan crystals were dissolved in 100 μL DMSO. The optical density was measured at 570 nm and at a reference wavelength of 650 nm using an absorbance microplate reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA). The 50% cytostatic concentration (IC50) was calculated based on a four-parameter logistic equation using GraphPad Prism 5 (GraphPad Software, Inc.).
Three different siRNA duplexes specific for STEAP4: A-siRNA (cat. no. SR312522A), B-siRNA (cat. no. SR312522B), C-siRNA (cat. no. SR312522C) and a Universal Scrambled negative control siRNA (NC-siRNA) duplex (cat. no. SR30004) were purchased from OriGene (OriGene, Rockville, MD). HCC-1954 cells were transfected with a final concentration of 10 nM siRNA, using siTRAN 2.0 transfection reagent (OriGene, Rockville, MD), according to the manufacturer’s protocol. Control cells were mock transfected with transfection reagents. HCC-1954 cells (2 x 105 per well) were seeded in 6-well plates and cultured until they reached 60% - 70% confluency. The cells were incubated with transfection mixtures containing 10 nM of STEAP4-siRNA or NC-siRNA (non-silencing of STEAP4) for 14 h and then the culture supernatant was replaced with fresh culture medium. The cells were harvested after 48 h and evaluated for their silencing efficiency using Western Blot, as described above.
To determine the proliferative ability of cells, HCC-1954 cells at a density of 5 × 103 cells per well were cultured in 96-well plates. The cells were initially transfected with STEAP4-siRNA or NC-siRNA. After 24, 48 and 72 h of transfection, the cell medium was removed and MTT solution (0.3 mg/mL in PBS) was added to cells for 3 h at 37°C. The optical density was measured at 570 nm and at a reference wavelength of 650 nm using an absorbance microplate reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA).
To investigate the effect of siRNA on Lapatinib IC50 values, HCC-1954 cells were seeded into 96-well plates (5 × 103 cells/well) and allowed to attach for 24 h (37°C, 5% CO2). Then, cells were treated with 10 nM of STEAP4-siRNA or NC-siRNA for 14 h at 37°C in a 5% CO2 incubator. After incubation, the culture supernatant was replaced with fresh culture medium containing serum and different concentrations of Lapatinib (10 nM-30 μM) for a total time of 72 h. After incubation, the cell viability was detected using the same MTT assay described above.
The results presented herein are expressed as mean ± SD. Correlations between STEAP4 expression and clinicopathological parameters were determined using the Chi-square test. Statistical analyses and calculation of all IC50’s were performed by GraphPad software. Each point was the result of three independent experiments performed in triplicate for statistical analysis. Statistical significance was determined using the Student’s t-test. A P value of less than 0.05 was considered significant.
Our key methodological principle was to increase the threshold of sensitivity to ensure that even low-abundant cell membrane proteins in aggressive subtypes of BC could be detected. Thus, our initial step was to perform an efficient subcellular fractionation of three well characterized BC epithelial cell lines: HCC-1954 (HER2 overexpressing), MDA-MB-231 (TNBC) and MCF-10A (benign control). Enriched membrane fractions were isolated and separated from the cytosolic fractions using a commercially available kit, whereas whole cell lysate fractions were prepared using the Ripa Buffer cell lysis protocol (
Experimental workflow for identification of candidate Breast Cancer (BC) biomarkers or drug targets using a strategy that combines a membrane enrichment protocol with GeLC-MS/MS technique.
Following the successful fractionation, a global proteomic profiling was performed in order to identify new pharmacological targets or biomarkers for HER2+ and TNBC. For such a demanding proteomic analysis we utilized the Global Discovery-based GeLC-MS/MS approach on a high resolution Orbitrap Mass analyzer. The technique is based on the initial separation of the protein mixture by one-dimensional SDS-page gel electrophoresis followed by in-gel tryptic digestion and finally on the Mass Spectrometric and Bioinformatic analysis of the peptide mixture (
In order to investigate the quantitative similarities and differences in protein expression resulting from malignant transformation of the breast epithelium, comparative proteomic analysis was performed among the described BC cell lines. The Venn diagrams in
Comparison of the membrane-associated proteins identified in breast cancer (HCC-1954 and MDA-MB-231) versus benign control (MCF-10A) cell lines by GeLC-MS/MS analysis. Proteins from 4 biological replicates of each breast cancer cell line were combined for this comparison.
Quantitative comparison of HCC-1954 (HER2+) and MDA-MB-231 (TNBC) versus MCF-10A cells based on the average Normalized Area of each protein entry revealed promising molecules to investigate further as potential specific biomarkers or drug targets for HER2+ BC or TNBC. To narrow down the list of candidate targets in HER2+ BC and TNBC the following criteria were applied:
(1) Candidate proteins were only detected in the cancerous cell lines (HCC-1954 and MDA-MB-231) compared to the normal control cell line (MCF-10A).
(2) Protein expression levels in the normal cell line were defined as those proteins that were identified in at least three replicates with a Normalized Area ≥ 70; Candidate proteins were highly abundant. The proteins with the highest Normalized Area were reported.
(3) The percent Coefficient of Variation (% CV) between replicates of each candidate protein was ≤ 40, based on statistical analysis. Applying these criteria led to the selection of the top 30 candidate protein targets in HER2+ BC and TNBC as shown in
Selected membrane associated proteins specifically identified in HCC-1954 compared to MCF-10A cells.
Uniprot accession no. | Gene names | Protein names | Average Normalized_Area | SD |
---|---|---|---|---|
P04626 | ERBB2 | Receptor tyrosine-protein kinase erbB-2 | 2103 | 225 |
Q687X5 | STEAP4 | Metalloreductase STEAP4 | 1214 | 176 |
Q96KA5 | CLPTM1L | Cleft lip and palate transmembrane protein 1-like protein | 638 | 92 |
P05534 | HLA-A | HLA class I histocompatibility antigen, A-24 alpha chain | 573 | 104 |
Q14451 | GRB7 | Growth factor receptor-bound protein 7 | 537 | 137 |
Q04826 | HLA-B | HLA class I histocompatibility antigen, B-40 alpha chain | 442 | 116 |
P11166 | SLC2A1 | Solute carrier family 2, facilitated glucose transporter member 1 | 433 | 65 |
Q14849 | STARD3 | StAR-related lipid transfer protein 3 | 403 | 81 |
Q03518 | TAP1 | Antigen peptide transporter 1 | 376 | 130 |
P30504 | HLA-C | HLA class I histocompatibility antigen, Cw-4 alpha chain | 374 | 93 |
O15533 | TAPBP | Tapasin | 359 | 107 |
Q03519 | TAP2 | Antigen peptide transporter 2 | 292 | 47 |
O95832 | CLDN1 | Claudin-1 | 249 | 74 |
P49788 | RARRES1 | Retinoic acid receptor responder protein 1 | 245 | 20 |
P43003 | SLC1A3 | Excitatory amino acid transporter 1 | 212 | 30 |
P48651 | PTDSS1 | Phosphatidylserine synthase 1 | 208 | 48 |
Q9NZ08 | ERAP1 | Endoplasmic reticulum aminopeptidase 1 | 199 | 3 |
Q6PI78 | TMEM65 | Transmembrane protein 65 | 199 | 20 |
Q8N5K1 | CISD2 | CDGSH iron-sulfur domain-containing protein 2 | 186 | 28 |
Q9P0I2 | EMC3 | ER membrane protein complex subunit 3 | 184 | 43 |
Q15392 | DHCR24 | Delta(24)-sterol reductase | 182 | 16 |
Q8WY22 | BRI3BP | BRI3-binding protein | 166 | 22 |
Q96HV5 | TMEM41A | Transmembrane protein 41A | 165 | 16 |
Q8NHH9 | ATL2 | Atlastin-2 | 164 | 26 |
P21397 | MAOA | Amine oxidase [flavin-containing] A | 162 | 39 |
Q92575 | UBXN4 | UBX domain-containing protein 4 | 159 | 39 |
P53621 | COPA | Coatomer subunit alpha | 156 | 33 |
Q6NUQ4 | TMEM214 | Transmembrane protein 214 | 156 | 24 |
P29317 | EPHA2 | Ephrin type-A receptor 2 | 153 | 18 |
Q9BPW9 | DHRS9 | Dehydrogenase/reductase SDR family member 9 | 153 | 44 |
Selected membrane associated proteins specifically identified in MDA-MB-231 compared to MCF-10A cells.
Uniprot accession no. | Gene names | Protein names | Average Normalized_Area | SD |
---|---|---|---|---|
P01892 | HLA-A | HLA class I histocompatibility antigen, A-2 alpha chain | 4575 | 1130 |
P10316 | HLA-A | HLA class I histocompatibility antigen, A-69 alpha chain | 4409 | 1138 |
Q95604 | HLA-C | HLA class I histocompatibility antigen, Cw-17 alpha chain | 4114 | 1126 |
P30479 | HLA-B | HLA class I histocompatibility antigen, B-41 alpha chain | 1382 | 367 |
Q9NZ08 | ERAP1 | Endoplasmic reticulum aminopeptidase 1 | 473 | 73 |
P42892 | ECE1 | Endothelin-converting enzyme 1 | 443 | 78 |
P04233 | CD74 | HLA class II histocompatibility antigen gamma chain | 412 | 162 |
P29966 | MARCKS | Myristoylated alanine-rich C-kinase substrate | 303 | 122 |
P29317 | EPHA2 | Ephrin type-A receptor 2 | 276 | 98 |
Q03518 | TAP1 | Antigen peptide transporter 1 | 267 | 76 |
P48960 | CD97 | CD97 antigen | 262 | 72 |
P13760 | HLA-DRB1 | HLA class II histocompatibility antigen, DRB1-4 beta chain | 246 | 49 |
O75976 | CPD | Carboxypeptidase D | 233 | 54 |
Q9NZB2 | FAM120A | Constitutive coactivator of PPAR-gamma-like protein 1 | 226 | 78 |
P35610 | SOAT1 | Sterol O-acyltransferase 1 | 215 | 29 |
Q9NRX5 | SERINC1 | Serine incorporator 1 | 210 | 80 |
P11717 | IGF2R | Cation-independent mannose-6-phosphate receptor | 198 | 43 |
P01903 | HLA-DRA | HLA class II histocompatibility antigen, DR alpha chain | 194 | 30 |
P30530 | AXL | Tyrosine-protein kinase receptor UFO | 191 | 50 |
Q9BYC5 | FUT8 | Alpha-(1,6)-fucosyltransferase | 180 | 45 |
Q15599 | SLC9A3R2 | Na(+)/H(+) exchange regulatory cofactor NHE-RF2 | 174 | 26 |
P01130 | LDLR | Low-density lipoprotein receptor | 171 | 63 |
Q9ULC5 | ACSL5 | Long-chain-fatty-acid–CoA ligase 5 | 170 | 32 |
P60059 | SEC61G | Protein transport protein Sec61 subunit gamma | 165 | 53 |
P08174 | CD55 | Complement decay-accelerating factor | 165 | 28 |
Q9Y371 | SH3GLB1 | Endophilin-B1 | 165 | 49 |
P13498 | CYBA | Cytochrome b-245 light chain | 164 | 74 |
Q15382 | RHEB | GTP-binding protein Rheb | 161 | 50 |
Q14739 | LBR | Lamin-B receptor | 160 | 63 |
P34810 | CD68 | Macrosialin | 158 | 29 |
Notably, among the list of identified candidates exclusively found in the HER2+ BC cell line (
With respect to TNBC, among the list of identified candidates specifically found in the TNBC cell line (
To further verify the expression pattern of the four selected candidate membrane protein targets, Western blot and Immunofluoresence (IF) analysis of HER2, STEAP4, EPHA2 and CD97 were performed from the whole cell lysates of the HCC-1954, the MDA-MB-231 and the MCF-10A cell lines. Results are depicted in
Verification of the expression pattern of the selected protein targets identified by GeLC-MS/MS with Western blot and Immunofluorescence (IF) analysis.
In conclusion, the differential expression levels of these proteins confirmed the findings derived from the proteomic data analysis. While the presence of the adhesion GPCR CD97 has been previously described in BC, the mitochondrial iron reductase STEAP4, to the best of our knowledge, has not been previously implicated in BC (HER2+ subtype). Thus, the novelty and abundance of STEAP4 prompted us to select it for further evaluation and pharmacological analysis.
To assess the clinical relevance of STEAP4 in BC and normal breast tissues, IHC analysis of STEAP4 was conducted initially on tissues derived from mice inoculated with the HCC-1954 and MDA-MB-231 cell lines and on two independent BC TMAs (BRC253, BRC961) containing a total of 18 benign and 42 malignant breast tissues.
Consistent with Western blot and IF results, the HCC-1954 xenograft tissues were positive for STEAP4, whereas the MDA-MB-231 xenograft tissues were negative as shown in
Immunohistochemical analysis of STEAP4 expression in human BC tissues. STEAP4 expression was assessed in 2 independent tissue microarray (TMA) slides, containing 6 cases of invasive ductal carcinoma and adjacent normal breast tissue (BR251c) and 36 cases of breast cancers and 12 cases of normal and benign breast tissues (BRC961). Representative IHC images (100x and 400x magnifications, scale bars = 40 μm) of
Association between STEAP4 expression and clinicopathological features derived from the two TMAs are merged and summarized in
Association of STEAP4 expression with patient’s clinicopathological features in the TMA slides.
Clinical pathological parameters | Case number | STEAP4 positive | STEAP4 negative | P-values |
---|---|---|---|---|
|
||||
Normal & Benign | 17/18 | 0 | 17 |
|
Cancer | 41/42 | 20 | 21 | |
|
||||
|
||||
> 40 | 24/42 | 14 | 10 | 0.1459 |
≤ 40 | 17/42 | 6 | 11 | |
|
||||
T2 | 24/42 | 15 | 9 | 0.0836 |
T3 | 10/42 | 3 | 7 | |
|
||||
II | 10/42 | 10 | 0 |
|
II~III | 8/42 | 4 | 4 | |
III | 12/42 | 4 | 8 | |
|
||||
Negative | 20/42 | 7 | 13 | 0.0578 |
Positive | 20/42 | 13 | 7 | |
|
||||
IIA | 10/42 | 5 | 5 | 0.3729 |
IIB | 20/42 | 9 | 11 | |
IIIA | 5/42 | 4 | 1 | |
|
||||
Negative | 18/42 | 7 | 11 | 0.8902 |
Positive | 17/42 | 7 | 10 |
Chi-square test was used to test statistical association. Statistical significant
Remarkably, due to its membrane-bound localization and its high over-expression in neoplastic compared to non-neoplastic tissues, STEAP4 constitutes an ideal pharmacological target for cancer therapy.
Based on our findings, STEAP4 could be used as a novel candidate therapeutic target for HER2+ BC. A recent study has shown that an FDA-approved iron chelator, Deferiprone (DFP), can inhibit the STEAP4 pathway (
Given the fact that Lapatinib is a dual EGFR/HER2 inhibitor, we chose the HER2 overexpressing BC cell line, HCC-1954, and the EGFR overexpressing benign control cell line, MCF-10A, for further evaluation. Moreover, STEAP4 expression levels were highly and consistently detectable in the HCC-1954 cell line whereas the MCF-10A cell line had undetectable levels of expression, based on our results. We therefore studied the sensitivity of the selected BC cell lines to Lapatinib and DFP, either alone or in combination after a 72-hour treatment. As shown in
To examine the silencing efficiency of siRNAs on STEAP4 protein, three siRNA duplexes (A-siRNA, B-siRNA and C-siRNA) and a negative control (NC-siRNA), which is absent in human genomes, were used for transfection of the HCC-1954 cells. The effects of the specific siRNAs on STEAP4 protein expression were evaluated by Western blot analysis, after the treatment of 10nM for each siRNA. The results indicated that STEAP4 expression was significantly decreased in the A-siRNA and B-siRNA treated groups compared with the C-siRNA, NC-siRNA treated groups and untreated group (
Effect of STEAP4 silencing on cell proliferation and Lapatinib inhibition in HCC-1954 (HER2+) cells.
After A-siRNA, B-siRNA and NC-siRNA of STEAP4 were transfected into HCC-1954 cells for 14 h, the cells were cultured for 24, 48, and 72 h and then treated with the MTT solution. The NC-siRNA was used as a negative control to compare nonspecific toxicity. Absorbance in the cells was detected respectively after which the cell growth curve was drawn (
Since A-siRNA had the most marked silencing effect among the three siRNAs assessed (
To investigate the effect of siRNA-A treatment on the chemosensitivity of HCC-1954 cells to Lapatinib treatment, cells were treated with 10 nM of A-siRNA or NC-siRNA for 14 h followed by treatment of different concentrations of Lapatinib. The IC50 values of Lapatinib following transfection were determined (
Our study was not limited to presenting a wide dynamic range, sensitive and robust approach for BC proteomic analysis, but it also advanced our understanding on the proteomic profile of BC with the discovery of potential biomarkers and therapeutic candidate targets for pharmacological intervention. The combination of a membrane enrichment protocol with the label-free GeLC-MS/MS quantitative proteomic profiling generated a total of 536, 641 and 604 reliable membrane-associated proteins from the HCC-1954, the MDA-MB-231 and the MCF-10A cell lines. Deep-dive proteomic data analysis showed that 110 and 191 proteins were exclusively present in the HCC-1954 and the MDA-MB-231 cell lines, respectively compared to the MCF-10A cell line. Several of the proteins identified in our study have been reported previously to be elevated in HER2+ BC and TNBC, suggesting that the GeLC-MS/MS methodology is a powerful screening tool for the identification of novel biomarkers for aggressive subtypes of BC. To our knowledge, this is the first time that the six-transmembrane epithelial antigen of prostate 4 (STEAP4) which emerged from our screening efforts is linked to BC, and provides a novel pharmacological target with potential clinical translation.
STEAP4 is an iron-metabolism-related mitochondrial protein that functions as a metalloreductase involved in cellular iron and coper homeostasis and its expression has been modulated in response to inflammation, oxidative stress and metabolism of fatty acids and glucose (
The overexpression of STEAP4 in the HER2+ BC cells which emerged for the first time in our screening efforts, in tandem with the IHC results in the TMAs, was the inflection point which triggered us to proceed to the pharmacological evaluation of the STEAP4 in HER2+ BC models.
A frequently used iron chelating drug, DFP, has been recently demonstrated to inhibit STEAP4 and as a result it could be used as a promising pharmacological regime against various types of cancer where STEAP4 overexpression is observed. Iron chelators have been previously explored as cancer chemopreventive and chemotherapeutic agents (
With respect to HER2+ BC, there have been significant therapeutic advances in the field over the last decade, with several potent targeted treatments clinically available, including trastuzumab and Lapatinib alone or in combination with chemotherapeutics and new agents targeting other related to HER2 pathways (
Consistent with our hypothesis, the dual drug inhibition of HER2 and STEAP4 by the combinational treatment of Lapatinib with the DFP significantly decreased cell proliferation in the HER2+ BC cell lines (HCC-1954, SKBR3, BT474) than either drug alone, suggesting a new pharmacological treatment scheme for HER2+ BC. Future preclinical studies on the appropriate dose adjustment during this combinational treatment may be needed to minimize untoward Lapatinib-related hepatotoxicities. Furthermore, silencing of STEAP4 using small interfering RNA also resulted in a significant reduction in cell growth and enhanced the inhibition potential of the Lapatinib in the HCC-1954 cells.
Based on our pharmacological findings
In conclusion, STEAP4 may represent a potential BC related biomarker and a promising new pharmacological target for the treatment of HER2+ BC. Further validation studies involving a larger clinical sample size would be necessary to clarify the association of STEAP4 with clinical parameters and its potential role in the tumorigenesis and treatment of HER2+ BC.
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: ProteomeXchange Consortium
All animal procedures were approved by the Bioethical Committee of BRFAA based on the European Directive 86/609.
I-MO designed aspects of the study, performed the experiments, analyzed and interpreted the data, and wrote the manuscript. CT conceived and designed the study, wrote and edited the paper and was the principal investigator of the study. OA prepared the xenograft tumor sections and contributed to the critical revision of the manuscript. AP designed the silencing studies. KV performed the mass spectrometry analysis. ST-B evaluated the clinical tissue microarray samples. OA, AP, ST-B, and KV participated in the manuscript revision and were involved in helpful suggestions and discussions. All authors contributed to the article and approved the submitted version.
The research work was supported by the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT), under the HFRI PhD Fellowship grant (GA. no. 2026). This work was further supported by the “Infrastructure for Preclinical and early-Phase Clinical Development of Drugs, Therapeutics and Biomedical Devices (EATRIS-GR)” (MIS 5028091), which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014- 2020) and co-financed by Greece and the European Union (European Regional Development Fund).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors would like to thank the BRFAA colleagues Dr Stamatis Pagakis and Demosthenes Mitrossilis for assistance with microscopic analysis and IF quantification, Ms Anna Agapaki for tumor tissue sectioning and Dr Manousos Makridakis for technical advice and guidance on the GeLC-MS/MS technique.
The Supplementary Material for this article can be found online at:
Verification of the STEAP4 expression by Western blot and Immunofluorescence (IF) analysis in HER2+ BC cells.
Immunohistochemical analysis of STEAP4 expression in human BC xenograft tissues. STEAP4 expression was examined in the HCC-1954 and MDA-MB-231 xenograft tissues. Representative IHC images (200x magnifications, scale bars = 40 μm) of
In vitro MTT cytotoxicity of Lapatinib compared to combined treatment of Lapatinib and Deferiprone in HER2+ BC cells.
Uncropped Western blot images from the main text (provided as a separate PDF file). Pictures depict membranes probed with antibodies as indicated on the blots. Black boxes represent final cropped regions shown in manuscript. All top to bottom images are different exposure from the same membrane.
Complete lists of membrane proteins found in the HCC-1954 cells and MCF-10A cells (provided as a separate excel file). Combined lists of all the membrane-associated proteins found in four replicates of the HCC-1954 cells compared to four replicates of the MCF-10A cells along with their Normalized Areas. Bold letter proteins represent the top 30 candidate protein targets shown in the Table 1 of the main text.
Complete lists of membrane proteins found in the MDA-MB-231 cells and MCF-10A cells (provided as a separate excel file). Combined lists of all the membrane-associated proteins found in four replicates of the MDA-MB-231 versus the MCF-10A cells along with their Normalized Areas. Bold letter proteins represent the top 30 candidate protein targets shown in the Table 2 of the main text.