Peptidomic analysis of breast cancer reveals a putative surrogate marker for estrogen receptor-negative carcinomas

Peptidomic analysis of breast cancer revealsa putative surrogate marker for estrogenreceptor-negative carcinomas

Frank Traub1, Marco Jost2, Rudiger Hess2, Karl Schorn2, Christoph Menzel2, Petra Budde2,Peter Schulz-Knappe2, Norbert Lamping2, Andreas Pich1, Hans Kreipe1 andHarald Tammen2

1Institute of Pathology, Medizinische Hochschule Hannover, Hannover, Germany and 2BioVisioN AG,Hannover, Germany

Estrogen-receptor status provides a major biomarker in breast cancer classification and has an importantimpact on prognosis and treatment options. The aim of this study was to investigate peptide profiles of invasivebreast cancer with positive (n¼ 39) and negative receptor status (n¼ 41). Peptide profiles were generated by‘Differential Peptide Display’, which is an offline-coupled combination of reversed-phase-HPLC and MALDImass spectrometry. Mass spectrometric data were correlated with the immunohistochemically determinedreceptor state. Identification of peptides of interest was carried out by additional mass spectrometric methods(eg MALDI-TOF-TOF-MS-MS). Approximately 3000–7000 signals were detected per sample and thymosin alpha-1, an asparaginyl endopeptidase generated cleavage product of the ubiquitous acidic protein prothymosin-alpha, was found to differentiate the tumor samples according to their receptor status with the highestspecificity. The concentration of Thymosin alpha-1 was found to be upregulated (n¼ 37) in estrogen-negativecancer samples and downregulated (n¼ 32) in estrogen-positive breast cancer samples. The expression of theprecursor protein (Prothymosin-alpha) has been discussed previously as a prognostic factor in breast cancer. Itis involved in the ER signal transduction pathway as an anti-coactivator-inhibitor. From our findings weconclude that Thymosin alpha-1 could serve as a surrogate marker in breast cancers and may indicate ERfunctionality.Laboratory Investigation (2006) 86, 246–253. doi:10.1038/labinvest.3700385; published online 6 February 2006

Keywords: breast cancer; differential peptide display; estrogen receptor; peptidomics; prothymosin alpha;thymosin alpha-1

In the Western world, breast cancer is the mostprevalent cancer in women and the second leadingcause of cancer death. In the past two decades, themolecular dissection of cancer has increased ourunderstanding of the pathways that are altered inneoplastic cells and that have a growing impact inoncologic therapy. Complex genetic and proteomicalterations induce cancer growth and progressionresulting in significant clinical heterogeneity amongindividual tumors and individual prognosis.

According to the College of American Patho-logists, expression of estrogen receptor-alpha (ER)and progesterone receptor (PgR) belongs to the firstcategory of prognostic factors besides tumor size,

histological grade and lymph node status. The ERmediates the stimulatory effects of estrogens andthe inhibitory effects of anti-estrogens in breastcancer and other hormone-dependent tissues. Forpatients with hormone-receptor (HR)-positive breastcancer (B75% ductal invasive carcinoma), endocrinetherapy (eg Tamoxifen) is the preferred treatment.Approximately 50–60% of patients with ER-positivetumors benefit from endocrine therapy.1 The ERexpression is gradually lost during tumor progres-sion, leading to a more undifferentiated state and soefficacy of endocrine therapy is limited. Panendo-crine resistance2 is almost universally in breastcancer negative for ER and PgR content but alsooccurs in a significant fraction of breast cancer casesthat express HR. PgR expression is ER-dependentand the presence of PgR in the breast indicatesprobable responsiveness to endocrine therapy.3,4

The underlying mechanism of endocrine resistanceis still obscure. Potentially critical links between the

Received 13 October 2005; revised and accepted 18 November2005; published online 6 February 2006

Correspondence: Dr H Tammen, MD, BioVisioN AG, Feodor-Lynen-Str. 5, D-30625 Hannover, Germany.E-mail: [email protected]

Laboratory Investigation (2006) 86, 246–253& 2006 USCAP, Inc All rights reserved 0023-6837/06 $30.00

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ER and cell-cycle or cell-survival pathways aredisrupted, inducing usage of alternative pathwaysto serve functions previously subject to masterregulation by the ER.

In recent years, peptidomic techniques have evol-ved, which allow the comprehensive analysis ofnative peptides in virtually every biological source.In this study, we applied a peptidomic tech-nique called differential peptide display (DPD) tocompare ER-positive and ER-negative tumor tissues(Table 1) in order to reveal specific profiles.

DPD5–8 is a peptidomic technique to generatecomprehensive peptide displays of usually 41500peptides from a variety of biological samplesincluding blood plasma, urine, cerebrospinal fluid,cell and tissue extracts or cell culture supernatants.For clarification, the size we refer to for the term‘peptides’ covers a mass range of 750–15 000 Da.

Materials and methods

Tissue Samples

Freshly frozen as well as formalin-fixed, paraffin-embedded tissue of 80 cases of invasive ductalcarcinomas were retrieved from the archive of theInstitute of Pathology, Hannover Medical School.The samples were collected according to the guide-lines of the local ethics committee.

Immunohistochemistry

Sections measuring 2 mm of carcinoma tissue werede-waxed by xylene, rehydrated by graded alcohol

and epitope retrieval was carried out in a microwaveoven (20 min; 1001C in 10 mM sodium citrate; pH6.0). Sections were stained using the Shandoncoverplate system in a Tecan Genesis Autostainer(ThermoShandon, Frankfurt a.M., Germany; Tecan,Deisenhofen, Germany). Tissue peroxidase activitywas blocked by incubation with 3% hydrogenperoxide for 8 min. The primary antibodies againstPgR (PgR 636), Her2neu (c-erbB-2), Ki67 (MIB-1)(all from Dako, Germany), p53 (DO-7) and for ER(6F11) (both from Nova Castra, Germany) were used.Biotinylated secondary antibodies were used for thecatalyzed signal amplification technique (CSA,Dako). New fuchsin served as chromogen, andhemalaun served as counterstain.

Differential Peptide Display

Peptide extractionPro analysi guanidinium chloride was obtainedfrom Merck KGaA (Darmstadt, Germany). Pro analy-si glycine was obtained from Carl Roth GbmH & Co(Karlsruhe, Germany). Microcon centrifugal filterunits with an MWCO of 50 000 were purchased fromMillipore (Bedford, MA, USA). A measure of 10 mgof each frozen sample was solubilized in extractionbuffer (6 M guanidinium chloride, 50 mM glycine,pH 2.6). The protein/peptide extraction was carriedout at 991C and 1100 r.p.m. in a thermomixer. Afterincubating for 10 min, the tubes were centrifuged at18 000 g for 30 min at 101C. The supernatants weretransferred to the ultra centrifugal filter units andcentrifuged (Z 233 MK-2, HERMLE LabortechnikGmbH, Wehingen, Germany) at 12 000� g in a fixedangle rotor for 30 min.

Peptide Display

The methodology has been described by Schulte etal,8 Tammen et al,9 and Rai et al10 in detail. Briefly,peptides are extracted from biological sources andfractionated by means of reversed phase chromato-graphy. Each fraction is subjected to MALDI-TOF-MS and the mass spectra of all 96 fractions arecombined, resulting in a two-dimensional displayof peptide masses named ‘Peptide Display’, wherethe abscissa displays the mass-to-charge ratio andthe ordinate is determined by the retention time onthe RP-HPLC and the signal intensity is depicted bythe color saturation. Specific software (Spectro-manias) has been designed to allow the visualiza-tion, processing and analysis of mass spectra.11 Forthe identification of peptides, peaks from individualHPLC fractions can be subjected to tandem MSidentification by means of nESI-qTOF-MS sequen-cing or MALDI-TOF-TOF-MS resulting in peptidefragment spectra. These spectra serve to identify thecorresponding peptide sequence by remote databasesearching.

Table 1 Patient stratification

Parameter ER+ (n¼ 39) ER� (n¼ 41)

Age 59.7 (s.d. 11.4) 56 (s.d. 15.1)PR 75% (n¼30) 0HER2 (0) 37.5% (n¼15) 53.6% (n¼ 22)HER2 (1+) 50% (n¼29) 24.4% (n¼ 10)HER2 (3+) 10% (n¼4) 22% (n¼ 9)HER2 (2+) 2.5% (n¼1)p53 22.1 (s.d. 22.8) 44.6 (s.d. 40.8)Ki67 22.6 (s.d. 14.3) 53.7 (s.d. 23.5)pT1b 5% (n¼2)pT1c 35% (n¼14) 31.7% (n¼ 13)a

pT2 57.5% (n¼23) 58.5% (n¼ 24)pT3 — 7.3% (n¼ 3)pN0 65% (n¼25) 56.4% (n¼ 22)b

pN1 25% (n¼10) 28.2% (n¼ 11)pN2 7.5 % (n¼3) 12.8% (n¼ 5)pN3 2.5% (n¼1) 2.6% (n¼ 1)G2 47.5% (n¼19) 9.8% (n¼ 4)G3 52.5% (n¼20)c 90.2% (n¼ 37)

Tabular listing of pathological metadata.aOne relapse.

bTwo tumors not analyzable.

cOne DCIS.

Peptidomic analysis of breast tumorsF Traub et al

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Laboratory Investigation (2006) 86, 246–253

Sample size and statistical data analysisFor this study, more than 200 peptide displays weregenerated from 80 different samples. Each displayconsisted of 96 MS spectra. Each sample wasmeasured at least twice either on the STR-Platformor on the ABI4700 TOF-TOF analyzer. Detection ofdifferentially expressed peptides was achieved bycalculation of subtractive peptide displays or corre-lation analysis (DPD) by referring to mass spectro-metric data. In addition, all mass spectra with thesame fraction number were averaged, assuming thatthey contain an identical subset of peptides, yield-ing an average peptide display. The averaged spectraof this display served as a template for the definitionof those peak coordinates, which can be clearlydistinguished from baseline noise. Signal intensitiesof all peaks derived from each group were exportedfor comprehensive correlation data analysis withcommercially available software. For quality assur-ance, each sample was spiked with a definedpeptide mixture (500 fmol per sample).

Peptide Identification

For MALDI-TOF/TOF-MS/MS identifications thearchived MALDI target plates from the peptidedisplay process were used. High accuracy surveyMALDI-TOF-MS spectra were acquired in a fullyautomated fashion in the reflector mode of a 4700proteomics analyzer (Applied Biosystems, Framing-ham, USA). All measurements were calibrated usinga daily updated default calibration. Peptides weresubsequently fragmented in the MS/MS mode usinga collision energy of 1 keV and an intermediate gas

pressure of approximately 3� 10–7 Torr of ambientair (Figure 1). MS/MS spectra were noise filtered,peak de-isotoped and transferred into the MASCOTgeneric file format using the MascotDistiller soft-ware tool (Matrix Science Ltd, UK). Cascadingsearches on a MASCOT database engine (MatrixScience) including several post-translational modi-fications were performed by the Mascot Daemonsoftware (Version 1.8 or higher) against the Swiss-Prot (Version 39 or higher, GeneBio, Geneva,Switzerland) database.

Western-blot analysisFrozen tissue homogenates were denatured inSDS–PAGE sample buffer at 951C for 5 min twice.A measure of 15 mg of protein (measured by Bradfordassay) of each sample was analyzed by SDS–PAGE(Tris-HCl Gels 10–20%, Bio-Rad, Germany), trans-ferred to 0.45 mm Nytran SPC (Schleicher undSchull, Germany), and subjected to Western-blotanalysis. The membrane was incubated with theprimary antibody 2F11 (Alexis, Germany) (diluted1:100) for 1 h at RT. After washing with PBS, theperoxidase-conjugated second antibody was addedand the protein was visualized by chemilumine-scence detection.

Results

In a first approach, samples were prepared in twodifferent concentrations (3 and 7 mg wet weightequivalent) and subjected to peptide profiling byDPD (Figure 2 and 3). Every data point (approxi-mately 1.4 million per display) of every display was

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Figure 1 MALDI-TOF/TOF-MS/MS spectrum of PTMA_HUMAN 1–28. Peaks depicting identified fragment ions are labeled according tothe standard fragment ion nomenclature. The peptide identification score (probability based MOWSE score using the MASCOT databaseengine) for this particular spectrum was highly significant (MOWSE score¼ 75).


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then correlated to the receptor state. This analysisrevealed that a peptide with m/z ratio of 3108exhibited the highest correlation value. This signalcould be identified as thymosin alpha-1 (prothymo-sin-alpha 1–28 with an acetylated N-terminus(N-acetylserine), THYA) by MALDI-TOF/TOF-MS-

MS. For further characterization of the samples, anoverview sequencing approach was applied (insertTable Figure 2). The analysis revealed extracellularas well as intracellular peptides.

To validate this first result, 60 samples were againobtained from the Institute of Pathology. A total

Figure 2 Peptide displays from tumor specimens (mean display of ER-negative and ER-positive tumors). The x-axis displays the mass-to-charge ratio, the y-axis depicts the retention time of the peptides on the RP-HPLC. The signal intensity is indicated by color saturation.Between 3000 and 7000 signals are visualized per peptide display. Sequenced peptides are marked with Arabic numerals, which refer tothe inserted table. Peptides no. 22 and 24 were added prior to processing of the samples for quality control purposes. Insert: The tabledepicts the precursor name, the putative origin of the peptide and the Swiss Prot accession number.

Figure 3 Mass spectrometric detection of THYA. The figure depicts a distinct region of peptide displays from ER-negative and ER-positive tumor samples. Each lane separates two different wet weight equivalents of tumor tissue. The signal of THYA is marked.Additionally, as a reference a signal corresponding to Thymosin beta 4 sulphoxid is tagged.


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of 50% of the samples were blinded for theprimary investigator. Blinded samples and sampleswith known receptor state were investigated espe-cially for the occurrence of Thymosin alpha-1 andmeasured at least twice. Based on the intensitylevels of Thymosin alpha-1 and visual inspectionof each spectrum, the blinded samples were assig-ned to the putative receptor state. After assignment,the results were transferred back to the Instituteof Pathology, logged and the results of the IHCanalysis were disclosed. After disclosure of theIHC results the assignment was found to be correctin 26 out of 30 cases. Figures 4 and 5 show theintensity levels of THYA in correlation to thereceptor state.

The detailed data analysis assign 69 samples to bein accordance to the IHC analysis, which means thatpositive correlation is seen between the occurrenceof THYA and negative estrogen receptor state. Inseven ER-positive breast cancer specimens, THYAwas detectable and in four ER-negative samplesTHYA was not detectable. These 11 samples werereanalyzed including de novo sampling and IHCanalysis (Figure 5, panel d) and findings wereconfirmed.

To validate the results Western blots for THYAwere performed. In most cases, the Western blot wasnot successful, resulting in non-detection of THYA

and its precursor. In successful attempts twobands were displayed, corresponding to Prothy-mosin-alpha (PTa) and THYA in ER-negative breastcarcinomas. In ER-positive breast carcinomas,exclusively, the precursor was detectable (Figure 6).

Discussion

The overview (sequencing) approach shed light on acommon problem in tumor sample composition,namely the tumor heterogeneity. Among the ex-tracted peptides, not only the tumor-associatedpeptides as Vimentin or Cytokeratin 8 were identi-fied, but also peptides with origin outside of tumorcells were sequenced. Multiple peptides derivedfrom hemoglobin as indicators of blood contamina-tion. For this reason, it is insufficient to use patternrecognition. Sequencing and identifying peptidesand protein associated with discriminating peaksobviate the heterogeneity problem and will improvebiomarker reproducibility.

Gene transcriptional activation by ER is enhancedor repressed by interaction with regulatory factors,which function in a positive fashion (coactivators)or negative fashion (corepressor) and are believed tobe interposed between the receptor and the basaltranscription complex. These co-regulators exist asa part of large complexes that can be recruited bythe ER and function as chromatin remodelingfactors. For that reason, this tripartite action of theER, involving the receptor, its ligands and co-regu-lator proteins, allows the precise regulation of thebiological effects of these HRs on gene expression.

The DPD analysis of ER-positive and ER-negativebreast cancer tissue samples revealed THYA as thepeptide with the highest power to differentiatebetween the two groups. THYA is upregulated inER-negative breast cancer tissue. A broad spreadin THYA signal expression from the same sampleduring independent analysis rounds can be seen.This may have multiple reasons: heterogeneity oftumors, insufficient extraction of peptides, loss ofmaterial during processing or sample mix-up. Tocurate such errors multiple measurements wereperformed and all data, even with confoundingresults, are shown. Nevertheless, the data showrather good robustness and high reproducibility. Onthe other hand, the validation by Western blottingwas not reproducible and error prone. It is estab-lished that highly conserved polypeptides like PTaand THYA are only poorly immunogenic. Further-more, these small molecules hardly bind to mem-branes used routinely for Western blots.12 Thesedifficulties become even more noticeable when theliterature is screened for optimized detection meth-ods. Only a few reports demonstrate immunogenicdetection of PTa. However, after optimizing theconditions as described by Rodriguez et al,13 insome cases a separation and blotting of THYA andPTa (the precursor) was possible.

Figure 4 The figure shows a zoom view representing a distinctchromatographic area and m/z region (x-axis) from peptidedisplays. Lanes of peptide displays of independent experimentalgroups are shown (separated by solid lines). The outcome of theIHC (separated by dashed lines) is depicted on the right side.THYA (arrow) is highly upregulated in ER-negative tumorsamples.


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A link between PTa and breast cancer was firstpublished in 1993.14 It is a highly acidic proteinpresent in virtually every cell.15 Its high conserva-tion in mammals and wide tissue distributionsuggests an essential biological role and several

descriptions16–18 point to its involvement in cellcycle. PTa expression, measured via radio immuno-assay, is higher in tumor samples compared tonormal breast tissue, and the expression levels ofPTa correlate with the proliferation status andmetastatic potential of tumors.19 The group ofKatzenellenbogen published results describing theinteraction of a repressor of estrogen activity (REA)with the liganded ER. REA suppresses ER-activatedgene transcription by estrogens, and competeswith coactivators for binding to ER.20–22 PTa wasidentified as a binding protein partner of REAand by binding to REA selectively activates ER.This interaction of PTa with REA enables coactivatorinteraction with ER, thus resulting in enhanced ERtranscriptional activity. Furthermore, it was foundthat gene expression of PTa is rapidly increased byestrogens, highlighting a new role for PTa and REA

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Figure 5 Intensity levels of THYA. Each graph depicts the signal intensities of THYA in different panels (a–d). Panel a corresponds to thediscovery set. Panel b contains redundant measurements (eight ER-positive cases and 13 ER-negative cases). Panel c represents MALDI-TOF-TOF data and Panel e corresponds to samples that show a positive correlation between positive estrogen receptor status andoccurrence of THYA. Open circles¼ER negative samples, solid squares¼ER positive samples.

Figure 6 Exemplary Western blot of estrogen-positive andestrogen-negative carcinoma.


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as ER activity-modulating proteins. The repressorREA20–22 is inhibited by binding of PTa. Thus, PTacan be regarded as an anti-coactivator-inhibitor.

Thymosin alpha-1, a truncated N-terminal form ofPTa is generated at least partially, by the lysosomalasparaginyl endopeptidase legumain.23 Breast can-cer tissues are found to be positive for legumain,24

but so far there are no studies that have determinedthe legumain activity or expression levels in breastcancers in correlation to ER receptor state. Biancoand co-workers25 reported that increased intracellu-lar PTa expression in the presence of estrogens isaccompanied by increased nuclear and decreasedcytoplasmic PTa localization if estrogens are pre-sent. As estrogen-dependent nuclear translocationin ER-negative tumors is diminished, PTa may bemore susceptible to lysosomal processing by legu-main in ER-negative breast cancers. This assump-tion may explain the processing of PTa leadingto the occurrence of THYA in ER-negative tumorsamples. This is supported by Sakabe et al26 whoreported that natural and environmental estrogensmodulate the production of THYA in ER-positiveTECs (thymus epithelial cells) in a negative fashion.Thus, the emersion of THYA may be a result of thedysfunction and not of the expression of the ER.

The analysis of the clinical data revealed anaccumulation of patients with negative progesteronestatus (Table 2) in the subgroup of patients withpositive ER status and THYA, which is indicativeof dysfunctional ER. Therefore, we conclude thatthe detection of THYA in estrogen-positive casesmay indicate steroid-insensitive breast cancers orbreast cancers with compromised response toregular steroid stimulation. Consequently, the ana-lysis of breast tumor tissue for these specificmolecules may be useful for stratification of patientswho are likely to profit from anti-estrogen therapiesor are in need of a more intensive therapy.

As peptides appear at the end of metabolic path-ways, they are likely to subsume genetic changes aswell as changes in protein expression, protein half-life and biochemical pathways. The half-life times ofpeptides is often increased as compared to proteins,as the number of sites for proteolytic degradation isalready drastically reduced. In addition, peptidesusually have higher permeability between tissuecompartments and therefore the chance of detectingpeptides apart from the tissue itself (eg in blood) is

higher compared to proteins and nucleic acids. Datafrom previous studies27 suggest that truncated formsof PTa are capable of migrating from cells into theperipheral blood compartment. Therefore, we arecurrently conducting studies to determine whetherTHYA is detectable at different levels in plasma ofbreast cancer patients in correlation to the ER state.The determination must be specific for THYA as PTamay cross-react in an immunoassay. The level ofTHYA may indicate ER-negative breast cancer or theconversion to hormone-insensitive breast cancerduring anti-estrogen (eg Tamoxifen) therapy.

Note

Peptidomics, Differential Peptide Display, Spectro-mania and BioVisioN are registered trademarks ofBioVisioN AG, Hannover, Germany.

Acknowledgement

This work was supported by Grant No. 203.19-32329-5-406 (Federal State of Lower Saxony).

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Table 2 Clinical metadata of patients grouped according to the ER state and the THYA content

Number ofcases

ER status THYA status Progesteronereceptornegative

Mean year ofdiagnostic

finding

s.d. Mean DOB s.d.

32 Positive Negative 7 (B22 %) 2001.3 1.7 1942 127 Positive Positive 4 (B57 %) 2002.0 1.9 1942 1137 Negative Positive 37 (100%) 2001.6 1.7 1945 154 Negative Negative 4 (100%) 2000.8 1.5 1941 15


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Peptidomic analysis of breast cancer reveals a putative surrogate marker for estrogen receptor-negative carcinomas