Abstract

Introduction

   Breast cancer is a complex heterogeneous disease and is a leading cause
   of death in women. Early diagnosis and monitoring progression of breast
   cancer are important for improving prognosis. The aim of this study was
   to identify protein biomarkers in urine for early screening detection
   and monitoring invasive breast cancer progression.

Method

   We performed a comparative proteomic analysis using ion count relative
   quantification label free LC-MS/MS analysis of urine from breast cancer
   patients (n = 20) and healthy control women (n = 20).

Results

   Unbiased label free LC-MS/MS-based proteomics was used to provide a
   profile of abundant proteins in the biological system of breast cancer
   patients. Data analysis revealed 59 urinary proteins that were
   significantly different in breast cancer patients compared to the
   normal control subjects (p<0.05, fold change >3). Thirty-six urinary
   proteins were exclusively found in specific breast cancer stages, with
   24 increasing and 12 decreasing in their abundance. Amongst the 59
   significant urinary proteins identified, a list of 13 novel
   up-regulated proteins were revealed that may be used to detect breast
   cancer. These include stage specific markers associated with
   pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS)
   samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early
   invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein
   C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer
   (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3
   potential markers (ECM1, MAST4 and filaggrin) identified was performed
   in breast cancer cell lines by Western blotting. One potential marker
   MAST4 was further validated in human breast cancer tissues as well as
   individual human breast cancer urine samples with immunohistochemistry
   and Western blotting, respectively.

Conclusions

   Our results indicate that urine is a useful non-invasive source of
   biomarkers and the profile patterns (biomarkers) identified, have
   potential for clinical use in the detection of BC. Validation with a
   larger independent cohort of patients is required in the following
   study.

Introduction

   Breast cancer (BC) is a major public health problem worldwide. Despite
   the widespread use of mammographic screening, which has contributed to
   reduced mortality, BC is still the most common form of cancer among
   women. It can only be detected using mammography if there is a visible,
   detectible abnormality with architectural distortion or calcification,
   which correlates with the presence of several hundred thousand tumor
   cells. Once BC has been biopsied and the diagnosis has been confirmed
   pathologically, the tumor is surgically excised. The complexity and
   heterogeneity of individual tumors play an important role in
   therapeutic decision making. Pathological examination is still the gold
   standard for diagnosis and assessment of prognostic indicators in BC
   which include tumor size, grade (degree of tumor cell differentiation),
   presence or absence of positive lymph nodes (metastases),
   immunohistochemical expression of key proteins such as estrogen
   receptor (ER), progesterone receptor (PR) and HER2 [[33]1].

   Although advances in BC diagnosis have been made in the last decade,
   there are still many BC patients who cannot be diagnosed in the early
   stages of disease or monitored adequately for tumor recurrence using
   current techniques. To reduce morbidity and mortality from BC, novel
   approaches must be considered for screening, early detection and
   prevention, as well as for monitoring cancer progression or recurrence.
   The early detection of ductal carcinoma in-situ (DCIS) or invasive
   breast cancer (IBC) may prevent the development of life threatening
   metastatic disease. Additionally, monitoring metastatic progression
   could identify early BC recurrence and help guide therapeutic decision
   making.

   Human urine is one of the most interesting and useful bio-fluids for
   clinical proteomics studies. Advances in proteomics, especially in mass
   spectrometry (MS) [[34]2,[35]3] have rapidly changed our knowledge of
   urine proteins which have simultaneously led to the identification and
   quantification of thousands of unique proteins and peptides in a
   complex biological fluid [[36]4,[37]5]. Proteomic studies of urine are
   highly informative, and have been successfully used to discover novel
   markers for cancer diagnosis and surveillance [[38]6–[39]9] as well as
   for monitoring cancer progression [[40]10,[41]11]. Technological
   development combined with the addition of urine screening would
   increase the knowledge about patient status and further assist
   assessment and treatment in clinical practice. Proteomic analysis of
   urine holds the potential to apply a non-invasive method to identify
   novel biomarkers of BC. However, investigation of urinary proteins from
   different stages of BC patients using a liquid chromatography tandem
   mass spectrometry (LC-MS/MS) proteomic approach has not been reported
   to date.

   In this study, we used a label free LC-MS/MS technique to test the
   feasibility of urine as a source for BC biomarkers and identify the
   urinary proteins for BC diagnosis and monitoring progression. One
   potential marker (extracellular matrix protein 1 (ECM1) previously
   identified and associated with BC), and two novel potential protein
   markers (MAST4-microtubule associated serine/threonine kinase family
   member 4 and filaggrin) identified from BC urine were validated in BC
   cell lines and MAST4 was validated in a small number of primary BC
   tissues and in the individual human BC urine samples, demonstrating the
   link of these proteins with BC. However, a larger cohort of BC
   patients’ samples is needed for the validation of the identified
   potential markers in the following studies. The proteins identified
   showed significant differences in abundance between the different BC
   disease stages which provide a useful reservoir of biomarkers for the
   detection of early and advanced BC.

Materials and Methods

Study design and ethics

   In this pilot study, all the female BC subjects had received detailed
   diagnostic procedures, i.e. a physical breast examination, mammography,
   ultrasound and biopsy or excision with a detailed pathological report
   on the cancer. Ethics approval for the collection of human urine and
   tissue samples was approved by the South Eastern Sydney Area Health
   Service Ethics Committee (SEA HRCE) (#07/71Li). The study was designed
   and conducted in accordance with the ethical principles and all
   participants signed informed consent forms. None of the subjects had
   received any prior treatment, either endocrine or chemotherapy. The
   healthy disease free control group (n = 20) were age matched with the
   BC patients (range 35–70 years, mean age, 51 ± 10.5 years). Urine
   samples were collected prior to surgery while BC tissues and normal
   part of breast tissues were collected after surgery (St George Private
   Hospital, Sydney, Australia). The collected samples were evaluated and
   grouped in the analysis according to histopathology report, after
   diagnosis. The breast carcinoma typing and grading were performed by a
   pathologist according to the World Health Organization criteria
   [[42]1]. The samples were grouped into 3 different BC stages: DCIS (n =
   6), early IBC (with or without axillary lymph node involvement, but no
   distant metastases, n = 8), and metastatic breast cancer (MBC) (distant
   metastases to viscera or bone, n = 6) along with a group of samples
   with benign breast disease (BBD) (n = 6). The histopathology
   characteristics and clinical features are summarized in [43]Table 1.

Table 1. The histopathology characteristics and parameters, of the patients
in this study.

   Patient Groups Number of Patients Tumour Size (mm) TumourGrade
   Histological Diagnosis DCIS LN +/- Biomarkers ER/PR status HER-2
   DCIS 6 25–48 3 DCIS
   IBC 5 9–32 2 or 3 Ductal or Lobular present + +/+ -
   2 or 3 Ductal or Lobular present - +/- -
   3 14–22
   MBC 6 10–23 2 or 3 Ductal or Lobular present + +/+ -
   2 or 3 Ductal or Lobular present + +/- +
   10–24
   BBD 6 - - Fibrocystic change, fibroadenoma - - - -
   [44]Open in a new tab

   Notes:LN: Lymph Node involvement; +/-: positive involved/ negative not
   involved; Tumour grade 1–3 [[45]1].

   Abbreviations: DCIS: ductal carcinoma in-situ; IBC: invasive BC; MBC:
   metastatic BC; BBD; benign breast disease; ER: oestrogen receptor
   status; PR: progesterone receptor status; HER2: human epidermal growth
   factor receptor status.

Urine sample collection and processing

   Clean catch (no skin contamination), midstream 30–50 mL urine samples
   were collected in a sterile tube and immediately transported on ice.
   The urine was centrifuged at 2000 x g (4000 rpm), at 4°C for 10 min to
   remove insoluble materials and cellular debris. The supernatants were
   aliquoted and frozen at -20°C and then transferred to -80°C for long
   term storage. All samples were handled by the same standard operating
   procedures and processed for storage within one hour of collection.

   All urine samples had protein concentration and urine creatinine levels
   measured, and abnormal samples were excluded from the study. The
   appropriate volume of urine samples was then pooled within the
   appropriate group to ensure the same total concentration of proteins
   for proteomics analysis. The pooled urine supernatants from each group
   were subjected to total protein precipitation by 1:8 sample-solvent
   ratio of ice-cold (-20°C) acetone, mixed and stored for 1 hour at
   −20°C, and then high speed centrifuged with high speed centrifugation
   (HSC), 11,000 x g at 4°C for 30 min. The supernatants were removed and
   the pellets were further air-dried.

   To further precipitate and concentrate the proteins, the pellets were
   resuspended in 2 mL of fresh TCA solution (concentrated: 10 g TCA in 10
   mL Milli-Q H[2]O) in a 4:1 sample-to-solvent ratio, vortexed, incubated
   at 4°C for 1 hour and then centrifuged with HSC at 4°C for 30 min.
   After carefully discarding the supernatants, protein pellets were
   washed twice with ice-cold acetone for 15 min, along with HSC at 4°C
   for 15 min. All pellets were air-dried as our published method
   [[46]12].

   All protein pellets were resuspended in 100 μL of rehydration buffer
   (RB) solution (2 M thiourea, 7 M urea, 40 mM Tris-base, 1% 3-[(3
   cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM DTT
   and 0.1% Bromothymol Blue) before use, and vigorously vortexed to
   ensure the pellets were completely dissolved. The protein
   concentrations of samples were determined with 2-D Quant Kit method (GE
   healthcare-Life sciences. Product code 80-6483-56) following the
   manufacturer’s instructions.

Urine sample protein clean-up and digestion

   The peptide fractions were enzymatically digested with trypsin.
   Lyophilized protein samples were reconstituted with 25 μL of 50 mM
   Ammonium bi-carbonate (AMBIC) (pH 8). Trypsin (12.5 ng/μL trypsin
   proteomic grade, Sigma-Aldrich, St. Louis, MO, USA) was added to a
   final enzyme-to-protein ratio of 1:100 (w/w) and was incubated at 37°C
   overnight. The reaction was stopped by acidifying the preparation to
   ~pH 3 using neat formic acid (FA). Samples were dried in a vacuum
   centrifuge to concentrate the samples which were stored at -20°C.
   Following trypsin digestion, the peptide samples were purified using
   Strong Cation exchange (SCX) and C18 StageTips (Thermo Scientific, USA)
   following the manufacturer’s instructions.

LC-MS/MS analysis of urine sample

   Label-free LC-MS/MS quantification was performed using an Orbitrap
   Velos (LTQ-Orbitrap, Thermo Scientific, USA). All urine samples were
   run in triplicate. Peptides were reconstituted in 10 μL of 0.1% FA and
   separated by nano-LC using an Ultimate 3000 HPLC and auto sampler
   (Dionex, Amsterdam, Netherlands). The samples (0.6 μL, 2 μg total load)
   were loaded onto a micro C18 pre-column (500 μm × 2 mm, Michrom
   Bio-resources, Auburn, CA, USA) with Buffer A at 10 μL/min (2% ACN and
   0.01% Heptafluorobutyric Acid (HFBA) in water). After a 4-min wash, the
   pre-column was switched (Valco 10 port valve, Dionex) into line with a
   fritless nano column (75 μm diameter × 12 cm) containing reverse phase
   C18 media (3 μm, 200Å Magic, Michrom Bio-resources). Peptides were
   eluted using a linear gradient of Buffer A to Buffer B (98% ACN, 0.01%
   HFBA in water) at 250 nL/min over 60 min. High voltage (2000 V) was
   applied to a low volume tee (Upchurch Scientific, Oak Harbor, WA, USA)
   and the column tip positioned ~0.5 cm from the heated capillary (T =
   280°C) of an Orbitrap Velos (Thermo Electron, Bremen, Germany) mass
   spectrometer. Positive ions were generated by electrospray and the
   Orbitrap was operated in data-dependent acquisition mode. A survey scan
   MS was acquired in the Orbitrap in the 350–1750 m/z range with the
   resolution set to a value of 30 000 at m/z = 400 (with an accumulation
   target value of 1 000 000 ions), with lock-mass enabled. The 10 most
   intense ions (>5000 counts) with charge states +2 to +4 were
   sequentially isolated and fragmented within the linear ion trap using
   CID with an activation q = 0.25 and activation time of 30 milliseconds
   (ms) at a target value of 30 000 ions. The m/z ratios selected for
   MS/MS were dynamically excluded for 30 seconds to prevent repetitive
   selection of the same peptide.

Label-free LC-MS quantitative profiling

   MS peak intensities were analyzed using Progensis QI, LC-MS data
   analysis software (version 4.1, Nonlinear Dynamics, Newcastle upon
   Tyne, UK). Ion intensity maps from each run were aligned to a reference
   map and ion feature matching was achieved by aligning consistent ion
   m/z and retention times. The peptide intensities were normalized
   against total intensity (sample specific log-scale abundance ratio
   scaling factor) and compared between groups by one-way analysis of
   variance (ANOVA, p ≤ 0.05 for statistical significance). Type I errors
   were controlled by False Discovery Rate (FDR) with q value set at 0.02
   [[47]13,[48]14].

   MS/MS spectra were searched and identified against the human protein
   database Uni-Prot database (downloaded January 2013) using the database
   search program MASCOT (Matrix Science, London, UK,
   [49]www.matrixscience.com). Parent and fragment ions were searched with
   tolerances of ± 6 ppm and ± 0.6 Da, respectively. Searched peptide
   charge states were limited to +2 to +4. Deamination (M), Oxidation.
   Phosphorylation was chosen as variable modifications. Only peptides
   with an ion score >25 were considered for protein identification.
   Proteins were considered to be significantly different at p < 0.05;
   fold change >3.

Generation of the heat map

   The area under curve (AUC) of all MS1 peaks generated from comparisons
   among different stages of BC in urine (Progensis data) was normalised
   to the mean of all AUC using TIBCo spotfire (Boston, MA, USA). The
   clustering method used is UPGMA and distance measure was Cosine
   correlation in logarithmic scale for rows. Columns were clustered using
   a Ward’s method with distance measured using Half Square Euclidean.

Cell lines and cell culture

   The human primary breast cancer cell line (BT-474) and metastatic cell
   lines (MDA-MB-231, MCF-7 and SK-BR-3) were obtained from the American
   Type Tissue Culture Collection (ATCC, Rockville, MD). All tissue
   culture reagents were supplied by Invitrogen Australia Pty Ltd
   (Melbourne, VIC, Australia), unless otherwise stated. The primary BC
   cell line (BT-474) was maintained in Improved Minimal Essential Medium
   (IMEM) supplemented with 10% heated-inactivated fetal bovine serum
   (FBS), 50 U/mL of penicillin and 50 μg/mL of streptomycin. Other three
   metastatic BC cell lines (MDA-MB-231, MCF-7 and SKBR-3) cell lines were
   cultured in RPMI-1640 supplemented with 10% FBS, 50 U/mL of penicillin
   and 50 μg/mL of streptomycin. All cell lines were maintained in a
   humidified incubator at 37°C and 5% CO[2]. Protein from each cell line
   was extracted. Briefly, cells with 80–90% confluency were rinsed twice
   with Dulbecco’s phosphate-buffered saline (DPBS) (pH7.2), and then
   lysed in a buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L
   Sodium chloride (NaCl), 0.1% SDS, 10 mmol/L NaF, 1 mmol/L Sodium
   orthovanadate (Na3VO4), 0.5% sodium deoxycholate, 1% Triton X-100, and
   1/12 (v/v) protease inhibitor cocktail (Sigma-Aldrich, Pty Ltd, Castle
   Hills, NSW, Australia). After a brief incubation for 5 min on ice, the
   lysates were collected and centrifuged at 14,000 rpm for 10 min at 4°C
   and the supernatants were collected and stored at -80°C for Western
   blotting experiment.

Western blotting

   Protein expression levels were determined by Western blotting analysis
   as previously described [[50]15]. Human urine protein pellets
   (following acetone/TCA extraction) were re-suspended in rehydration
   buffer (25 mmol/L Tris-HCl (pH 8.0), 0.5% SDS). Briefly, total proteins
   were measured using a BCA assay kit (Thermo Scientific, Rockford, IL,
   USA). Proteins from human BC cell lines and human urine samples were
   resolved on 4–20% Tris-Nupage gels (Invitrogen Australia Pty Ltd,
   Melbourne, VIC, Australia) and transferred to polyvinylidene fluoride
   membranes. Membranes were blocked with 5% bovine serum albumin (BSA)
   (Sigma-Aldrich, Sydney, NSW, Australia) in Tris-buffer with 0.1%
   Tween-20 (TBS-T) for 1 h, and then incubated with mouse anti-secretory
   glycoprotein (ECM1) monoclonal antibody (MAb) (1:500 dilution, Abcam,
   USA), rabbit anti-MAST4 polyclonal antibody (PAb) (1:1000 dilution,
   Abcam, USA) and rabbit anti-filaggrin PAb (1:1000 dilution, Abcam, USA)
   at 4°C overnight (o/n), followed by incubation in horseradish
   peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse
   secondary antibodies (1:2000 dilution). Immunoreactive bands were
   detected using enhanced chemiluminescence (ECL) substrate (Pierce
   Chemical Co, Rockford, USA), and imaged using the ImageQuant LAS4000
   system (GE Health care, USA). To confirm equal loading of protein
   lysates, membranes were stripped (Restore Western Blot Stripping
   Buffer, Pierce) and re-probed using housekeeping antibodies GAPDH (MAb:
   EDM Millipore) or β-tubulin (MAb: Sigma-Aldrich Pty Ltd, Australia),
   then processed as above. Images were processed in Adobe Photoshop.

Immunohistochemistry

   Standard immunoperoxidase procedures were used to visualize MAST4
   expression using our published method [[51]16]. Briefly, paraffin
   sections including BC tissues and normal breast tissues were
   deparaffinised in xylene, followed by a graded series of alcohols
   (100%, 95%, and 75%) and re-hydrated in water followed by Tris-buffered
   Saline (TBS) (pH 7.5). Slides were subsequently immersed in boiling 0.1
   M citrate buffer (pH 6.0) for 30 min to enhance antigen retrieval,
   treated with 3% hydrogen peroxide and then incubated with primary
   rabbit anti-MAST4 PAb (1:100 dilution) o/n at 4°C. After washing with
   TBS, slides were incubated with goat anti-rabbit IgG (Dako, North
   Sydney NSW, Australia) secondary antibody (1:100 dilution) for 45 min
   at room temperature. Sections were finally developed with 3,3’
   diaminobenzidine (DAB) substrate solution (Sigma-Aldrich, Pty Ltd,
   Castle Hills, NSW, Australia) as a chromogen, then counterstained with
   hematoxylin and blued with Scotts Bluing solution. Control slides were
   treated in an identical manner, and stained with an isotype matched
   non-specific immuno-globulin as a negative control. MDA-MB-231 cell
   line was used as positive control.

Assessment of immunostaining

   Staining intensity (0–3) was assessed using light microscopy (Leica
   microscope, Germany) at a x 40 objective as—(negative), + (weak), ++
   (moderate), and +++ (strong) using our previously published method
   [[52]16]. Evaluation of tissue staining was done, independently, by two
   experienced observers (JB and YL). All specimens were scored blind and
   an average of grades was taken. If discordant results were obtained,
   differences were resolved by joint review and consultation with a third
   observer, experienced in immunohistochemical pathology.

Results and Discussion

Proteomic discovery of circulating urinary markers in human BC

   Label-free LC-MS/MS quantification was used to characterize the
   differential expression of urinary proteins in various human BC stages.
   The urine samples were analysed from patients with DCIS, IBC, MBC, BBD
   and normal healthy control subjects (ANOVA p< 0.05; q<0.02). A reverse
   database was also searched to determine protein level FDR. Using
   Progenesis software to compare protein expression between all the
   samples, we identified a total of 166 proteins with 1% FDR (for protein
   identification, determined by searching a reverse database).

   Using the raw urine data and Tibco-spotfire software Inc. 2014, a
   biological heat map of clusters from different stages of BC patients
   and normal health control subjects was produced. The representative
   data are shown in [53]Fig 1. This analysis demonstrates datasets as
   clustered patterns which show an overview of the distribution of urine
   proteins represented according to their expression.

Fig 1. Heat map analysis of urine proteins from BC patients and conrol
subjects.

   [54]Fig 1
   [55]Open in a new tab

   Heat map and dendogram of the raw data of urinary proteins identified
   up or down expression of urine proteins from different stages of BC
   patients and normal health control subjects.

   The data obtained with Progenesis LC-MS analysis was then applied to
   calculate the fold change (FC) as a normalised ratio for disease
   compared to healthy control subjects. This statistical analysis
   revealed 59 significant urinary proteins in BC with >3-fold change
   relative to the normal healthy control subjects. These protein profiles
   are all recorded in [56]Table 2 and [57]S1–[58]S3 Tables. A review of
   the literature demonstrates that the 59 significant proteins changing
   in abundance have not been detected in human urine in either BC or BBD.
   Several of these proteins identified were previously reported in blood,
   tissue and human BC cell lines (associated references are shown in