Identification and Functional Validation of RAD23B as a Potential

Jun 4, 2014 - UV excision repair protein RAD23 homologue B (RAD23B) was found among the downregulated proteins in highly invasive breast cancer cell ...
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Identification and Functional Validation of RAD23B as a Potential Protein in Human Breast Cancer Progression Annett Linge,*,†,§ Priyanka Maurya,† Katrin Friedrich,‡ Gustavo B. Baretton,‡ Shane Kelly,† Michael Henry,† Martin Clynes,† Annemarie Larkin,†,∥ and Paula Meleady†,∥ †

National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland Institute of Pathology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany

‡

S Supporting Information *

ABSTRACT: Identification of protein targets that play a role in breast cancer invasion may help to understand the rapid progression of cancer and may lead to the development of new biomarkers for the disease. In this study, we compared two highly invasive and two poorly invasive breast cancer cell lines using comparative label-free LC−MS profiling in order to identify differentially expressed proteins that may be linked to the invasive phenotype in vitro. Forty-five proteins were found to be upregulated, and 34 proteins, downregulated. UV excision repair protein RAD23 homologue B (RAD23B) was found among the downregulated proteins in highly invasive breast cancer cell lines. In poorly invasive breast cancer cell lines, siRNA-mediated downregulation of RAD23B subsequently led to an increase in invasion and adhesion in vitro. Immunohistochemistry analysis of 164 specimens of invasive breast cancer showed that having a high percentage (>80%) of RAD23B positive nuclei was significantly associated with histopathological grades 1 and 2 breast cancer and with low mitotic activity. In addition, a high staining intensity for RAD23B in the cytoplasm was significantly associated with histopathological grade 3 breast cancer. This study suggests a potential role of RAD23B in breast cancer progression and may further imply a tumor suppressor role of nuclear RAD23B in breast cancer. KEYWORDS: Biomarker, breast cancer, invasion, label-free LC−MS proteomics, RAD23B

1. INTRODUCTION Breast cancer is the most commonly diagnosed cancer in women and is the leading cause of cancer deaths in women age 20−59 years. The 5 year survival rate is close to 98% when the cancer is confined to the breast. However, when cancer cells metastasize to other organs, this rate is reduced to less than 25%.1 Having greater insight into the molecular basis of breast cancer invasion and metastasis may further improve our understanding of the disease and potentially lead to the development of novel drug therapies. A limited number of proteomic studies have been carried out to date using clinical samples to gain a deeper understanding of the processes of invasion and metastasis.2−6 Breast cell lines have also been used to study invasion and metastasis using two main model systems: one approach involves the use of isogenic cell line models of cancer progression and metastasis,7−12 and the second main approach involves the comparison of different breast cell lines with varying invasive phenotypes.13−15 These studies have provided a large number of protein targets that may be useful biomarkers for the invasive phenotype, although the clinical use of a large number of these target proteins remains, as yet, unknown. Further work to validate these targets in vivo using © XXXX American Chemical Society

serum or breast cancer tissue, either individually or as panels of proteins, remains extremely challenging.16 Also, in the majority of microarray and proteomic studies of breast cancer invasion and metastasis, functional analysis of differentially regulated proteins involved in these processes has been limited, and as a result, the significance of a functional role for these proteins in breast cancer invasion remains poorly understood. In this study, we employed quantitative label-free LC−MS analysis profiling to analyze and compare the cellular proteome of four different breast cancer cell lines and to generate a list of protein targets that may be involved in the invasive phenotype of breast cancer: (a) highly invasive, triple-negative Hs 578T and MDA-MB-231 breast cancer cell lines and (b) poorly invasive, luminal MCF7 and T-47D breast cancer cell lines. This resulted in the identification and verification of RAD23B as a potential protein target involved in breast cancer progression. RAD23B is part of the nucleotide excision repair (NER) process17 and has also been shown to interact with the 26S proteasome through its N-terminal ubiquitin-like domain (UbLR23).18 Therefore, Received: December 13, 2013

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2.3. Mass Spectrometry Using LC−MS/MS

RAD23B creates a link between DNA repair and proteasome pathways.18 Recently, a number of DNA repair capacity proteins have been analyzed for SNPs, and RAD23B rs 10739234 was found to be associated with breast cancer risk.19 RAD23B protein has been previously shown to be expressed in breast tissue;20 however, very little is known about a potential role for this protein in breast cancer progression or in cancer in general. In this study, functional analyses were carried out to investigate the role of RAD23B in the invasive phenotype of breast cancer in vitro. RAD23B protein expression was also analyzed in 164 formalin-fixed, paraffin-embedded specimens of invasive breast carcinoma using tissue microarrays and correlated with clinicopathological features.

Nano LC−MS/MS analysis was carried out using an Ultimate 3000 nanoLC system (Dionex, Thermo Fisher Scientific, Hemel Hempstead, Hertfordshire, UK) coupled to a hybrid linear ion trap/Orbitrap mass spectrometer (LTQ Orbitrap XL; Thermo Fisher Scientific). One microgram of digest per sample was loaded onto a C18 trap column (C18 PepMap, 300 μm i.d. × 5 mm, 5 μm particle size, 100 μm pore size; Dionex) and desalted for 10 min using a flow rate of 25 μL/min in 0.1% TFA with 2% acetonitrile (ACN). The trap column was then switched online with the analytical column (PepMap C18, 75 μm i.d. × 500 mm, 3 μm particle and 100 μm pore size; Dionex), and peptides were eluted with the following binary gradients of solvents A and B: 0−20% solvent B in 280 min and 20−98% solvent B in a further 20 min, where solvent A consisted of 2% ACN and 0.1% formic acid in water and solvent B consisted of 80% ACN and 0.08% formic acid in water. Column flow rate was set to 350 nL/min. Data were acquired with Xcalibur software, version 2.0.7 (Thermo Fisher Scientific). The mass spectrometer was operated in data-dependent mode and externally calibrated. Survey MS scans were acquired in the Orbitrap in the 400−1800 m/z range with the resolution set to a value of 60 000 at m/z 400. Up to seven of the most intense ions (1+, 2+, and 3+) per scan were CID-fragmented in the linear ion trap. A dynamic exclusion window was applied within 40 s. All tandem mass spectra were collected using a normalized collision energy of 35%, an isolation window of 3 m/z, and one microscan.

2. MATERIALS AND METHODS 2.1. Cell Culture

All breast cancer cell lines were obtained from the American Type Culture Collection (ATCC). Genomic DNA was prepared from all cell lines according to manufacturer’s instructions (Wizard Genomic DNA Purification Kit; Promega, Madison, WI, USA), and the identity of all cell lines was confirmed by DNA fingerprinting by the Heflin Genomics Core Facility, Department of Genetics, University of Alabama at Birmingham, USA. The following media were used to culture the breast cancer cell lines: MCF7 and T-47D were cultured in DMEM (SigmaAldrich Company Ltd., Irvine, Ayrshire, UK) supplemented with 10% FBS (PAA Laboratories GmbH, Pasching, Austria), Hs 578T was cultured in DMEM supplemented with 10% FBS and 10 μg/mL bovine insulin (Sigma-Aldrich), MDA-MB-231 was cultured in RPMI-1640 (Sigma-Aldrich) supplemented with 10% FBS and 1% sodium pyruvate (Gibco, Life Technologies, Paisley, UK), and MCF10A was cultured in proprietary mammary epithelial growth medium (MEGM) supplemented with a growth factor kit (containing bovine pituitary extract, hEGF, insulin, hydrocortisone, and transferrin) (Lonza, Slough, UK) and 100 ng/mL cholera toxin (Sigma-Aldrich). Cells were cultured without antibiotics and incubated at 37 °C in an atmosphere of 5% CO2. All cell lines were free of Mycoplasma contamination, as tested monthly with the indirect Hoechst staining method.

2.4. Label-Free LC−MS Quantitative Profiling

Label-free LC−MS analysis was carried out using Progenesis label-free LC−MS software, version 4.1 (NonLinear Dynamics Ltd., Newcastle upon Tyne, UK) as previously described21 and essentially as recommended by the manufacturer (see www. nonlinear.com for further background to alignment, normalization, calculation of peptide abundance, etc.). A number of criteria was used to filter the data before exporting the MS/MS output files to MASCOT (www.matrixscience.com) for protein identification; peptide features with ANOVA < 0.01 between experimental groups, mass peaks (features) with charge states from +1 to +3, and greater than three isotopes per peptide. All MS/MS spectra were exported from Progenesis software as a MASCOT Generic file (mgf) and used for peptide identification with MASCOT (version 2.2) searched against the UniProtKB-SwissProt database (taxonomy, Homo sapiens, 20 307 entries, downloaded in January 2013). The search parameters used were as follows: peptide mass tolerance set to 20 ppm, MS/MS mass tolerance set at 0.5 Da; up to two missed cleavages were allowed, carbamidomethylation set as a fixed modification, and methionine oxidation set as a variable modification. Only peptides with ion scores of 350 and above were considered and reimported back into Progenesis LC−MS software for further analysis. From Progenesis LC−MS analysis, a number of criteria was applied to assign a differentially expressed protein as identified and used for quantification between the experimental groups (i.e., highly invasive cell lines compared to poorly invasive cell lines); proteins with ≥3 peptides matched, a ≥2.0-fold difference in abundance between experimental groups, an ANOVA between experimental groups of ≤0.05, and an ion score (confidence score) ≥105 (which is the sum of individual ion scores for each individual peptide used for quantification and identification).

2.2. Sample Preparation for Label-Free LC−MS Analysis

Cell pellets were lysed with lysis buffer containing 6 M Urea, 2 M Thiourea, 4% CHAPS, and 10 mM Tris for 1 h at room temperature followed by centrifugation at 19 200g for 10 min. The supernatant was then cleaned up using the Ready Prep 2-D clean up kit (Bio-Rad Laboratories GmbH, Hercules, CA, USA), and precipitated protein was resuspended in 6 M Urea, 2 M Thiourea, and 10 mM Tris. Protein concentration was determined using the Quick Start Bradford assay (Bio-Rad Laboratories GmbH). Ten micrograms of protein sample was resuspended in 50 mM ammonium bicarbonate. Reduction was performed by adding DTT to a final concentration of 5 mM and incubation at 56 °C for 20 min, and samples were allowed to cool to room temperature for approximately 15 min. They were then alkylated by adding iodoacetamide to a final concentration of 15 mM for 30 min in the dark at room temperature. Digestion with sequence-grade Lys-C (Promega) was carried out at a ratio of 1:20 Lys-C/protein at 37 °C for 6 h followed by a second digestion using sequence-grade trypsin (Promega) at a ratio of 1:25 trypsin/protein at 37 °C overnight. Trifluoroacetic acid (TFA) was added to a final concentration of 0.5% to stop the trypsin digestion.

2.5. Molecular Function Enrichment within Lists of Differentially Expressed Proteins

To assess significant enrichment of gene ontology (GO) molecular function within the lists of differentially expressed B

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2.9. Invasion and Motility Assays

proteins, DAVID was used (http://david.abcc.ncifcrf.gov/). Enrichment was considered to be significant when the Bonferroni p-value adjustment was ≤0.05.

Invasion and motility assays were carried out as previously described.24,25 In brief, Boyden chambers with an 8 μm pore size (BD Biosciences, Bedford, MA, USA) were coated with 100 μL of Matrigel (BD Biosciences) and incubated overnight at 4 °C. Cell suspensions were prepared in serum-free basal media at a concentration of 2 × 105 cells/mL. Seven hundred and fifty microliters of complete media was then added to the lower chamber of the insert in the 24-well plate. Five hundred microliters of the cell suspension was added to the upper chamber and then incubated for 48 h at 37 °C and 5% CO2. Noninvading cells were removed by wiping the inner side of the insert with a PBS-soaked cotton swab. The outer side of the insert was stained with 0.25% crystal violet for 10 min. Excess stain was rinsed off with sterile water. Motility assays were carried out in a similar manner except that uncoated inserts (8 μm pore size) were used. All assays were subjected to statistical analysis using Student’s t-tests (two-tailed, two-sample unequal variance).

2.6. Western Blotting

Protein samples were prepared in Laemmli sample buffer (Sigma-Aldrich), heated at 95 °C for 5 min, and cooled on ice prior to loading onto 4−12% NuPAGE Bis-Tris gels (Invitrogen, Life Technologies). Electrophoretic transfer, blocking, and development of western blots were carried out as described previously.22 The following primary antibodies were used: polyclonal mouse anti-RAD23B (dilution 1:750, v/v; Abnova, Taipei, Taiwan), monoclonal mouse anti-p84 (clone 5E10, dilution 1:1000, v/v; GeneTex, Inc., Irvine, CA, USA [GTX70220]), monoclonal mouse anti-GAPDH (clone 6C5, dilution 1:10 000, v/v; Abcam, Cambridge, UK), monoclonal mouse anti-β-Actin (clone AC-15; Sigma-Aldrich). Bound antibodies were detected using horseradish peroxidaseconjugated secondary antibodies (anti-mouse; dilution 1:5000, v/v; Dako, Glostrup, Denmark).

2.10. Adhesion Assays

2.7. Nuclear and Cytoplasmic Protein Fractionation

Human fibronectin, human collagen IV, and ultrapure mouse laminin (entactin-free) were dissolved following the manufacturer’s instructions (BD Biosciences). Twenty-four-well plates were coated with fibronectin (2 μg/well in PBS) or collagen IV (2 μg/well in 0.05 N HCl) for 1 h at room temperature or with laminin (10 μg/well in DMEM) at 4 °C overnight. Wells were rinsed twice with PBS and blocked with 2% sterile-filtered, heat-inactivated BSA in PBS for 1 h at 37 °C in order to prevent nonspecific adhesion of the cells.26,27 Wells were washed twice with PBS, and 500 μL of serum-free cell suspension was added (2.5 × 104 cells/mL) into each well and incubated for 15 min (fibronectin) or 60 min (collagen IV and laminin) at 37 °C with the 24-well plate lid removed. Wells were then gently rinsed twice with PBS to remove unbound cells. Adherent cells were stained with 0.25% crystal violet for 10 min followed by two washes in sterile water. Plates were allowed to dry, and dye was eluted with 33% glacial acetic acid. Absorbance was read at 570 nm with a reference wavelength of 620 nm.

Nuclear and cytoplasmic protein fractions were prepared using the Nuclear Extract Kit according to the manufacturer’s instructions (Active Motif, Rixensart, Belgium). Briefly, cells were collected in ice-cold phosphate buffered saline (PBS) without Ca2+ and Mg2+, and the cell pellet was then resuspended in ice-cold hypotonic buffer, which causes swelling and fragility of the cytoplasmic membrane. After addition of a detergent, the cytoplasmic protein fraction was collected. The pelleted nuclei were then lysed using the lysis buffer provided with the kit. Nuclear and cytoplasmic protein fractions were stored at −80 °C until required. 2.8. Transient siRNA Transfection

Two independent predesigned Silencer Select siRNA molecules (nos. s11731 and s11732) (Applied Biosystems, Life Technologies) were used for knockdown of RAD23B. Silencer Select siRNAs are chemically modified siRNAs that are meant to reduce overall off-target effects by up to 90% without compromising potency. For each set of siRNA transfections, scrambled siRNA (no. AM4390843) (Applied Biosystems) treated cells were used as a control, but untreated cells and transfection reagent-treated cells were also monitored. siRNA against Kinesin (Applied Biosystems) was used as a positive control to assess the efficiency of the siRNA transfection.23 Solutions of siRNAs at a final concentration of 25 nM per well of a 6-well plate were prepared in 50 μL/well of OptiMEM (Invitrogen). The transfection reagent, NeoFX (Applied Biosystems), was prepared in OptiMEM in parallel and incubated at room temperature for 10 min. An equal volume of this NeoFX solution was then added to each siRNA solution and incubated for a further 10 min at room temperature. One hundred microliters of siRNA/NeoFX complex or NeoFX solution was added to each relevant well. Cells were then added to achieve a cell density per well of 1.5 × 105 cells/well for MCF7, T-47D, and MCF10A cells. The plates were mixed gently and incubated at 37 °C. After 24 h, the culture medium was replaced with fresh medium. After a total of 72 h of transfection, the cells were seeded into invasion and motility chambers for 48 h to assess the effect of siRNA transfection on invasion and motility of the cell lines. In parallel, cells were also collected for protein extraction to verify the effect of siRNA transfection on the RAD23B protein level by western blot analysis.

2.11. Immunohistochemistry

The expression of RAD23B, HER-2/neu, estrogen receptor, and progesterone receptor (see Supporting Information Table 1 for details on antibodies and dilutions used) was analyzed in 164 formalin-fixed, paraffin-embedded (FFPE) specimens of invasive breast carcinoma and in 11 FFPE noncancerous breast specimens using tissue microarrays. Immunohistochemical staining was performed as previously described25 using the Dako REAL EnVision Detection System (Dako). The sections were counterstained with Mayer’s hematoxylin, dehydrated in graded alcohols, and glass mounted using DPX (Sigma-Aldrich). All steps were performed at room temperature. All staining results were scored semiquantitatively by considering the percentage of cells stained and the staining intensity. The percentage of nuclei with RAD23B expression and the cytoplasmic staining intensity were analyzed. The cutoff for the percentage of positive nuclei in a case was the median value of 80%. If the cytoplasmic staining for RAD23B was either missing or detectable in all cells of a case, then the cytoplasmic staining intensity was analyzed by stratification in negative/weak and moderate/strong positive cases. The mitotic count was performed on H&E stained sections, as was histopathological grading, according to Elston and Ellis.28 Chi-squared test was used to compare RAD23B expression and histopathological grade, and Student’s t-test was C

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Table 1. Clinicopathological Features of Human Breast Cancers Studied no. of cases Histological Type invasive ductal invasive lobular other Tumor Stage pT1 pT2 pT3 pT4 Lymph Node Stage pN0 pN1−3 pNx Histopathological Grade grade 1 grade 2 grade 3 Hormone Receptor Expression estrogen receptor negative estrogen receptor 1−9% positive estrogen receptor >9% positive progesterone receptor negative progesterone receptor 1−9% positive progesterone receptor >9% positive HER-2/neu negative positive unknown

133 15 16 79 72 7 6

Figure 1. Comparative analysis of the invasion levels of the four breast cancer cell lines. The total number of invading cells was determined by counting the number of cells per field in 15 fields per chamber within a grid at 200× magnification. The average count was multiplied by the conversion factor of 140 (growth area of membrane/field area viewed at 200× magnification, which was calibrated using a microscope graticule) to determine the total number of invading cells per chamber. Error bars represent standard deviation from data obtained from three separate experiments (n = 3), which included three technical repeats within each of the three separate experiments.

81 71 12 18 86 60

two poorly invasive cell lines were grouped together. They were then compared to each other in order to try to identify proteins potentially involved in the invasive phenotype of breast cancer and to reduce cell line-specific differences. Following Progenesis LC−MS analysis, peptide features with ANOVA ≤ 0.05 and 1+, 2+, and 3+ charge states were subjected to MASCOT database searching. The resultant MASCOT mgf files were then resubmitted to the Progenesis software in order to yield a list of identified proteins. From these lists, proteins with less than three peptides matched and a fold change < 1.5 as well as nonstatistically significant proteins were removed. A total of 45 proteins were found to be upregulated (Table 2) and 34 proteins were found to be downregulated (Table 3) when comparing the highly invasive Hs 578T and MDA-MB-231 cell lines to the poorly invasive MCF7 and T-47D cell lines. All of the listed proteins had peptides present from the differential analysis that were unique only to that protein and did not conflict with related protein family members. Full protein ID information describing the abundance levels for each cell line and identified protein with at least three peptides matched is shown in Supporting Information Table 2A (upregulated proteins in highly invasive compared to poorly invasive breast cancer cell lines) and in Supporting Information Table 2B (downregulated proteins in highly invasive breast cancer cells compared to poorly invasive breast cancer cell lines). Figure 2 shows a representative output from Progenesis LC−MS software of RAD23B identified as being downregulated in highly invasive compared to poorly invasive breast cancer cell lines. We also examined molecular function enrichment among the differentially expressed protein in highly and poorly invasive breast cancer cell lines using DAVID and GO analysis (Table 4). In the upregulated protein list from highly invasive breast cancer cell lines, molecular function enrichment was found relating to the cytoskeleton and cell−cell junctions, and the downregulated protein list showed enriched proteins relating to single-stranded DNA binding. From the literature, the involvement of the cytoskeleton in invasion is wellknown.31 Therefore, we decided to follow up on RAD23B, which came up in the single-stranded DNA binding group. Very little is known about its potential function in breast cancer progression to date.

39 2 123 58 11 94 140 19 5

performed to compare RAD23B expression and mitotic index. The clinicopathological features are outlined in Table 1. Ethical approval for the study was granted from the research ethics committee of the Dresden University Hospital “Carl Gustav Carus” (no. 59032007). 2.12. Statistical Analysis

Statistical analysis was performed using SPSS-Statistics 17 (SPSS, Munich, Germany) for survival analysis (Kaplan−Meier statistics). Excel 2007 was used for comparison of RAD23B expression with clinicopathological features (chi-squared test) and mitotic activity (Student’s t-test). All comparisons were considered to be significant at p < 0.05.

3. RESULTS 3.1. Analysis of Differential Expression of Proteins Using Quantitative Label-Free LC−MS Profiling

To identify cellular proteins that might play a role in breast cancer invasion and metastasis, two poorly invasive luminal breast cancer cell lines (MCF7 and T-47D) and two highly invasive triple-negative breast cancer cell lines (Hs 578T and MDA-MB-231) were chosen. Their invasion status is shown in Figure 1 and is in line with previously published results.29,30 Four biological replicates from each of the two poorly and two highly invasive breast cancer cell lines were separated and analyzed by LC−MS using 5 h reverse gradients per sample. The resultant LC−MS data were transferred to Progenesis LC−MS software to compare the protein expression between the poorly and highly invasive cell lines. The LC−MS runs from the two highly invasive cell lines were grouped together, and the LC−MS runs from the D

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Table 2. List of Proteins with Increased Expression in Highly Invasive Breast Cancer Cell Lines Hs 578T and MDA-MB-231a

a

accession

gene name

protein description

fold

ANOVA (p value)

ion score

% coverage

no. peptides matched

O00154 O00159 O00299 O43707 O75083 O75369 P04083 P05556 P08670 P11388 P13797 P15121 P16070 P17655 P18206 P21333 P24844 P26038 P26639 P27816 P30740 P35579 P35580 P40121 P46013 P46821 P46940 P78417 P78527 Q01082 Q01518 Q07020 Q07065 Q09666 Q13813 Q14315 Q15149 Q16222 Q16555 Q6NZI2 Q96HC4 Q9BUF5 Q9NZM1 Q9P2E9 Q9Y490

ACOT7 MYO1C CLIC1 ACTN4 WDR1 FLNB ANXA1 ITGB1 VIM TOP2A PLS3 AKR1B1 CD44 CAPN2 VCL FLNA MYL9 MSN TARS MAP4 SERPINB1 MYH9 MYH10 CAPG MKI67 MAP1B IQGAP1 GSTO1 PRKDC SPTBN1 CAP1 RPL18 CKAP4 AHNAK SPTAN1 FLNC PLEC UAP1 DPYSL2 PTRF PDLIM5 TUBB6 MYOF RRBP1 TLN1

Cytosolic acyl coenzyme A thioester hydrolase Unconventional myosin-Ic Chloride intracellular channel protein 1 Alpha-actinin-4 WD repeat-containing protein 1 Filamin-B Annexin A1 Integrin beta-1 Vimentin DNA topoisomerase Plastin-3 Aldose reductase CD44 antigen Calpain-2 catalytic subunit Vinculin Filamin-A Myosin regulatory light polypeptide 9 Moesin Threonine−tRNA ligase, cytoplasmic Microtubule-associated protein 4 Leukocyte elastase inhibitor Myosin-9 Myosin-10 Macrophage-capping protein Antigen KI-67 Microtubule-associated protein 1B Ras GTPase-activating-like protei Glutathione S-transferase omega-1 DNA-dependent protein kinase catalytic subunit Spectrin beta chain, nonerythrocytic 1 Adenylyl cyclase-associated protein 1 60S ribosomal protein L18 Cytoskeleton-associated protein 4 Neuroblast differentiation-associated protein Spectrin alpha chain, nonerythrocytic 1 Filamin-C Plectin UDP-N-acetylhexosamine pyrophosphorylase Dihydropyrimidinase-related protein 2 Polymerase I and transcript release factor PDZ and LIM domain protein 5 Tubulin beta-6 chain Myoferlin Ribosome-binding protein 1 Talin-1

3.62 2.77 3.07 2.69 2.90 3.14 14.70 6.85 5.04 6.93 4.43 48.37 13.27 10.03 3.00 2.90 3.64 87.40 2.91 4.32 12.32 5.69 4.80 19.49 26.15 23.23 6.37 4.62 7.87 8.02 2.69 2.54 3.02 4.29 2.69 34.91 3.01 14.35 19.21 8.44 25.51 4.00 8.49 5.96 9.28

1.33 × 10−6 1.03 × 10−4 1.44 × 10−6 3.40 × 10−4 1.18 × 10−7 5.20 × 10−3 1.72 × 10−8 1.41 × 10−10 1.37 × 10−10 2.30 × 10−4 7.85 × 10−4 2.57 × 10−3 5.46 × 10−11 1.23 × 10−8 3.05 × 10−4 1.41 × 10−3 8.72 × 10−4 1.46 × 10−9 1.46 × 10−8 1.91 × 10−8 4.01 × 10−5 4.38 × 10−5 8.53 × 10−3 4.97 × 10−7 5.20 × 10−6 4.24 × 10−8 4.99 × 10−5 2.53 × 10−4 4.46 × 10−3 1.09 × 10−8 4.93 × 10−4 3.02 × 10−4 2.45 × 10−4 2.49 × 10−4 5.48 × 10−10 3.05 × 10−10 8.99 × 10−6 6.23 × 10−7 3.91 × 10−9 1.25 × 10−8 2.87 × 10−10 6.86 × 10−9 3.00 × 10−4 1.46 × 10−8 2.14 × 10−9

229.97 186.48 136.31 621.62 145.81 175.12 505.19 152.85 916.24 248.46 162.05 133.73 277.66 164.34 584.36 204.56 149.61 422.04 228.64 270.85 162.20 1198.40 180.34 158.15 308.03 476.85 941.63 126.02 428.79 1017.14 147.77 162.15 423.28 232.04 833.05 424.06 2408.98 190.19 97.51 213.72 175.25 330.91 338.14 460.35 689.26

13.4 3.6 12 12.8 8.6 1.4 27.5 3.8 33.3 4.9 5.1 9.2 6.1 6 9.8 2.4 17.4 10.7 5.1 5.6 8.2 11.5 2.4 11.5 2.1 4.1 13.6 14.9 2.3 8.8 6.5 19.7 13.6 1.3 7.2 3.9 10.5 9.2 11.4 9 9.1 13 4.5 6.4 5.8

4 3 3 11 4 4 8 3 14 5 3 3 4 3 10 5 3 8 3 5 3 20 4 3 6 8 19 3 8 17 3 3 6 6 15 8 43 4 4 3 4 5 7 6 12

Compared to those of poorly invasive breast cancer cell lines MCF7 and T-47D.

3.2. Validation of RAD23B Protein Levels by Western Blot Analysis

western blot analysis. RAD23B showed a trend for decreased cytoplasmic but increased nuclear localization in poorly invasive breast cancer cell lines compared to that in highly invasive breast cancer cell lines (Figure 3B). GAPDH served as an internal control for cytoplasmic fractions, and p84, as a marker for nuclear fractions. Coomassie staining was also used to show equal loading (not shown).

RAD23B expression was validated by western blot analysis in highly invasive Hs 578T and MDA-MB-231 and poorly invasive MCF7 and T-47D breast cancer cell lines. RAD23B was found to be upregulated in whole-cell lysates of poorly invasive MCF7 and T-47D breast cancer cell lines compared to its level in the highly invasive Hs 578T and MDAMB-231 cell lines (Figure 3A). To assess the cellular distribution of RAD23B in breast cancer cell lines, cytoplasmic and nuclear fractions of poorly invasive MCF7 and T-47D cells as well as those of highly invasive Hs 578T and MDA-MB-231 cells were prepared and subjected to

3.3. Downregulation of RAD23B Protein Levels Using siRNA Results in Increased Invasion of MCF7 and T-47D cells

Western blot analysis of whole-cell lysates showed a trend for increased expression of RAD23B in poorly invasive breast cell lines MCF7 and T-47D compared to its level in highly invasive cell lines Hs 578T and MDA-MB-231, as shown in Figure 3A. E

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Table 3. List of Proteins with Decreased Expression in Highly Invasive Breast Cancer Cell Lines Hs 578T and MDA-MB-231a accession

gene name

O75131 O75390 P00505 P00966 P02786 P04792 P05783 P05787 P06396 P07384 P08727 P11586 P20073 P22314 P30084 P31939 P40926 P42704

CPNE3 CS GOT2 ASS1 TFRC HSPB1 KRT18 KRT8 GSN CAPN1 KRT19 MTHFD1 ANXA7 UBA1 ECHS1 ATIC MDH2 LRPPRC

P48735 P49327 P49588 P53004 P54727 P54886 P55786 P58107 Q02790 Q06323 Q12931 Q13263 Q14566 Q16719 Q86VP6 Q96AE4

IDH2 FASN AARS BLVRA RAD23B ALDH18A1 NPEPPS EPPK1 FKBP4 PSME1 TRAP1 TRIM28 MCM6 KYNU CAND1 FUBP1

a

protein description Copine-3 Citrate synthase, mitochondrial Aspartate aminotransferase, mitochondrial Argininosuccinate synthase Transferrin receptor protein 1 Heat shock protein beta-1 Keratin, type I cytoskeletal 18 Keratin, type II cytoskeletal 8 Gelsolin Calpain-1 catalytic subunit Keratin, type I cytoskeletal 19 C-1-tetrahydrofolate synthase, cytoplasmic Annexin A7 Ubiquitin-like modifier-activating enzyme 1 Enoyl-CoA hydratase, mitochondrial Bifunctional purine biosynthesis protein Malate dehydrogenase, mitochondrial Leucine-rich PPR motif-containing protein, mitochondrial Isocitrate dehydrogenase [NADP], mitochondrial Fatty acid synthase Alanine−tRNA ligase, cytoplasmic Biliverdin reductase A UV excision repair protein RAD23 homologue B Delta-1-pyrroline-5-carboxylate synthase Puromycin-sensitive aminopeptidase Epiplakin Peptidyl-prolyl cis−trans isomerase FKBP4 Proteasome activator complex subunit 1 Heat shock protein 75 kDa, mitochondrial Transcription intermediary factor 1-beta DNA replication licensing factor MCM6 Kynureninase Cullin-associated NEDD8-dissociated protein 1 Far upstream element-binding protein 1

fold

ANOVA (p value)

ion score

% coverage

no. peptides matched

2.82 3.86 2.91 57.51 5.00 10.27 34.39 43.65 9.54 3.15 20.37 2.51 2.76 3.31 3.76 3.43 2.74 2.52

9.92 × 10−6 7.45 × 10−7 2.31 × 10−7 1.01 × 10−7 8.33 × 10−6 1.99 × 10−4 8.58 × 10−4 1.18 × 10−10 2.81 × 10−5 1.20 × 10−3 9.17 × 10−8 1.73 × 10−4 3.07 × 10−4 2.38 × 10−9 4.57 × 10−6 2.47 × 10−7 1.80 × 10−7 1.90 × 10−4

288.29 206.53 420.32 102.27 153.28 126.27 413.72 555.30 132.27 140.79 291.44 595.88 173.60 242.16 153.09 423.30 226.50 200.57

8.9 10.9 16 5.8 4.2 22.9 18.1 26.5 4.7 4.5 13.8 11.6 8.4 5.9 11.7 16.2 12.7 2.9

5 5 6 3 3 3 10 12 3 3 6 10 4 5 3 8 4 5

7.12 6.19 3.59 3.01 2.80 2.74 7.69 23.64 7.34 2.83 4.36 3.20 2.86 12.19 2.97 2.77

2.21 × 10−7 2.61 × 10−7 2.69 × 10−6 4.99 × 10−5 2.14 × 10−4 1.03 × 10−6 5.11 × 10−6 5.80 × 10−10 1.89 × 10−10 4.46 × 10−4 3.11 × 10−8 4.31 × 10−4 1.30 × 10−3 1.18 × 10−11 3.44 × 10−9 2.25 × 10−3

591.11 1447.19 264.55 210.69 201.84 196.09 559.94 394.87 393.01 169.37 223.82 272.32 199.51 284.05 176.01 320.34

25.7 11.4 4.6 16.6 10.3 4.2 12.1 1.9 16.1 13.3 8.9 5.9 4.1 10.5 5 9.8

9 25 4 4 4 3 11 8 7 3 5 5 3 5 5 5

Compared to those of poorly invasive breast cancer cell lines MCF7 and T-47D.

Figure 2. Representative output from Progenesis LC−MS label-free software. RAD23B expression is decreased in highly invasive breast cancer cell lines Hs 578T and MDA-MB-231 (left) compared to its level in poorly invasive breast cancer cell lines MCF7 and T-47D (right). The graph shows average normalized abundance volumes of the peptides identified from RAD23B. The horizontal axis represents the four individual biological replicates of (1) each of the highly invasive breast cancer cell lines, Hs 578T and MDA-MB-231, and (2) each of the poorly invasive breast cancer cell lines, MCF7 and T-47D (n = 4). The vertical axis represents normalized abundance volumes (log).

To study the potential effect of RAD23B on breast cancer invasion, RAD23B downregulation experiments using siRNA were performed in the MCF7 and T-47D cell lines. Two independent siRNA molecules were used to reduce the expression of RAD23B protein in MCF7 and T-47D cell lines,

and this was confirmed by western blot analysis, as shown in Figure 4i. Following siRNA transfection, invasion and migration assays were performed to investigate potential effects associated with the reduction of RAD23B expression in MCF7 and T-47D cell lines. The total number of cells invading the membrane of the F

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cells, which are known to be estrogen-receptor negative,32 to try to clarify whether the effect seen by reduction of RAD23B expression and associated increase in invasiveness of T-47D and MCF7 cells is potentially independent of estrogen-receptor status. We found that reduction of RAD23B expression in MCF10A significantly increased the invasion of MCF10A cells, and this is shown in Supporting Information Figure 2.

Table 4. GO Molecular Function Enrichment for Differentially Expressed Proteins in Highly Invasive Breast Cancer Cell Linesa molecular function Upregulated GO: 0005856 (cytoskeleton) GO: 0043228 (nonmembranebounded organelle) GO: 0043232 (intracellular nonmembrane-bounded organelle) GO: 0015629 (actin cytoskeleton) GO: 0044430 (cytoskeletal part) GO: 0043292 (contractile fiber) GO: 0005938 (cell cortex) GO: 0042995 (cell projection) GO: 0044449 (contractile fiber part) GO: 0005829 (cytosol) GO: 0030863 (cortical cytoskeleton) GO: 0030016 (myofibril) GO: 0016323 (basolateral plasma membrane) GO: 0005912 (adherens junction) GO: 0044448 (cell cortex part) GO: 0070161 (anchoring junction) GO: 0030017 (sarcomere) Downregulated GO: 0003697 (single-stranded DNA binding)

count

p value

adjusted

27 31

2.9 × 10−16 1.5 × 10−13

5.9 × 10−14 2.6 × 10−11

31

1.5 × 10−13

2.6 × 10−11

14 16 8 8 13 7 16 5 6 7

8.6 × 10−13 7.4 × 10−8 8.2 × 10−8 3.0 × 10−7 8.6 × 10−7 1.3 × 10−6 5.4 × 10−6 1.6 × 10−5 2.3 × 10−5 3.7 × 10−5

1.5 × 10−10 1.3 × 10−5 1.5 × 10−5 5.3 × 10−5 1.5 × 10−4 2.2 × 10−4 9.7 × 10−04 2.8 × 10−03 4.1 × 10−3 6.5 × 10−3

6 5 6 5

1.2 × 10−4 1.3 × 10−4 1.9 × 10−4 2.4 × 10−4

2.0 × 10−2 2.0 × 10−2 3.0 × 10−2 4.0 × 10−2

4

3.6 × 10−4

5.0 × 10−2

3.4. Downregulation of RAD23B Protein Levels Using siRNA Increases Adhesion to Fibronectin of Breast Cancer Cells

In order to assess if RAD23B is involved in adhesion of breast cancer cells, adhesion assays were carried out using 24-well plates that were pretreated with fibronectin, collagen IV, or laminin. Downregulation of RAD23B protein expression resulted in a significant increase in the adhesion of MCF7 (1.22-fold increase for siRNA no. 1, p < 0.001, and 1.21-fold increase for siRNA no. 2, p < 0.001, compared to that of scrambled siRNA-treated cells) and T-47D cells (1.42-fold increase for siRNA no. 1, p < 0.001, and 1.49-fold increase for siRNA no. 2, p < 0.001, compared to that of scrambled siRNA-treated cells) to fibronectin (Figure 4iii). Adhesion to collagen IV and laminin was not altered after knockdown of RAD23B in MCF7 and T-47D cell lines (not shown). 3.5. Decreased Nuclear RAD23B Expression in High-Grade Breast Cancers

To assess the role of RAD23B in breast cancer in vivo, RAD23B expression was also examined in 164 FFPE specimens of invasive breast cancer. All breast cancers showed nuclear expression of RAD23B. This percentage ranged from 5 to 90%, with a median value of 80%. Cytoplasmic RAD23B expression was detected in 142 cases, with different staining intensities: 80 cases with weak, 56 with moderate, and 6 cases with strong staining intensity. The number of grade 3 cases with at least 80% RAD23B positive nuclei was significantly lower (28 out of 60 cases) than the number of grade 1 and 2 cases with such a high percentage of RAD23B positive nuclei (73 out of 104 cases) (p = 0.0302; Table 5) (Figure 5A,B). The mitotic activity was lower in cases with RAD23B expression in more than 80% of nuclei compared to cases with RAD23B expression in a lower percentage of nuclei (12.2 ± 8.2 vs 8.8 ± 7.9; p = 0.009; Table 5). Moderate or high cytoplasmic expression was significantly associated with high histological grade 3 compared to that for lower histological grades 1 and 2 breast cancer (29 out of 60 cases vs 33 out of 104 cases; p = 0.0376) (Table 6). No correlation between nuclear RAD23B and estrogen receptor expression (p = 0.3192) or between cytoplasmic RAD23B and estrogen receptor expression was observed (p = 0.9313). Furthermore, there was no correlation between other clinicopathological features (such as histological type, tumor and lymph node stage, hormone receptor expression, and HER-2/neu) and RAD23B expression (chi-squared test). In noncancerous breast epithelium, RAD23B was detected in both nuclei and cytoplasm, with an increased staining intensity in the nuclei (Figure 5C).

a Enrichment was considered significant upon observation of a p value of ≤0.05 and a Bonferroni adjusted p value ≤0.05. Count corresponds to the overlap between proteins on the list and a particular GO category.

Figure 3. Western blot analysis of (A) whole-cell extracts and (B) cytoplasmic and nuclear fractions. Proteins were separated by SDSPAGE and electrophoretically transferred to PVDF membranes followed by incubation with anti-RAD23B antibody. β-Actin served as internal loading control for whole-cell lysates; p84 and GAPDH served as internal controls for nuclear and cytoplasmic fractions, respectively. Representative data from one out of three independent experiments is shown (C, cytoplasmic fraction; N, nuclear fraction).

invasion chamber was significantly increased following siRNA transfection in MCF7 cells (2.65-fold increase, p < 0.001, for siRNA no. 1 and 2-fold increase for siRNA no. 2; p < 0.001, compared to that for scrambled siRNA-treated cells) as well as in T-47D cells (1.8-fold increase for siRNA no. 1, p < 0.001, and 1.74-fold increase for siRNA no. 2, p < 0.001, compared to that for scrambled siRNA-treated cells) (Figure 4ii). In both cell lines, RAD23B downregulation did not significantly affect migration (Supporting Information Figure 1). Proliferation assays were also carried out following siRNA knockdown in both cell lines, and there was no effect on the proliferative rates of the siRNA-treated cell lines compared to those of the scrambled controls (not shown). Because both T-47D and MCF7 cell lines are estrogenreceptor positive, we transfected RAD23B siRNAs into MCF10A

4. DISCUSSION In our comparative proteomic study, we compared breast cancer cell lines with differing invasive phenotypes: poorly invasive and estrogen receptor-positive MCF7 and T-47D breast cancer cell lines and highly invasive but estrogen receptor-negative Hs 578T and MDA-MB-231 breast cancer cell lines. It is possible that the proteins we identified are not only related to the invasiveness of G

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Figure 4. Effect of transient siRNA-mediated knockdown of RAD23B on (i) the protein level, (ii) invasion, and (iii) adhesion of poorly invasive (left) MCF7 and (right) T-47D cell lines. Two independent siRNA molecules of RAD23B were transfected into the cells. (i) Untreated cells (control), transfection reagent-treated cells (NeoFX), scrambled siRNA control (scrambled), RAD23B siRNA no. 1 and RAD23B siRNA no. 2 treated cells were harvested 72 h post-transfection and subjected to western blot analysis. GAPDH served as an internal loading control. (ii) Total number of cells invading through the membrane of invasion chambers after siRNA transfection. (iii) siRNA-transfected cells were allowed to adhere to fibronectin. Error bars represent standard deviation from data obtained from three separate experiments (n = 3), which included three technical repeats within each of the three separate experiments. *, p < 0.05 and ***, p < 0.001 compared to the assay results from the scrambled controls.

line studies on breast cancer invasion and metastasis, such as KRT8,14,31,33 KRT19,31 VIM,31 HSPB1,9,13,14 ITGB1,34 CD44,35 CapG,36 and CLIC1.11,12,14 Other identified proteins, such as ANX7,37 AKR1B1,38 and FLNA,39 have been previously linked to playing a role in cancer. RAD23B protein was also among the differentially expressed proteins and has previously been reported to be expressed in primary breast cancer tissue.20 However, to date, most studies on RAD23B have been carried out in yeast, and very little is known on the functions of its human homologue and its potential role in human cancer. RAD23B is involved in nuclear excision repair (NER), which is a versatile repair mechanism that repairs a variety of DNA lesions that are caused by ultraviolet or ionizing radiation as well as exogenous agents such as mutagenic and carcinogenic chemicals.40 It is also involved in other cellular processes such as cell cycle progression.41 Here, we show that poorly invasive breast cancer cell lines express higher levels of RAD23B compared to that in highly invasive breast cancer cell lines. We have also shown that knockdown of RAD23B expression using transient siRNA assays increased the invasiveness of estrogen-positive breast cancer cell lines, MCF7 and T-47D, and the estrogen-negative breast cell line, MCF10A. Our immunohistochemical analysis of 164 FFPE invasive breast

Table 5. Nuclear RAD23B Expression in Human Breast Cancera nuclear RAD23B expression RAD23B ≤ 80% Histopathological Grade grades 1 and 2 grade 3 Mitotic Index mean value (standard deviation)

RAD23B > 80%

p value

31/104 28/60

73/104 32/60

0.0302b

12.25 (8.18)

8.81 (7.87)

0.0090c

a

A median cutoff of 80% was used for the percentage of positive nuclei stained with RAD23B in low- and high-grade breast cancers. Significant results of immunohistochemical analysis on tissue microarrays are shown. bStatistical analysis was performed using the chi-squared test; a p value < 0.05 was considered to be significant. cStatistical analysis was performed using the Student’s t-test; a p value < 0.05 was considered to be significant.

the cell lines examined but also to their estrogen receptor status or a combination of both. However, our comparative study yielded several differentially expressed proteins that were found to overlap with recent cell H

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Figure 5. Immunohistochemical staining of RAD23B in invasive breast cancer and noncancerous breast epithelium. (A) Low-grade breast cancer, (B) high-grade breast cancer, and (C) noncancerous breast epithelium. Low-grade breast cancer showed predominantly nuclear RAD23B expression compared to that of high-grade breast cancer, which shows cytoplasmic and nuclear RAD23B expression.

histopathological grades 1 and 2 cancers with low mitotic activity could be due to the association of RAD23B with XPC and Centrin 2 in the DNA damage recognition complex.46,47 Centrin was first identified as a small acidic calcium-binding protein in the flagellar apparatus of unicellular green algae.48 Centrin 2 is one of the Centrin homologues and is ubiquitously expressed;49 it has been found to play an important role in centriole duplication.49,50 Together with Centrin 2, RAD23B stabilizes XPC in vitro, linking DNA repair and cell cycle progression.51 A cell cycledependent distribution of RAD23B was also reported by Katiyar and Lennarz,41 who showed a predominant nuclear localization of RAD23B during interphase and a cytoplasmic localization during mitosis in HeLa cells. We further examined the correlation between RAD23B expression and estrogen receptor status of these breast cancer specimens, but no positive correlation was found. This further strengthens the notion that RAD23B is linked to breast cancer invasion and not to estrogen receptor status itself. There was also no correlation between RAD23B and other clinicopathological features assessed. We further demonstrated that RAD23B downregulation induces invasion and adhesion in poorly invasive MCF7 and T-47D breast cancer cell lines in vitro. No correlation between RAD23B expression and invasion/lymph node metastasis was observed in vivo. This seems to be in disagreement, but it could be due to a lot of features, such as the impact of the tumor microenvironment and cell−cell contacts in breast cancer tissues as well as tumor heterogeneity.52−55 Taken together, our studies show that RAD23B is not only differentially expressed in highly invasive versus poorly invasive breast cell lines but also shows a different distribution within the cells between low- and high-grade breast cancers in vivo. This indicates an involvement of RAD23B in breast cancer progression. A low percentage of RAD23B positive nuclei in highgrade breast cancer may also imply a tumor suppressor role of RAD23B. However, more investigations have to be carried out in order to improve our understanding of the roles of RAD23B. In conclusion, this study demonstrates that a proteomic approach using label-free proteomic analysis and mass spectrometry to identify proteins that may be related to the invasive phenotype can yield proteins that have a functional effect on the invasive phenotype in vitro.

Table 6. Cytoplasmic RAD23B Expression in Human Breast Cancera cytoplasmic RAD23B expression negative/weak Histopathological Grade grades 1 and 2 71/104 grade 3 31/60

moderate/strong

p value

33/104 29/60

0.0376b

a

Cytoplasmic expression of RAD23B was either missing or detectable in all cells of a case. The cytoplasmic staining intensity was therefore analyzed by stratification in negative, weakly, moderately, or strongly positive cases. Significant results of immunohistochemical analysis on tissue microarrays are shown. bStatistical analysis was performed using the chi-squared test; a p value < 0.05 was considered to be significant.

cancers has shown that high cytoplasmic RAD23B expression is significantly associated with histopathological grade 3. In addition, breast cancers with histopathological grades 1 and 2 more frequently displayed a high percentage (>80%) of RAD23B positive nuclei than that in grade 3 cancers. It has been reported that breast cancer evolution is closely related to DNA damage repair defects or defects in cell-cycle checkpoints that allow damaged DNA to be maintained, resulting in the accumulation of abnormal or damaged proteins and an increase in proteasomal degradation.42,43 Medicherla et al. identified RAD23 as an essential component of endoplasmic reticulum-associated degradation (ERAD), in which aberrant proteins in the ER are recognized by the protein quality control machinery and translocated to the cytosol for degradation by the proteasome.44 Thus, the increased cytoplasmic localization of RAD23B in histopathological grade 3 breast cancers may reflect this involvement of RAD23B in proteasomal degradation, making it more efficient. However, the prevention of an accumulation of abnormal proteins (and thereby apoptosis) may lead to a growth advantage of tumor cells and help to maintain the invasive phenotype, driving breast cancer progression. Interestingly, Ortolan et al. demonstrated that RAD23 inhibits degradation of proteasome substrates.45 They further proposed that the UbL domain of RAD23 can negatively regulate degradation of DNA repair factors after DNA damage, which may help to promote NER.45 Increased RAD23B expression and subsequently enhanced DNA repair has also been found to cause cisplatin resistance in A549 human lung cancer cells.46 Recently, RAD23B was among a number of DNA repair capacity proteins that have been analyzed for eight singlenucleotide polymorphisms in terms of breast cancer development. It was found that RAD23B rs 10739234 is significantly associated with breast cancer risk.19 This further supports the potential role of RAD23B in breast cancer progression. From the immunohistochemical analysis, the association of RAD23B expression in a high percentage of nuclei in

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ASSOCIATED CONTENT

S Supporting Information *

Figure 1: Effect of transient siRNA-mediated knockdown on migration in poorly invasive MCF7 and T-47D cells. Figure 2: Effect of transient siRNA-mediated knockdown of RAD23B on (i) the protein level and (ii) invasion in MCF10A cells. Table 1: Antibodies used for immunohistochemical analysis of human I

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of label-free and SILAC-based quantification with shotgun, directed, and targeted MS approaches. J. Proteome Res. 2013, 12, 4627−4641. (7) Mbeunkui, F.; Metge, B. J.; Shevde, L. A.; Pannell, L. K. Identification of differentially secreted biomarkers using LC−MS/MS in isogenic cell lines representing a progression of breast cancer. J. Proteome Res. 2007, 6, 2993−3002. (8) Leth-Larsen, R.; Lund, R.; Hansen, H. V.; Laenkholm, A. V.; Tarin, D.; Jensen, O. N.; Ditzel, H. J. Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry. Mol. Cell. Proteomics 2009, 8, 1436− 1449. (9) Kreunin, P.; Yoo, C.; Urquidi, V.; Lubman, D. M.; Goodison, S. Proteomic profiling identifies breast tumor metastasis-associated factors in an isogenic model. Proteomics 2007, 7, 299−312. (10) Ho, J.; Kong, J. W.; Choong, L. Y.; Loh, M. C.; Toy, W.; Chong, P. K.; Wong, C. H.; Wong, C. Y.; Shah, N.; Lim, Y. P. Novel breast cancer metastasis-associated proteins. J. Proteome Res. 2009, 8, 583−594. (11) Lau, T. Y.; Power, K. A.; Dijon, S.; de Gardelle, I.; McDonnell, S.; Duffy, M. J.; Pennington, S. R.; Gallagher, W. M. Prioritization of candidate protein biomarkers from an in vitro model system of breast tumor progression toward clinical verification. J. Proteome Res. 2010, 9, 1450−1459. (12) Xu, S. G.; Yan, P. J.; Shao, Z. M. Differential proteomic analysis of a highly metastatic variant of human breast cancer cells using twodimensional differential gel electrophoresis. J. Cancer Res. Clin. Oncol. 2010, 136, 1545−1556. (13) Nagaraja, G. M.; Othman, M.; Fox, B. P.; Alsaber, R.; Pellegrino, C. M.; Zeng, Y.; Khanna, R.; Tamburini, P.; Swaroop, A.; Kandpal, R. P. Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: comprehensive profiles by representational difference analysis, microarrays and proteomics. Oncogene 2006, 25, 2328− 2338. (14) Lai, T. C.; Chou, H. C.; Chen, Y. W.; Lee, T. R.; Chan, H. T.; Shen, H. H.; Lee, W. T.; Lin, S. T.; Lu, Y. C.; Wu, C. L.; Chan, H. L. Secretomic and proteomic analysis of potential breast cancer markers by two-dimensional differential gel electrophoresis. J. Proteome Res. 2010, 9, 1302−1322. (15) Imai, K.; Ichibangase, T.; Saitoh, R.; Hoshikawa, Y. A proteomics study on human breast cancer cell lines by fluorogenic derivatizationliquid chromatography/tandem mass spectrometry. Biomed. Chromatogr. 2008, 22, 1304−1314. (16) Paulovich, A. G.; Whiteaker, J. R.; Hoofnagle, A. N.; Wang, P. The interface between biomarker discovery and clinical validation: the tar pit of the protein biomarker pipeline. Proteomics: Clin. Appl. 2008, 2, 1386− 1402. (17) Watkins, J. F.; Sung, P.; Prakash, L.; Prakash, S. The Saccharomyces cerevisiae DNA repair gene RAD23 encodes a nuclear protein containing a ubiquitin-like domain required for biological function. Mol. Cell. Biol. 1993, 13, 7757−7765. (18) Schauber, C.; Chen, L.; Tongaonkar, P.; Vega, I.; Lambertson, D.; Potts, W.; Madura, K. Rad23 links DNA repair to the ubiquitin/ proteasome pathway. Nature 1998, 391, 715−718. (19) Perez-Mayoral, J.; Pacheco-Torres, A. L.; Morales, L.; AcostaRodriguez, H.; Matta, J. L.; Dutil, J. Genetic polymorphisms in RAD23B and XPC modulate DNA repair capacity and breast cancer risk in Puerto Rican women. Mol. Carcinog. 2013, 52, 127−138. (20) Chen, L.; Madura, K. Evidence for distinct functions for human DNA repair factors hHR23A and hHR23B. FEBS Lett. 2006, 580, 3401− 3408. (21) Meleady, P.; Gallagher, M.; Clarke, C.; Henry, M.; Sanchez, N.; Barron, N.; Clynes, M. Impact of miR-7 over-expression on the proteome of Chinese hamster ovary cells. J. Biotechnol. 2012, 160, 251− 262. (22) Meleady, P.; Henry, M.; Gammell, P.; Doolan, P.; Sinacore, M.; Melville, M.; Francullo, L.; Leonard, M.; Charlebois, T.; Clynes, M. Proteomic profiling of CHO cells with enhanced rhBMP-2 productivity following co-expression of PACEsol. Proteomics 2008, 8, 2611−2624.

breast cancer specimens. Table 2A: Upregulated proteins in highly invasive breast cancer cell lines Hs 578T and MDA-MB231 compared to those in poorly invasive breast cancer cell lines MCF7 and T-47D. Table 2B: Downregulated proteins in highly invasive breast cancer cell lines Hs 578T and MDA-MB-231 compared to those in poorly invasive breast cancer cell lines MCF7 and T-47D. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 0049 351 458 5299. Fax: 0049 351 458 5716. Present Address §

(A.Li.) German Cancer Consortium (DKTK), Dresden, Germany; Department of Radiation Oncology and OncoRay − National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany; Helmholtz-Zentrum Dresden − Rossendorf; and German Cancer Research Center (DKFZ), Heidelberg, Germany.

Author Contributions ∥

A.La. and P.M contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Florian Goerl (MD) for the composition and preparation of the tissue microarrays. This work was supported by funding from the Higher Education Authority Programme for Research in Third Level Institutions (Cycle 3), a Career Start Grant from Dublin City University awarded to Paula Meleady, and a Deutsche Forschungsgemeinschaft research fellowship to Annett Linge (GZ Li 1900/1-1). The funding sources were not involved in the study design, collection, analysis, and interpretation of data, in the writing of the manuscript, or in the decision to submit the manuscript for publication.

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REFERENCES

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dx.doi.org/10.1021/pr4012156 | J. Proteome Res. XXXX, XXX, XXX−XXX