A targeted glycoproteomic approach identifies cadherin-5 as a novel biomarker of metastatic breast cancer

Cancer Letters 328 (2013) 335–344

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A targeted glycoproteomic approach identifies cadherin-5 as a novel biomarker of metastatic breast cancer Simon A. Fry a,⇑, John Sinclair b, John F. Timms b, Anthony J. Leathem b, Miriam V. Dwek a a b

Against Breast Cancer Research Unit, Dept. of Molecular and Applied Biosciences, University of Westminster, London W1W 6UW, UK EGA Institute for Women’s Health, University College London, London WC1E 6BT, UK

a r t i c l e

i n f o

Article history: Received 2 August 2012 Received in revised form 2 October 2012 Accepted 9 October 2012

Keywords: Breast cancer Metastasis Biomarker Glycosylation

a b s t r a c t Aberrant glycosylation has long been recognised as a hallmark of cancer, and is increasingly being exploited in biomarker discovery studies. Helix pomatia agglutinin (HPA) is known to bind aberrant glycans associated with metastatic breast cancer, and was used here to isolate glycoproteins from pooled breast cancer serum samples of (i) patients with recurrent breast cancer and (ii) patients with no sign of recurrence 5 years after diagnosis of their primary tumour. Pregnancy zone protein, the polymeric immunoglobulin receptor and cadherin-5 emerged as potential markers of metastasis following proteomic identification of HPA binding glycoproteins. ELISAs were developed to verify these findings, and to assess protein glycosylation, in individual patient sera. The cadherin-5 ELISA discriminated serum samples of patients with recurrent breast cancer from those with no sign of recurrence, and analysis of cadherin5 glycosylation by HPA also showed a significant difference between the two sample groups. The targeted glycoproteomic and validatory approach developed here has shown that when taking into account both the protein levels and HPA binding, serum cadherin-5 discriminated patients with recurrent breast cancer from those with no sign of recurrence with 90% specificity. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Breast cancer is the most common form of cancer in women, with a worldwide mortality rate that has increased at an annual rate of 1.8% over the past three decades [1]. In 2009 breast cancer was reported in more than 1.2 million women and accounted for approximately 500,000 female deaths [2]. The main cause of mortality after breast cancer is metastatic dissemination of the primary tumour to distant sites in the body. Whilst there have been many efforts to define the likelihood of this happening, new markers capable of identifying metastatic breast cancer are required to aid clinical decision making for individual patients [3,4]. The prognostic tests in current clinical use require tumour tissue to be obtained by biopsy or other surgical approaches. It is desirable to minimise such invasive procedures, and new validated serum/ plasma biomarkers are urgently required. Whilst several serum biomarkers have been evaluated over the past three decades, including CA15.3, carcinoembryonic antigen (CEA), CA27.29, tissue polypeptide antigen, tissue polypeptide specific antigen and the shed form of human epidermal growth factor receptor 2 (HER-2),

⇑ Corresponding author. Tel.: +44 0207 9115000x64410. E-mail addresses: [email protected] (S.A. Fry), [email protected] (J. Sinclair), [email protected] (J.F. Timms), [email protected] (A.J. Leathem), [email protected] (M.V. Dwek). 0304-3835/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2012.10.011

none of these possess sufficient accuracy in predicting recurrence [5], an event which occurs in approximately 30–40% of patients diagnosed with primary breast cancer [3,6]. Although several studies suggest that increasing marker concentrations can offer a lead time over clinical detection of breast cancer metastasis, none have the strength to inform the appropriate therapeutic regimen [6]. Despite recent advances in biomarker discovery technologies including proteomics, CA15.3, remains the most widely used serum marker for breast cancer [7], the main utility of which is in monitoring therapy in patients [5]. The CA15.3 sandwich-assay employs the monoclonal antibody 115D8, which binds to a glycosylated epitope on the tandem repeat of the mucin MUC1 [8]. Indeed, many widely used serum cancer biomarker tests recognise glycoproteins, including CA-125 in ovarian cancer, carcinoembryonic antigen in colorectal cancer and prostate-specific antigen (PSA) [9]. Glycans are synthesised by the cooperative action of multiple genes and an estimated 1% of the human genome involved in glycan biosynthesis [10], therefore, glycoproteins can potentially reflect multiple genetic anomalies in a single molecule. Aberrations in glycosylation are a common feature in a diverse range of tumour types [11,12], and represent a facet of cancer biology that could be further exploited to identify sensitive and specific cancer biomarkers. Breast cancer is a heterogeneous disease and as such studying potential biomarkers at the protein level without considering one of the most common forms of post-translational modification is

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counterintuitive in terms of maximising biomarker sensitivity and specificity. PSA is a good example in this context. Compared to measuring protein levels alone, PSA lectin binding analyses enhance the ability to distinguish prostate cancer patient blood samples from individuals with benign prostatic hypertrophy [13]. In recent years numerous approaches have been taken to identify alterations in glycosylation of serum proteins associated with breast cancer. Whole serum N-glycome analysis utilising HPLC, exoglycosidase digestion and mass spectrometry has revealed distinct alterations in glycosylation between breast cancer patients and disease-free controls [14,15]. Multi-lectin affinity chromatography (MLAC) has been used to isolate the serum glycoproteome in order to subsequently identify proteins with altered abundance and glycosylation that may act as biomarkers of breast cancer [16,17]. Such investigations have revealed important aspects of breast cancer glycobiology, expanding upon the previous understanding of glycosylation alterations associated with the disease. In this investigation lectin affinity chromatography was used in conjunction with 2-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography – tandem mass spectrometry (LC-MS/MS) in order to identify serum markers of metastatic breast cancer. Aberrant glycosylation known to be associated with metastatic breast cancer was specifically targeted. Helix pomatia agglutinin (HPA) is a lectin that displays nominal binding specificity for terminal a-N-acetylgalactosamine (GalNAc), as found in the tumour associated Tn antigen (GalNAca-Ser/Thr), and also shows affinity for a heterogeneous array of structures in glycan array analysis [18,19]. Not surprisingly, HPA recognises a diverse range of glycoproteins, many of which are involved in key pathways associated with cancer cell proliferation and metastasis [20,21], whilst HPA binding to primary breast tumours is associated with poor prognosis [22,23] and metastasis [24–26]. Although there is some ambiguity concerning the precise structure of the glycans to which HPA binds, it is evident that at least a proportion of these are associated with breast cancer cells that have acquired a metastatic phenotype. In order to identify potential biomarkers for further validation we identified the HPA binding glycoproteins in the serum of patients with metastatic breast cancer and compared these with glycoproteins in the serum of patients with no sign of metastasis.

2. Materials and methods 2.1. Patient samples Serum samples were selected from the DietCompLyf study, an ongoing multicentre observational study based in the UK, approved by the UCL/UCLH Committees on the Ethics of Human Research (ref. 96/3433 and 98/0090). All patients consented to their participation in the study, and the publishing of data derived from the study. Breast cancer patients were recruited onto the study 9–15 months postdiagnosis and annual blood samples were collected for 5 years post recruitment. Venipuncture was performed and blood was collected into 6 mL vacutainers (BD Bioscience, New Jersey, USA). The blood was left to stand for 1 h to clot, centrifuged at 1200g for 15 min and the resulting supernatants divided into 0.5 mL aliquots and frozen. Serum samples were selected from patients originally diagnosed with grade 2/3 ductal carcinomas with vascular invasion and no local or regional recurrence (Table 1). Equal volumes of individual patient sera were pooled in order to obtain sufficient material for subsequent analysis. The first pool contained 25 serum samples, each taken from a patient whose primary tumour was lymph node positive and had metastasised to a distant site 1–30 months post-recruitment to the DietCompLyf study. The patient sera collected closest to the time of diagnosis of distant metastasis was used, as this was considered likely to contain the highest concentration of potential blood markers of metastatic disease. The second pooled sample contained sera from 23 lymph node negative patients that had no sign of distant metastasis for at least 5 years since recruitment onto the DietCompLyf study. The earliest serum sample provided by these patients (i.e., 9–15 months post-diagnosis) was used in order to best represent non-metastatic disease. To determine the blood group of each patient a 10 lL aliquot of serum was used in an agglutination reaction with erythrocytes of blood groups A and B.

Table 1 Clinical pathological features of breast cancer patients from which samples were taken. NSR Range Number of cases Age at diagnosis Tumour size (mm) Grade 2 Grade 3 Lymph node +ve (%) Follow-up (months)

23 52.3 17 13 10 0 48

REC Range 33–73 6–28

25 52.6 32 6 19 36 12.6

ER +ve ve

19 4

15 10

PR +ve ve

10a 5

8b 10

ERBB2 +ve ve

3c 7

8d 11

9 2 2 9

10 2 3 9

Blood groups A B AB O

30–74 7–62

6–97 1–30

Serum was collected from patients 9–15 months post-diagnosis of their primary tumour, and patients were followed up for a further 4 years, providing annual blood samples. Serum samples provided 9–15 months after diagnosis from patients who had no sign of recurrence (NSR) 5 years post-diagnosis of their primary tumour were compared to serum samples from patients that developed a distant metastatic recurrence (REC). The annual blood sample taken chronologically closest to the diagnosis of secondary spread was used for each REC serum sample. Mean average values are shown where relevant. a Unknown receptor status for n = 8. b Unknown receptor status for n = 7. c Unknown receptor status for n = 13. d Unknown receptor status for n = 6.

2.2. HPA affinity chromatography Lectin affinity chromatography employing HPA was used to isolate potential biomarkers of metastatic breast cancer. The two pooled samples contained a comparable distribution of ABO blood groups (Table 1) to ensure HPA enrichment was not biased by a disproportional presence of a particular blood group antigen [27]. The pooled serum from patients with no sign of recurrence (NSR), (6.9 mL) and the pooled serum from patients with metastatic recurrence (REC), (5.9 mL) were each diluted 1:1 in equilibration buffer: 20 mM Tris Base, 400 mM NaCl, 0.02% (w/v) NaN3, pH 7.3 and passed through a 0.22 lm MillexÒ GP filter (Millipore, Massachusetts, USA) preconditioned with 5 mL of equilibration buffer to remove particulate matter. The filtrate was loaded onto an Affisep-HPA affinity chromatography column, 4.6  100 mm, 1.6 mL (GALAB, Geesthacht, Germany) fitted to the following HPLC equipment; two pumps (305 programmable pump and 306 unit), a manometric module and a dynamic mixer (Gilson, Wisconsin, USA). A constant flow rate of 0.8 mL/min was maintained throughout and UV absorbance at 280 nm was used to monitor protein elution. Bound proteins were eluted using a step gradient of freshly prepared 10 mM N-acetylgalactosamine (GalNAc) in equilibration buffer over a period of 8 min. Each 6.4 mL fraction was desalted by solvent exchange against HPLC grade water using an Amicon Ultra Centrifugal Filter Device (10,000 molecular weight cut off. Millipore, Massachusetts, USA) and concentrated to 5–10 lL by vacuum centrifugation. The yield of protein from the pooled sera samples were evaluated using the BCA Protein Assay. 2.3. 2D-DIGE analysis The internal standard method of 2D-DIGE analysis was employed for quantification and spot matching to enable comparison of the HPA binding proteins from the serum of NSR and REC breast cancer patients [28]. The bound fractions from the affinity chromatography separation were prepared in 2D lysis buffer: 8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.5% NP-40 (w/v), 10 mM Tris–HCl pH 8.3, and labelled using 4 pmol N-hydroxysuccinimidyl-cyanine (NHS-Cy) dye per lg of protein, on ice for 30 min in the dark. 49 lg of proteins from NSR and REC were labelled in triplicate using a random combination of Cy3 and Cy5 (both synthesised in house). 73.5 lg each of NSR and REC protein samples were mixed to create an internal standard and labelled with Cy2 (GE Healthcare, Bucks, UK). Labelling reactions

S.A. Fry et al. / Cancer Letters 328 (2013) 335–344 were quenched by the addition of a 20-fold molar excess of L-lysine to Cy dye and incubation on ice for 10 min in the dark. 49 lg of differentially labelled (Cy3 and Cy5) NSR and REC proteins were mixed and added to a 49 lg aliquot of the Cy2 labelled pool (to give 147 lg total protein). Samples were reduced by the addition of dithiothreitol (DTT) to a final concentration of 65 mM. Carrier ampholines/ Pharmalyte mix was added to a final concentration of 2% (v/v) and bromophenol blue to 0.001% (v/v). The volume was adjusted to 450 lL with 2D lysis buffer and used for rehydration of Immobiline Dry Strip IEF gels (IPG strips, 24 cm pH3– 10NL, GE Healthcare, Bucks, UK) in the dark overnight according to the manufacturer’s guidelines. Isoelectric focusing was performed using a Multiphore II apparatus (GE Healthcare, Bucks, UK) for a total of 80 kV h at 16 °C. IPG strips were equilibrated for 15 min in equilibration buffer (6 M urea, 30% (v/v) glycerol, 50 mM Tris–HCl pH 6.8, 2% (w/v) SDS) containing 65 mM DTT, and then for a further 15 min in equilibration buffer containing 240 mM iodoacetamide. Equilibrated strips were overlaid onto 24  20 cm  1 mm 12% polyacrylamide gels (cast between lowfluorescence glass plates and bonded to the inner plate with bind saline) in 0.5% (w/v) agarose in running buffer containing bromophenol blue. Proteins were separated by electrophoresis in an Ettan Dalt 12 gel tank at 16 °C and 2.2 W/gel until the dye front had run off the bottom of the gels. The three gels were scanned on a Typhoon 9400 multi-wavelength fluorescence imager (GE Healthcare, Bucks, UK). Scanned images were cropped in ImageQuant software and exported for image analysis using Decyder V5.0 software (GE Healthcare, Bucks, UK). In this approach spots were matched and quantified across the multiple samples and gel images. The presence of the internal standard on each gel facilitated spot matching and allowed spot by spot standardisation for spot quantification. The average standardised abundance of different proteins from three replicate samples of NSR and REC HPA eluted fractions were compared. Spots of interest had to display a> 1.5 average-fold difference in abundance, with a significance threshold of P < 0.01 over triplicate gels (as determined by Student t-test). Pick lists of spots of interest were created from Sypro Ruby stained images of the gels, which were matched with the CyDye images in Decyder. Spots were excised using an Ettan spot picking robot with a 2 mm picking head (GE Healthcare, Bucks, UK).

2.4. Trypsin digestion Gel pieces of excised spots were washed three times in 50% (v/v) acetonitrile, dried in a vacuum centrifuge, reduced in 10 mM DTT in 5 mM ammonium bicarbonate pH 8.0 for 45 min at 50 °C and alkylated with 50 mM iodoacetamide in ammonium bicarbonate for 1 h at room temperature in the dark. Gel pieces were washed twice in 50% acetonitrile, vacuum dried, and then 50 ng sequence grade modified trypsin (Promega, Southampton, UK) in 5 mM ammonium bicarbonate was added to each dried gel piece. After allowing gel pieces to re-swell for 5 min, 5 lL of 5 mM ammonium bicarbonate was added and gel pieces were incubated at 37 °C for at least 18 h. Tryptic peptides were extracted three times with 50% (v/v) acetonitrile containing 5% (v/v) trifluoroactetic acid. Extracts from each gel piece were pooled and vacuum centrifuged to dryness. Peptides were finally suspended in 5 lL of 0.1% (v/v) formic acid and stored at 20 °C prior to mass spectrometric analysis.

2.5. Liquid chromatography tandem mass spectrometry (LC-MS/MS) Analysis of tryptic peptides from digested DIGE gel spots was performed by nanoflow capillary reversed-phase LC linked to an LTQ Orbitrap XL mass spectrometer (Thermo). Typically, 5 lL of sample was injected onto a 300 lm i.d. 5 mm C18 PepMap guard column (5 lm bead size, 100 Å pore size, LC Packings, Netherlands) and washed for 3 min with 95% solvent A (water + 0.1% FA) at a flow rate of 25 mL/ min using an Ultimate 3000 system (Dionex). Reversed-phase chromatographic separation was then carried out on a 75 mm i.d. 150 mm C18 PepMap nano-LC column (3 lm, bead size, 100 Å pore size, LC Packings, Netherlands) with a linear gradient of 5–50% solvent B (water/ACN 20%:80% v/v + 0.1% FA). The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap MS and MS/MS acquisition Survey full scan MS spectra (from m/z 400–2000) were acquired in the Orbitrap with a resolution of 60,000 at m/z 400. The top six most intense ions were selected for collision induced dissociation. Target ions that had been selected for MS/MS were dynamically excluded for 60 s. For accurate mass measurement, the lock mass option was enabled using the polydimethylcyclosiloxane ion (m/z 455.12003) as an internal calibrant. For peptide identification, raw data files produced in Xcalibur software (Thermo Scientific) were processed in Mascot Distiller (V2.2) and searched against the IPI human database (version 20100213; 87,130 sequences). For searching, the MS tolerance was set to ±10 ppm and the MS/MS tolerance to 0.8 Da. One missed cleavage was allowed and carbamidomethylation (C) was set as a fixed modification. Methionine oxidation, acetylation (protein N-terminal), Glu?pyro-Glu (N-term Q) and deamidation (NQ) were set as variable modifications. Only peptides with Mascot scores >30 were accepted using a significance threshold of 0.05 and protein identifications had to have at least two unique peptides matched per protein.

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2.6. Validation of potential markers by ELISA 2D-DIGE/MS analysis revealed that amongst the proteins altered in metastatic breast cancer, pregnancy zone protein, cadherin-5 and polymeric immunoglobulin receptor were significantly increased in abundance in the pooled sera from the metastatic breast cancer patients. To assess the utility of these proteins as biomarkers for recurrent breast cancer, enzyme-linked immunosorbent assays (ELISAs) were developed and used to test the individual serum samples that had been combined to form the NSR and REC pools above. Optimal reagent concentrations, incubation times and wash steps were determined empirically. See Supplementary Fig. 2 for details of ELISA validation. Pregnancy Zone Protein (PZP): 96 well plates (Immuno MaxiSorp, Thermo Scientific, Rockford, USA) were coated with 50 lL goat polyclonal anti-human PZP (N-19, Insight Biotechnology, Middlesex, UK) at 5 lg/mL in 0.1 M carbonate/bicarbonate buffer, pH 9.6, by incubation at 4 °C overnight. All subsequent steps were undertaken with gentle shaking of the plate at room temperature. Wells were washed five times with 200 lL PBS + 0.1% (v/v) Tween 20 (PBS/T), blocked by the addition of 200 lL Carbofree Solution (Vector Labs, Peterborough, UK) for 30 min, and then washed five times with 200 lL PBS/T. Patient serum was diluted 1 in 15.6, 1 in 62.5 and 1 in 250 in PBS, to a final volume of 25 lL, and added to wells in duplicate. This was followed by the addition of 50 lL Carbofree solution to each well, before incubating the plate for 2 h. The plate was washed five times with PBS/ T and 50 lL of biotinylated goat polyclonal anti-human PZP (Y-20, Insight Biotechnology, Middlesex, UK) prepared at 1 lg/mL in Carbofree Solution was added for 1 h. Wells were washed four times with PBS/T and 50 lL of streptavidin conjugated to poly-HRP (Thermo Scientific, Rockford, USA) diluted 1:4000 in Carbofree Solution was added for 1 h. Wells were washed a further three times with PBS/T and three times with deionised water prior to the addition of 100 lL 3,30 ,5,50 -tetramethylbenzidine (TMB) microwell peroxidase substrate (Insight Biotechnology, Middlesex, UK). The reaction was quenched by the addition of 100 lL 1 M phosphoric acid. The absorbance was read at 450 nm in a Wallac 1420 Victor 2 plate reader (Perkin Elmer, Bucks, UK). Cadherin-5 (CDH5): The same protocol was used as for PZP, with the following changes. Wells were coated with 50 lL mouse monoclonal anti-human CDH5 (MM0012-8A03, Insight Biotechnology, Middlesex, UK) at 0.2 lg/mL in 0.1 M carbonate/bicarbonate buffer, pH 9.6, by incubation at 4 °C overnight. 50 lL biotinylated goat polyclonal anti-human CDH5 (N-14, Insight Biotechnology, Middlesex, UK) prepared at 0.5 lg/mL in Carbofree solution was employed as the detection antibody. Polymeric Immunoglobulin Receptor (pIgR): The same protocol was used as for PZP, with the following changes. Antibodies against the extracellular portion of pIgR (i.e. the secretory component) were employed in this assay. Wells were coated with 50 lL mouse monoclonal anti-human secretory component (clone 2A11, HyTest Ltd., Turku, Finland) at 5 lg/mL in 0.1 M carbonate/bicarbonate buffer, pH 9.6, by incubation at 4 °C overnight. Patient serum was diluted 1 in 31.25, 1 in 125 and 1 in 500 in PBS, to a final volume of 25 lL, and added to wells in duplicate. 50 lL biotinylated mouse monoclonal anti-human secretory component (clone 5D8, HyTest Ltd., Turku, Finland) prepared at 0.5 lg/mL in Carbofree solution was employed as the detection antibody. 2.7. HPA binding assays To assess the O-linked glycosylation status of the proteins captured by the ELISAs above, refined assays were developed incorporating the same capture antibodies, but employing the lectin HPA as the detection reagent rather than an antibody. Plates were coated with capture antibodies as described above. As these antibodies are glycosylated they will cross-react with HPA, giving rise to high background readings. To avoid this problem, the antibodies were oxidised by the addition of 75 lL 20 mM periodic acid to each well for 30 min at 4 °C. The wells were washed five times with PBS/T and blocked with Carbofree solution as described previously. Serum was then applied as above and incubated for 2 h. After washing the wells five times with PBS/T, 50 lL biotinylated HPA (Sigma, Poole, UK) diluted to 20 lg/mL in Carbofree solution was added to each well and incubated for 1 h. The wells were washed a further three times with PBS/T prior to the addition of streptavidin conjugated to poly-HRP as before. The HPA binding assays were performed with standard serum diluted 1 in 2.5 in PBS, using biotinylated HPA pre-incubated with 0–10 mM GalNAc or L-fucose (Sigma, Poole, UK) for 1 h at room temperature prior to applying to the 96 well plate, in order to demonstrate carbohydrate specificity of HPA binding. 2.8. Sample analysis For each ELISA described above, twofold serial dilutions of pooled serum from healthy individuals were used to produce a standard curve. Standard curves were measured in duplicate on each 96 well plate, together with 13 patient serum samples. A mixture of samples from patients with NSR and REC were included on each plate. Four or five plates per assay were required to analyse all samples. The mean values for each duplicate standard curve and the standard deviation was calculated. The average coefficient of variation (CV) was determined in order to assess intraassay variation. ‘‘Master’’ standard curves were produced by taking the mean value

S.A. Fry et al. / Cancer Letters 328 (2013) 335–344

of each of the standard curves on the four or five plates used for each individual ELISA. The inter-assay CV was determined using the mean and standard deviation across these eight or ten standard curves used to generate the ‘‘master’’ standard curve. Breast cancer patient serum samples were measured against ‘‘master’’ standard curves at three dilutions in duplicate. The average ‘‘master’’ blank reading (wells containing all reagents but no serum) was subtracted from each of the standard curves on each plate and the patient serum absorbance value. The patient serum absorbance reading that fell within the linear range of the ‘‘master’’ standard curve was used to infer the relative protein level, taking into account any dilution factor. The Mann Whitney test was used to analyse differences between inferred protein levels, relative HPA binding and ratio values of protein levels versus HPA binding in NSR and REC sample groups.

A

Abs (280nm)

338

Rec

3. Results HPA is an a-GalNAc-binding lectin whose interaction with primary breast tumours is associated with poor prognosis [22,23] and metastasis [24–26]. Glycoproteins secreted or cleaved from breast cancer cells will have been exposed to the same glycosylation machinery that generates aberrant glycans recognised by HPA on the cell surface. Thus, we sought to enrich the HPA-binding fraction of the breast cancer serum proteome in order to identify novel markers of metastatic disease.

NSR 20

40

60

80

100

120

140

Time (min)

B

3.1. HPA-affinity chromatography 5.9 mL of pooled sera from patients with metastatic breast cancer (REC) was loaded onto an HPA column and the bound proteins eluted with 10 mM GalNAc. This fraction contained 221 lg of protein, whilst 6.9 mL of pooled sera from patients with no sign of recurrence (NSR) gave an elution fraction containing 256 lg of protein (Fig. 1A). NSR and REC pooled sera thus contained 37.1 lg/mL and 37.5 lg/mL of HPA binding glycoproteins, respectively. This demonstrates that changes in serum protein glycosylation associated with metastatic breast cancer and HPA binding occur on minor components of the serum glycoproteome. 3.2. Proteomic identification of HPA-binding glycoproteins elevated in REC sera 2D-DIGE was employed to compare the proteins in the HPA binding fractions from sera of patients with no sign of recurrence (NSR) and metastatic breast cancer (REC) (Fig. 1B). Pooled samples were separated by 2D electrophoresis in triplicate alongside an internal standard composed of equal amounts of the two samples. A total of 31 spots with increased average abundance of more than 1.5-fold (P < 0.01) in the HPA elution fraction from the REC group compared to NSR were detected. These protein spots were excised from gels for identification by LC-MS/MS. Several spots yielded multiple identifications resulting in the identification of 9 unique proteins from 26 gel spots, with 5 spots failing to yield reliable identifications (see Supplementary information, Fig. S1 and Table S1). Of these, vitamin K-dependent protein S (PROS1) was not considered for further analysis following assessment for possible O-linked glycosylation sites (NetOGlyc 3.1 server: www.cbs.dtu.dk/services/NetOGlyc) [29] and due to a lack of availability of suitable antibodies for validation work. Pregnancy zone protein (PZP), cadherin-5 (CDH5) and polymeric immunoglobulin receptor (pIgR) were selected for further study (Table 2) as the spots from which they were identified displayed increased levels in the pooled serum from patients with recurrent breast cancer. PZP and CDH5 were both predicted to be O-glycosylated by the NetOGlyc 3.1 server, and have been investigated as serum biomarkers in studies with other cancers [30–32]. Whilst pIgR has no predicted O-glycosylation sites, the extracellular region of this protein, known as the secretory component (SC), can be cleaved and released from the surface of epithelial cells. The SC associates

Fig. 1. HPA affinity chromatography and 2D-gel electrophoresis. (A) HPA binding proteins were isolated from pooled breast cancer patient serum samples by lectin affinity chromatography. Pooled serum samples were diluted 1: 1 in equilibration buffer, filtered and loaded onto an HPA column. Protein elution was detected by measuring optical density at 280 nm. Bound glycoproteins were eluted with 10 mM GalNAc in equilibration buffer. (B) 2D-gel image showing positions of HPA bound glycoproteins on a pH 3–10 NL IPG, 24 cm  24 cm  1 mm 12% polyacrylamide gel. Pooled samples were run in triplicate against an internal standard composed of equal amounts of the two samples for enhanced spot matching and quantitative accuracy. Annotated spots indicate positions of proteins with >1.5 average-fold increase in abundance in the REC sera HPA binding fraction, relative to that of NSR.

with polymeric IgA1, which is O-glycosylated and has been shown to bind HPA [33]. pIgR is produced in the normal breast, and has also been shown to be abnormally expressed in malignant epithelia [34,35]. The association between IgA (including glycosylation) and metastatic breast cancer is subject to an ongoing research programme in our laboratory, and was therefore excluded from further analysis here. 3.3. Verification of discovery analysis The discovery phase of this work used pooled serum samples enabling sufficient amounts of HPA-bound material to be isolated and compared. However, pooling will mask inter-individual variation and may be skewed by samples with extreme alterations in glycosylation. To evaluate the variation in protein abundance and HPA binding across the individual serum samples, higher

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S.A. Fry et al. / Cancer Letters 328 (2013) 335–344 Table 2 HPA binding proteins showing an increased abundance in the pooled serum of patients with recurrent breast cancer. Spot No.

Protein name

461 526 608 608 609 613

Pregnancy zone protein (PZP) Polymeric immunoglobulin receptor Polymeric immunoglobulin receptor Cadherin-5 (CDH5) Polymeric immunoglobulin receptor Polymeric immunoglobulin receptor

(pIgR) (pIgR) (pIgR) (pIgR)

IPI Accession No.

MW (Da)

No. peptides

% Protein coverage

Mascot score

Average ratio REC versus NSR

t-Test

IPI00025426 IPI00004573 IPI00004573 IPI00012792 IPI00004573 IPI00004573

165,242 84,429 84,429 87,804 84,429 84,429

6 12 25 9 21 21

4.3 16 32.6 11.7 27.5 29.8

105 62 276 49 250 221

1.57 1.63 2.74 2.74 2.55 2.51

0.0047 0.0066 0.000027 0.000027 0.000028 0.00074

2D-gel spots that displayed >1.5 average-fold increase in abundance (n = 3; P < 0.01) in the HPA elution fraction of pooled REC sera were selected using DeCyder Software and identified by LC-MS/MS.

throughput and more sensitive assays were required. To this end, ELISAs were developed in order to verify the levels of pIgR, PZP and CDH5 proteins and to assess their HPA-binding properties across the samples used for the proteomic analysis. We have previously developed a similar assay for measuring the glycosylation of prostate specific antigen [13]. 3.3.1. pIgR ELISA assays The observation that pIgR HPA binding was elevated in pooled sera from patients with recurrent breast cancer was of considerable interest as serum IgA levels have been shown to be elevated in breast cancer [36] and IgA1 from breast tumours (but not normal tissue) bind HPA [33]. A sandwich ELISA was developed in order to measure the secretory component extracellular portion of pIgR in serum. Relative levels of pIgR were inferred by measurement against a standard curve (R2 value = 0.999) formed from twofold serial dilutions of pooled sera from healthy individuals (standard sera) (Fig. 2A). Extensive method development resulted in an inter-assay coefficient of variation (CV) of 9%, and an intraassay CV of 7%. The sandwich ELISA was modified to incorporate HPA in place of the detection antibody in order to assess the glycosylation of the captured SC-pIgR. Using the same standard sera, a

standard curve was constructed (R2 value = 0.999) against which patient sera were compared (Fig. 2B). This pIgR-HPA ELISA had average inter- and intra-assay CVs of 22% and 7%, respectively. The HPA binding was shown to be glycosylation dependent as evidenced by monosaccharide inhibition with GalNAc, but not fucose (Fig. 2C). All samples tested had detectable levels of pIgR, but no significant difference in pIgR protein levels were observed for the sera from patients with NSR versus REC breast cancer (Fig. 2D). The same finding was observed with a secretory component ELISA kit obtained commercially (Cusabio Biotech Co.; data not shown). HPA binding to pIgR was detected in just 7 out of the 46 samples tested (Fig. 2E and F), precluding a meaningful comparison between the glycosylation of pIgR in serum from patients with NSR and REC breast cancer, a finding perhaps consistent with a lack of predicted O-glycosylation in pIgR. 3.3.2. PZP ELISA assays PZP was identified as being potentially O-glycosylated with 2 predicted O-glycans. Patient serum samples were measured against standard curves prepared for the PZP ELISA and PZP-HPA ELISA assays (Fig. 3A and B). Pre-incubation of HPA with GalNAc and fucose showed the selective inhibition of the lectin and

Fig. 2. pIgR ELISA. (A) Standard curves prepared on each 96 well plate were combined to form a ‘‘master’’ standard curve for the pIgR sandwich ELISA. (B) A ‘‘master’’ standard curve was prepared in the same manner to measure HPA binding to (antibody) captured pIgR in the refined ELISA assay. Pooled serum from healthy individuals was serially diluted twofold in order to produce standard curves. Error bars show the standard deviation at each point on the standard curve. Precision is indicated by measurement of inter-assay variation (+) and average intra-assay variation ( ) at each point on the standard curve. (C) HPA binding to (antibody) captured pIgR was specifically inhibited by N-acetylgalactosamine (GalNAc) in the refined ELISA. O = GalNAc; X = Fucose. ‘‘Master’’ standard curves were used to infer the relative levels of pIgR protein (D) and HPA binding to antibody captured pIgR (E) in individual patient serum samples. (F) Ratio of pIgR levels to HPA binding. The median value for NSR and REC samples is indicated in each case.

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Fig. 3. PZP ELISA. (A) Standard curves prepared on each 96 well plate were combined to form a ‘‘master’’ standard curve for the PZP sandwich ELISA. (B) A ‘‘master’’ standard curve was prepared in the same manner to measure HPA binding to (antibody) captured PZP in the refined ELISA assay. Pooled serum from healthy individuals was serially diluted twofold in order to produce standard curves. Error bars show standard deviation at each point on the standard curve. Precision is indicated by measurement of interassay variation (+) and average intra-assay variation ( ). (C) HPA binding to (antibody) captured PZP was specifically inhibited by N-acetylgalactosamine (GalNAc) in the refined ELISA. O = GalNAc; X = Fucose. ‘‘Master’’ standard curves were used to infer the relative levels of PZP protein (D) and HPA binding to (antibody) captured PZP (E) in individual patient serum samples. (F) Ratio of PZP levels to HPA binding. The median value for NSR and REC samples is indicated in each case.

demonstrated glycosylation dependent binding of HPA (Fig. 3C). The PZP protein ELISA (R2 value = 0.998) had an average inter-assay CV of 15% and intra-assay CV of 5%. The PZP-HPA ELISA (R2 value = 0.998) had an average inter-assay CV of 13% and an intraassay CV of 6%. Of the patient sera evaluated, six serum samples from patients with NSR and four from patients with REC breast cancer had PZP protein levels outside the range of the assay, presumably because their PZP levels (5–40 lg/mL in healthy controls [31,37]) were below the limit of detection of the assay. A single serum sample from a patient with no sign of recurrence was below the detection level in the HPA ELISA. When the relative PZP protein levels and HPA-binding levels were compared between sera from patients with NSR and REC, the data showed a trend toward increased PZP-HPA interaction, but the results were not statistically significant (Fig. 3D–F). 3.3.3. CDH5 ELISA assays Increased expression of CDH5 has been observed in a range of cancers and so the proteomic identification in this study was particularly intriguing. CDH5 ELISA and CDH5-HPA ELISAs were developed as described above (Fig. 4A and B) with HPA binding to CDH5 shown to be specifically inhibited by GalNAc (Fig. 4C). The CDH5 protein ELISA (R2 value = 0.991) had an average inter-assay CV of 15% and an average intra-assay CV of 5%. The CDH5-HPA ELISA (R2 value = 0.999) had an average inter-assay CV of 13%, and an average intra-assay CV of 6%. A serum sample from one patient with NSR failed to give a reading in both the CDH5- and CDH5HPA ELISA, whilst all other samples fell within the linear detection range. CDH5 levels were elevated in sera from patients with REC breast cancer compared to those with NSR (P = 0.019; Fig. 4D). The CDH5 protein level relative to HPA binding was also elevated in sera from patients with recurrent breast cancer (P = 0.024; Fig. 4F), although no statistically significant difference in the levels of HPA binding alone to CDH5 was observed between the two sample groups (Fig. 4E). The sensitivity and specificity of serum CDH5 for recurrent breast cancer was determined. The relative CDH5 protein levels

in patient sera determined in the ELISA were converted to a percentage scale, with the sample giving the highest measured level assigned a value of 100%. A receiver operating characteristic (ROC) curve was plotted and the optimal sensitivity and specificity of the assay for identifying REC breast cancer was determined using the Youden index (J). J occurs at the point on the ROC curve that maximises the differentiating ability of CDH5 (between NSR and REC serum samples) giving equal weight to sensitivity and specificity. Using J, the CDH5 assay demonstrated a sensitivity and specificity of 83% and 62%, respectively (Fig. 5). Similar calculations were performed using the CDH5 to HPA binding ratio values, where J occurred at a sensitivity of 50% and a specificity of 90% (Fig. 5). A previous study of HPA binding glycoproteins in breast cancer serum samples found that HPA binding to serum glycoproteins was increased in individuals of blood groups A and AB, reflecting the presence of a terminal GalNAc in the blood group A antigen [27]. In order to establish whether a similar pattern was being detected in the CDH5-HPA ELISA, the data were considered with reference to patient blood group. Fig. 6A shows that there was no correlation between HPA binding to CDH5 and patient blood group. However, HPA binding to CDH5 was significantly increased in blood group A patients with recurrent breast cancer compared to those with no sign of recurrence (Fig. 6B). Likewise, blood group A (and to a lesser extent blood group O) patients with REC breast cancer had elevated serum CDH5 levels compared to patients with NSR (Fig. 6C). In summary, serum CDH5 levels discriminated between individuals with no sign of recurrence and those with recurrent breast cancer with a higher degree of significance when blood groups B and AB individuals are excluded from analysis (compare Figs. 4D and 6C).

4. Discussion HPA-affinity chromatography was used as a means of specifically mining the breast cancer serum proteome for proteins

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Fig. 4. CDH5 ELISA. (A) Standard curves prepared on each 96 well plate were combined to form a ‘‘master’’ standard curve for the CDH5 sandwich ELISA. (B) A ‘‘master’’ standard curve was prepared in the same manner to measure HPA binding to (antibody) captured CDH5 in the refined ELISA assay. Pooled serum from healthy individuals was serially diluted twofold in order to produce standard curves. Error bars show standard deviation at each point on the standard curve. Precision is indicated by measurement of inter-assay variation (+) and average intra-assay variation ( ). (C) HPA binding to (antibody) captured CDH5 was specifically inhibited by N-acetylgalactosamine (GalNAc) in the refined ELISA. O = GalNAc; X = Fucose. ‘‘Master’’ standard curves were used to infer the relative levels of CDH5 (D) and HPA binding to (antibody) captured CDH5 (E) in individual patient serum samples. (F) Ratio of PZP levels to HPA binding. The median value for NSR and REC samples is indicated in each case. The Mann Whitney test was used to analyse differences between inferred protein levels, relative HPA binding and ratio values of HPA binding to protein level between the two sample groups. The P value is indicated where statistically significant differences were observed.

Fig. 5. Sensitivity and specificity of CDH5. CDH5 levels and the ratio of CDH5 levels to HPA binding were converted to a percentage scale with the top reading given a value of 100% and all other readings adjusted accordingly. Sensitivity (true positives/true positives + false negatives) and specificity (true negatives/true negatives + false positives) were calculated at all cut-off points that gave a unique pair of values, and plotted on ROC curves. The Youden index (J) was used to determine the optimal sensitivity and specificity for discriminating patients with REC breast cancer from those with NSR according to CDH5 levels (d) and the ratio of CDH5 levels to HPA binding (s).

post-translationally modified by the attachment of glycans potentially associated with breast cancer metastasis. HPA-binding proteins were enriched from pooled serum collected from breast cancer patients with distant metastasis and compared to those from patients with no sign of distant metastasis for at least 5 years. Both of these pooled samples contained approximately 37 lg/mL of HPA binding glycoproteins, representing <0.1% of total serum protein. The proteins that were found at elevated levels in pooled

serum from patients with recurrent breast cancer were verified in ELISA assays, using the individual serum samples that had been pooled for discovery analysis. HPA-enrichment strategies have been applied by others in small-scale biomarker discovery studies using breast cancer serum and plasma samples. In a comparison of the HPA binding proteins of serum from three patients with metastatic breast cancer and three patients with non-metastatic breast cancer, the only significant differences in the SDS–PAGE protein patterns observed related to blood group [27]. A quantitative comparison of HPA-binding glycoproteins by LC-MS/MS revealed 7 plasma proteins elevated by >3-fold in a sample from a breast cancer patient relative to normal pooled human plasma [38]. However, as this comparison was made with a single plasma sample from a patient with ductal carcinoma the significance of this finding still needs to be determined. HPA has also been shown to be a prognostic indicator in colorectal cancer and proteomic analysis revealed that integrin av/a6 and annexin A2/A4, involved in cell adhesion and migration, are the major HPA binding proteins in metastatic colorectal cancer cells [20]. The ELISAs employed to verify the proteomic work were designed to use commonly available equipment in order to facilitate further validation work and adoption by other laboratories. Sensitive assays are required to measure serum CDH5, which typically circulates at low ng/mL levels. Whilst absolute levels of CDH5 were not determined here, less than 20 lL of patient serum was sufficient to measure relative CDH5 protein levels and glycosylation. In early reports related to CDH5 expression, this protein was found primarily on endothelial cells, however, there is growing evidence that CDH5 is aberrantly expressed in a variety of tumours, including invasive breast carcinoma [39], non-metastatic renal cell carcinoma [40], aggressive melanoma [41] and osteosarcoma [42]. We show here that serum CDH5 levels were elevated in patients with metastatic breast cancer compared to patients that have been free of distant metastases for at least 5 years (Fig. 4D). Elevated circulating CDH5 has been detected in colorectal cancer [32] and

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Fig. 6. CDH5 blood group analysis. (A) There was no correlation between patient blood group phenotype and HPA binding to CDH5. (B) HPA binding to CDH5 was significantly increased in blood group A patients with REC breast cancer relative to blood group A patients with NSR. (C) Serum CDH5 levels were significantly increased in blood group A patients with REC breast cancer relative to blood group A patients with NSR. Blood groups A and O patients with no sign of metastasis could be distinguished from blood groups A and O patients with recurrent breast cancer. The median value for NSR and REC samples is indicated in each case. The Mann Whitney test was used to analyse differences between CDH5 levels and relative HPA binding between the two sample groups. The P value is indicated where statistically significant differences were observed.

myeloma [43], as well as patients with Behçet’s (chronic multisystem vasculitis) disease [44] and women with severe ovarian hyperstimulation syndrome [45]. However, this is the first report suggesting that serum CDH5 levels are increased in metastatic breast cancer. The glycosylation of CDH5 (as measured by binding to HPA) was not significantly different in patients with metastatic breast cancer compared to those free of metastases (Fig. 4E). This interesting finding may be consistent with reports that the endothelial cells of the tumour microvasculature of breast [46] and hepatocellular [47] carcinomas up-regulate CDH5 expression promoting tumour angiogenesis [48], an important step in metastatic dissemination. In our system the CDH5 ELISA was 83% sensitive in identifying serum from patients with metastatic breast cancer compared to those with no sign of metastasis. Whilst elevated serum CDH5 has been associated with other cancer types [32,43] and diseases [44,45], the aim of this work was to investigate its utility as a marker of metastatic breast cancer recurrence. As such, an assay of high specificity is desired as this indicates a low false positive rate. The CDH5 protein: HPA binding ratio discriminated between nonrecurrent and recurrent cases with a specificity of 90%. In comparison, CA15.3, the most clinically useful marker in the diagnosis of metastatic breast cancer, has a reported specificity greater than 95% and sensitivity ranging from 32% to 87% [49,50]. Measuring CDH5 protein levels and HPA binding thus gives a biomarker with potential for monitoring breast cancer progression in patients diagnosed with a primary tumour. This finding now warrants further

validation using additional serum samples collected from the 3000 breast cancer patients that have been recruited onto the DietCompLyf observational study. Further validation is also required to address a limitation of the current assay, namely that the specificity of the antibodies employed must be determined. Although patient sample numbers were small, the binding of HPA to CDH5 was significantly increased in blood group A patients with recurrent breast cancer compared to those with no sign of recurrence. CDH5 serum levels distinguished between the two sample groups with increased significance when blood groups B and AB patients were excluded from analyses. Although the cause of these correlations with blood group is unclear, considering that 86% of the UK population have a blood groups A or O phenotype [51], these are potentially significant findings which require further corroboration. There are several factors that may have contributed to the disparity observed between the proteomic discovery analyses and the validatory ELISAs. CDH5 and PZP were initially identified from gel spots that contained a multitude of proteins. It was not possible to determine which specific protein(s) in the proteomic analysis were specifically responsible for the increase in abundance levels of protein(s) in sera from patients with REC compared to sera from patients with NSR. In addition, the proteomic analysis was performed on HPA binding glycoforms of serum proteins, whilst in the ELISA validation work, all isoforms of the proteins would have been assessed. As HPA-affinity chromatography was used to enrich pooled serum prior to proteomic identification, any proteins

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detected at higher levels in the serum of patients with metastatic breast cancer would be expected to be modified with HPA binding glycans. The analyses performed here, however, suggest that this is not necessarily the case. Indeed, it may be the case that proteins other than the HPA binding glycoproteins eluted from the chromatography step if they are present in complex with HPA binding glycoproteins. This appears to be the case for pIgR, which was elevated in the pooled serum from patients with metastatic breast cancer despite an absence of O-linked glycans as predicted by NetOGlyc 3.1. HPA binding to pIgR was detected in just two serum samples from patients with REC breast cancer and five with NSR, a finding that conflicts with the proteomic data. It is therefore likely that the pIgR binding partner in this case is IgA1, which is known to bind to HPA and is highly abundant in serum. Alternatively, the covalent immobilisation of HPA in an affinity chromatography column may reduce the structural flexibility of the lectin and alter its binding properties compared to when it is free in solution, as in the refined ELISA assay. REC breast cancer serum PZP levels were similar to those from patients with NSR. Although a trend toward increased PZP-HPA interaction in REC breast cancer sera was observed in the assays, the results were not statistically significant. This again may reflect subtle differences in HPA binding when immobilised compared to when in solution. In conclusion, lectin-affinity chromatography was used to isolate the <0.1% of total serum proteins that bind to HPA. Following separation in two dimensions by 2D-DIGE, several proteins that were present at altered levels in this fraction from patients with metastatic recurrence compared with patients that have been recurrence-free for 5 years were identified by LC-MS/MS. Three candidate biomarkers elevated in the serum of patients with metastatic breast cancer were verified by the development of in-house ELISA assays in order to measure both protein levels and HPA binding. CDH5 emerged as a potential marker of metastatic breast cancer with both protein levels and HPA binding contributing to a test that is comparable to CA15.3 in terms of specificity. As evidenced by the CDH5 data, the glycoproteomic and validatory approach employed here has the capacity to identify novel markers of breast cancer metastasis. Acknowledgements This work was supported by the Against Breast Cancer charity (Registered Charity No. 1121258) and was partly undertaken at UCLH/UCL who received a proportion of funding from the Department of Health’s NIHR Biomedical Research Centres funding scheme. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.canlet.2012. 10.011. References [1] M.H. Forouzanfar, K.J. Foreman, A.M. Delossantos, R. Lozano, A.D. Lopez, C.J. Murray, M. Naghavi, Breast and cervical cancer in 187 countries between 1980 and 2010: a systematic analysis, Lancet 378 (2011) (1980) 1461–1484. [2] J.R. Benson, I. Jatoi, M. Keisch, F.J. Esteva, A. Makris, V.C. Jordan, Early breast cancer, Lancet 373 (2009) 1463–1479. [3] B. Weigelt, J.L. Peterse, L.J. van‘t Veer, Breast cancer metastasis: markers and models, Nat. Rev. Cancer 5 (2005) 591–602. [4] M.T. Weigel, M. Dowsett, Current and emerging biomarkers in breast cancer: prognosis and prediction, Endocr. Relat. Cancer 17 (2010) R245–R262. [5] M.J. Duffy, D. Evoy, E.W. McDermott, CA 15–3: uses and limitation as a biomarker for breast cancer, Clin. Chim. Acta 411 (2010) 1869–1874. [6] A.M. Gonzalez-Angulo, F. Morales-Vasquez, G.N. Hortobagyi, Overview of resistance to systemic therapy in patients with breast cancer, Adv. Exp. Med. Biol. 608 (2007) 1–22.

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