Download Identification of Candidate Biomarker Proteins Released by Human

Identification of Candidate Biomarker Proteins Released by Human
Endometrial and Cervical Cancer Cells Using Two-Dimensional
Liquid Chromatography/Tandem Mass Spectrometry
Hongyan Li,†,‡ Leroi V. DeSouza,†,‡,§ Shaun Ghanny,†,‡ Wei Li,†,‡ Alexander D. Romaschin,|,⊥
Terence J. Colgan,⊥,O and K. W. Michael Siu*,†,‡,§
Department of Biology, Centre for Research in Mass Spectrometry, and Department of Chemistry, York
University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3, Division of Clinical Biochemistry, St.
Michael’s Hospital, 30 Bond Street, Toronto, Ontario, Canada M5B 1W8, Department of Laboratory Medicine
and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1L5, and Pathology and Laboratory
Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5
Received February 14, 2007
Candidate biomarker proteins, including chaperonin 10 and pyruvate kinase, previously discovered
and identified using mass-tagging reagents with multidimensional liquid chromatography and tandem
mass spectrometry (DeSouza, L.; et al. J. Proteome Res. 2005, 4, 377-386) have been identified in
serum-free media of cultured endometrial cancer (KLE and HEC-1-A) and cervical cancer (HeLa) cells.
These and other cancer-associated proteins were released by the cultured cells within 24 h of growth.
A total of 203 proteins from the KLE cells, 86 from HEC-1-A, and 161 from HeLa are reported.
Keywords: Endometrial cancer • Cell released proteins • Candidate biomarkers • Two-dimensional liquid
chromatography/tandem mass spectrometry
Introduction
Cells release proteins to its extracellular space by multiple
means, including secretion, ectodomain shedding of proteins,
and shedding of membrane-derived vesicles.1,2 Many of these
proteins represent main classes of bioactive molecules, including growth factors,3,4 cytokines,5 proteases, protease inhibitors,6,7 transmembrane receptors, and cell adhesion molecules.8,9 The dynamic change and interaction of these proteins
with the extracellular-matrix (ECM) molecules constitutes a
microenvironment for maintaining cell growth and tissue
development in a controlled way.10 Over the past decade, focus
on the tumor microenvironment has not only led to a better
understanding of tumorigenesis, but even brought an evolution
in our thinking of cancer development.11 Tumors are now
considered as complex tissues containing transformed cells and
co-evolving “normal” neighboring cell types.12 There is increasing evidence for the significant roles played by cell-released
proteins, for example, heparin-binding epidermal growth factor-like growth factor,13 macrophage inhibitory cytokine-1,14
and matrix metalloprotease,15 in communications between
diseased cells and their surrounding microenvironment, and
* To whom correspondence should be addressed: Prof K. W. Michael
Siu, Department of Chemistry, York University, 4700 Keele Street, Toronto,
Ontario, Canada M2J 1P3. Tel: (416) 650-8021; Fax: (416) 7365936; E-mail:
[email protected].
†
Department of Biology, York University.
‡
Centre for Research in Mass Spectrometry, York University.
§
Department of Chemistry, York University.
|
St. Michael’s Hospital.
⊥
University of Toronto.
O
Mount Sinai Hospital.
10.1021/pr0700798 CCC: $37.00
 2007 American Chemical Society
consequently on the final pathology. Studies on tumor cellreleased proteins have also shed light on the discovery of
biomarkers for early detection of cancer and intervention.11,16
Proteins that are secreted, or shed from cell membranes, have
been identified in blood or other bodily fluids of patients
afflicted with a variety of cancers. As sampling of bodily fluids
is relatively straightforward and is typically of minimal invasiveness; these proteins are potentially useful as biomarkers,
if the same proteins are not released (or released to the same
extent) from healthy cells. Indeed, blood- or bodily fluid-based
assays are the preferred methods for disease diagnosis and
prognosis, the blood test of prostate-specific antigen for
prostate cancer being the prime example.17
Endometrial cancer (EmCa) is the fourth most-common
malignancy in Canadian women with a 2% lifetime risk. At
present, there are no biomarkers available for diagnostic testing
of this disease. Women with symptoms typical of EmCa,
perimenopausal, postmenopausal, or abnormal uterine bleeding and/or discharge, must undergo an invasive procedure:
endometrial biopsy, curettage, and/or hysteroscopy to provide
a sample for pathological analysis. Availability of blood-borne
EmCa biomarkers would significantly enhance the ability to
diagnose the disease, perhaps in an earlier stage, and the
effectiveness of treatment. Our earlier work performed with
tissue homogenates from EmCa patients, using mass-tagging
reagents iTRAQ and cICAT with multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS),
resulted in a panel of candidate biomarkers.18 A number of
these differentially expressed proteins have recently been
verified in an iTRAQ-labeling study involving 40 samples.19 In
Journal of Proteome Research 2007, 6, 2615-2622
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Published on Web 05/25/2007
research articles
addition, the most sensitive and specific of these candidate
biomarkers have been independently validated on a second
cohort comprising 148 patients, using immunohistochemistry
in a tissue-microarray format.20
Here, we report results of an investigation into whether any
of the candidate biomarkers discovered and identified in these
previous studies can potentially be secreted or shed into the
extracellular environment. We expect that proteins isolated
from the serum-free media of cultured endometrial cancer and
HeLa cell lines would be a first indication of their possible
presence in actual bodily fluids.
Materials and Methods
Cell Culture and Collection of Culture Media. KLE, HEC1-A, and HeLa cells were obtained from American Type Culture
Collection. All cell types are of uterine and epithelial origin:
KLE cells were derived from poorly differentiated endometrial
carcinoma, HEC-1-A cells from moderately well-differentiated
endometrial carcinoma, while HeLa cells were from cervical
cancer. KLE and HeLa cells were grown in Dulbecco’s modified
Eagle’s medium, while HEC-1-A cells were grown in McCoy’s
5A medium (Wisent Inc.); both media were supplemented with
10% fetal bovine serum (FBS, HyClone) and 1 unit/mL penicillin-streptomycin (Invitrogen, Inc.). Cells were all grown under
37 °C and in a humidified atmosphere with 5% CO2. For
conditioned-medium collection, cells were grown to 60-80%
confluence in 100 mm dish (SARSTEDT). The culture medium
was aspirated, and the plates were rinsed four times with
phosphate-buffered saline (Sigma) and once with the corresponding growth medium without FBS. This medium was
collected for 0-h control. The cells were then incubated in the
appropriate medium free of FBS for 24 h. At the end of the
incubation time, the serum-free medium was collected and
filtered through a 0.2-µm nylon filter (SARSTEDT) to remove
any suspended cells. The medium was then frozen immediately
and stored at -80 °C until further processing. Media were
collected from 100-mm plates (a total of 50) of each cell type
for analysis.
Culture-Medium Protein Preparation. Proteins in the culture medium were isolated using 0.02% sodium deoxycholate
(Sigma) and 10% trichloroacetic acid (Sigma). Following 2-h
precipitation on ice, the samples were centrifuged for 30 min
at 11 000g and washed twice with ice-cold acetone. The
precipitated proteins were resuspended in 50 mM ammonium
bicarbonate. Protein concentrations were determined using the
Bradford assay (Bio-Rad). Proteins were digested with trypsin
using a modification of a literature method.21 Briefly, the
resuspended protein samples were heated to 60 °C for 1 h in
the presence of 5 mM dithiothreitol. The samples were allowed
to cool to room temperature and then alkylated by incubation
with 10 mM iodoacetamide for 1 h in the dark. Sequencinggrade trypsin (Promega, Madison, WI) at 1:20 (w/w) in 50 mM
ammonium bicarbonate was then added, and the samples were
incubated at 37 °C overnight. The digested samples were then
dried in a speed vacuum and resuspended in 10 µL of 0.1%
formic acid.
LC-MS/MS Analysis. Samples were analyzed by online twodimensional LC-MS/MS. The nanobore LC system and MS/
MS setup used for these analyses have been described previously.18,19 Briefly, the liquid chromatograph was an LC Packings
Ultimate (Amsterdam, The Netherlands), and the mass spectrometer was a QSTAR Pulsar-i hybrid quadrupole/time-offlight (TOF) instrument (Applied Biosystems/MDS SCIEX,
2616
Journal of Proteome Research • Vol. 6, No. 7, 2007
Li et al.
Foster City, CA). The tryptic peptides were first separated in
the first dimension using a strong cation exchange (SCX)
column (LC Packings: BioX-SCX cartridge, 500 µm × 15 mm).
One microliter of sample was loaded onto the SCX column and
was eluted in 10 fractions using 10-µL solutions of increasing
ammonium acetate concentration (10, 50, 100, 150, 200, 250,
300, 350, and 500 mM and 1 M) directly onto a C18 reversephase precolumn (LC Packings: 300 µm × 5 mm) for subsequent reverse-phase chromatography. Separation was effected
by a nonlinear binary gradient: eluent A consisting of 94.9%
deionized water, 5.0% acetonitrile, and 0.1% formic acid (pH
≈ 3); and eluent B consisting of 5.0% deionized water, 94.9%
acetonitrile, and 0.1% formic acid. During the first 5 min of
the LC run, eluent A at a flow rate of 50 µL min-1 was used to
load peptides salted out from the SCX column onto the C18
precolumn, after which the SCX column was switched out of
line. Desalting continued for 2 additional min. At the seventh
minute, the C18 precolumn was switched inline with the
reverse-phase analytical column (75 µm × 150 mm packed in
house with 3-µm Kromasil C18 beads with 100 Å pores, The
Nest Group); separation was performed at 200 nL min-1 using
a 90-min binary gradient shown below. Note that the “0” timepoint corresponds to the beginning of elution from the SCX
onto the C18 precolumn; the actual time at which the precolumn was brought inline with the analytical column was at the
seventh minute.
MS data were acquired in information-dependent acquisition
(IDA) mode with Analyst QS 1.1 and Bioanalyst Extension 1.1
software (Applied Biosystems/MDS SCIEX). MS cycles comprised a TOF MS survey scan with an m/z range of 400-1500
Th for 1 s, followed by five product-ion scans with an m/z range
of 80-2000 Th for 2 s each. The collision energy (CE) was
automatically controlled by the IDA CE Parameters script.
Switching criteria were set to ions with m/z g400 and e1500
Th, charge states of 2-4, and abundances of g10 counts.
Former target ions were excluded for 30 s, and ions within a
6-Th window were ignored. Additionally, the IDA Extensions
II script was set to “no repetition” before dynamic exclusion
and to select a precursor ion nearest to a threshold of 10 counts
on every fourth cycle.
Database Searching and Criteria. LC-MS/MS data were
searched using an in-house version of Mascot (Matrix Science,
U.K.) against an NCBI nr database (downloaded June 1, 2006
with 3 682 060 sequences) with the taxonomy selected for
mammals (which contained 446 729 sequences), and tolerances
set for 0.3 Da for peptide matches and 0.2 Da for MS/MS
fragment matches.
A second search was performed using ProteinPilot software
(Applied Biosystems, Foster City, CA) and a Celera human
protein database (CDS KBMS 20041109) containing 178 239
protein sequences to verify the results obtained with Mascot.
The cutoff for significance used for this search was set for a
score of 1.3, which corresponds to a confidence score of 95.
These peptides identified were compared with those reported
by the Mascot search in order to verify the identifications.
Bioinformatics. Identified proteins were analyzed for secreted protein features using the Signal Peptide Predictor
(http://www.cbs.dtu.dk/services/SignalP)22 and non-classical
and leaderless protein secretion (http://www.cbs.dtu.dk/
Biomarkers from Human Endometrial and Cervical Cancer Cells
research articles
Figure 1. Subcellular locations of proteins released by the endometrial and cervical cancer cell lines. Percentages of the proteins are
given. ER, endoplasmic reticulum; ECM, extracellular matrix.
services/SecretomeP-2.0/).23 Signal Peptide Predictor incorporates neural network and hidden Markov model algorithms to
detect signal peptides from input protein sequences. SecretomeP utilizes a neural network combining six protein features
to predict whether a protein sequence undergoes non-classical
secretion or not; these features are the number of atoms,
number of positively charged residues, presence of transmembrane helices, presence of low-complexity regions, presence of pro-peptides, and subcellular localization. The Gene
Ontology Consortium tool was applied for subcellular and
functional annotation analysis.24
Results and Discussion
Cell-Culture Condition Optimization. Culture cells are
typically grown in serum-supplemented media; however, highabundance serum proteins can interfere with the subsequent
detection of secreted proteins by mass spectrometry.25 To
circumvent this interference, serum-free media were used in
this study. The drawbacks, however, were that cell growth in
such media is typically slower and the rate of cell death higher,
thus, increasing the chance of cell autolysis and nonspecific
release of cytoplasmic proteins. For these reasons, we monitored closely the viability and the death rate of the cells in the
serum-free media. To seed the serum-free media with cells of
high viability, we grew cells in serum-containing media up to
a certain confluence and then switched to serum-free media:
HeLa and HEC-1-A cells up to 60% and KLE 75% confluence,
before they were washed and inoculated onto the serum-free
media. After 24 h, all cell lines reached about 85% confluence,
and the media were collected and processed for released
proteins. Harvesting the media after 24 h minimizes the extent
of cell stress and autolysis. The strategy of using serum-free
medium for the final growth stage and minimizing the duration
of this stage is similar to that in studies published within the
last year.26-28
Identification of Proteins Released by the Three Cancer
Cell Lines. For every cell line, we analyzed the proteomic
profiles of the serum-free media for the 0-h and 24-h incubations. Bovine proteins appearing in both profiles, including
bovine albumin and γ-globin, were considered as media
proteins and were removed from the lists. This resulted in lists
of nonredundant proteins totaling 160 for the HeLa, 198 for
the KLE, and 87 for the HEC-1-A cell lines. These are given in
Supplementary Tables 1s-3s in Supporting Information. Sixtyfive percent of the proteins were identified with two or more
peptides. The MS/MS spectra of proteins identified with single
peptides were verified by manual inspection and were accepted
only when a series of a minimum of four b- or y-type ions was
matched with a Mascot score >35. For these proteins, the
sequences of the peptides identified are also given in Supplementary Tables 1s-3s in Supporting Information. The extent
of overlap between the peptides identified in the Mascot and
those identified in the ProteinPilot search is summarized in
Supplementary Table 4s in Supporting Information. The number of unique peptides reported with ProteinPilot that are not
observed with Mascot appears to be high as the former includes
non-tryptic variations/degradation products of tryptic peptides.
Thirty-five percent of the identified proteins contain a
predicted signal-peptide sequence. As proteins can be exported
into the extracellular matrix without a classical N-terminal
Journal of Proteome Research • Vol. 6, No. 7, 2007 2617
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Li et al.
Figure 3. Overlaps of proteins identified in the culture media of
KLE, HEC-1-A, and HeLa cells.
Figure 2. (a) Molecular weight distributions and (b) isoelectrical
point distributions of identified proteins released by the endometrial and cervical cancer cells.
signal peptide, we analyzed the identified proteins also for
prediction of nonclassical and leaderless secretion. This revealed that 59% of the proteins could be secreted extracellularly
by nonclassical secretory pathways. Categorizing the proteins
according to their subcellular locations using GoMiner tool
(Figure 1) showed that on average 27% of identified proteins
were known to be secretory or extracellular, and 13% were
membrane-associated. These percentages compare favorably
with those reported in other studies on secretory proteins,26-28
and are consistent with our expectation of locating secreted
and released proteins in the cell-culture media.
The identified proteins of the three investigated cell lines
showed similar molecular-weight distributions. Fifty percent
of the proteins are 20-40 kDa (Figure 2a). Eighty-six percent
of the identified proteins from the HEC-1-A and HeLa cells had
molecular weights <80 kDa. There were a few more highmolecular-weight proteins (>100 kDa) identified from the KLE
cells, resulting in 70% of the identified proteins having molecular weights <80 kDa. Figure 2b shows the isoelectric point
(pI) distributions of the identified proteins from the three cell
lines. Proteins with calculated pIs from 3.35 (14-3-3 γ) to 11.96
(splicing coactivator subunit SRm300) were identified in this
study. Approximately 60% of the proteins identified were acidic
with pIs in the range 4-7 with the remainder having pIs in the
range 7-12. There are significant differences in the proteins
identified from the three cell lines (Figure 3), despite the fact
that two, HEC-1-A and KLE, were from uterine tumors, and
HeLa originated from cervical cancer. Sample complexity and
irreproducible sampling of the peptides cannot be the only
reasons for these differences, as replicate analyses of the same
sample resulted in an average of 89% of the same proteins
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Journal of Proteome Research • Vol. 6, No. 7, 2007
identified from each cell line. We also analyzed the protein
samples separated by one-dimensional liquid chromatography
(data not shown); the results reveal an overlap of 75-81%
(depending on the cell lines) of identified proteins with those
observed in two-dimensional LC. As pointed out earlier, the
KLE cell line was derived from poorly differentiated endometrial
carcinoma, while the HEC-1-A cell line was from moderately
well-differentiated endometrial carcinoma. Thus, the differences that we observe here may partially reflect actual differences in the nature of released proteins in these cell lines of
endometrial cancer origin. HeLa cells were from cancer of the
cervix, an epithelial cancer of a different part of the uterus.
Potential Biomarker Proteins Released by Endometrial
Cancer Cells. We had previously analyzed homogenates of
endometrial tissue in search of biomarkers for EmCa using
iTRAQ and cICAT labeling and tandem mass spectrometry.
Those studies resulted in the identification of a number of
differentially expressed proteins that appeared promising.
These included chaperonin 10, pyruvate kinase M1/M2 isozyme
(PK-M1/M2), alpha-1-antitrypsin (AAT), calgizzarin, and macrophage migration inhibitory factor (MIF).18 One of the complications resulting from these studies with homogenates of
snap-frozen endometrial tissue samples is that the homogenates contained not only endometrial epithelium, but also
supportive stroma, blood vessels, and secretions. The question
of localization has recently been addressed, and the validity of
some of these proteins confirmed by an immunohistochemical
analysis in a tissue microarray format involving 148 patient
samples, including 63 cases of pathological and 85 cases of
benign endometria.20 A panel of chaperonin 10, PK-M2, and
AAT satisfactorily differentiate endometrial cancer from benign
endometrium with a sensitivity of 0.85 and a specificity of 0.93.
To address whether any of the previously identified biomarker
proteins can be released extracellularly by tumor cells, we
pursued this study using cultured endometrial cancer cells and
HeLa cells. Encouragingly, we have identified chaperonin 10,
calgizzarin, PK-M1/M2, AAT, and MIF as being released by at
least one of the two endometrial cancer cell lines investigated
(Table 1). We have also identified clusterin in all three cell lines
and WAP four-disulfide core domain 2 protein (WFDC2 or HE4)
in KLE cell lines. Both proteins were reported as potential
endometrial cancer markers in a recent study.19 Taken together,
they support our optimism that some of these proteins may
be observable in blood and may serve as cancer biomarkers
for a blood-based diagnostic test. Significantly, AAT is a wellestablished abundant serum protein;29 in addition, other
independent, large-scale studies on serum have reported the
presence of many of the proteins listed in Table 1.21,30-34 Other
identified proteins of note are also listed in Table 1. Details of
research articles
Biomarkers from Human Endometrial and Cervical Cancer Cells
Table 1. Significant Proteins Released by Human Endometrial and Cervical Cancer Cells
protein name
Alpha-1 antitrypsin
Calgizzarin (S100 calcium-binding
protein A11)
Chaperonin 10
Macrophage migration inhibitory factor
Pyruvate kinase isozymes M1/M2 (EC
2.7.1.40)
WAP four-disulfide core domain 2
Clusterin isoform 1
Prostate differentiation factor
Mesothelin precursor
Insulin-like growth factor-binding
protein 2
Insulin-like growth factor-binding
protein 3
Insulin-like growth factor binding
protein 4
Insulin-like growth factor binding
protein 6
Insulin-like growth factor binding
protein 7
Insulin-like growth factor-binding
protein 10
Reticulon 4
Kallikrein 10
Kallikrein 6 precursor
Cystatin B
Cystatin C
Tissue inhibitor of metalloproteinase 1
Tissue inhibitor of metalloproteinase 2
Cell division cycle 5-like protein, polo-like
kinase
Secreted protein acidic and rich in
cysteine, Osteonectin
Osteopontin precursor
a
abbreviated
name
AAT
UNIPROT
accession no.
KLEa
HEC-1-Aa
HeLaa
+, #
+, #
+, #
+, #
+, #
description
Q13747
P31949
+, #
MIF
PK-M1/M2
Q53 × 54
Q6FHV0
P14618
+, #
+
+, #
WFDC2, HE4
Clusterin
GDF15, MIC-1
MSLN
IGFBP2
Q6IB27
Q2TU75
Q9BWA0
Q4VQD5
P18065
+, #
+, #
+
+, #
+, #
IGFBP3
P17936
+, #
IGFBP4
Q5U012
+, #
IGFBP6
Q5U012
+, #
+, #
IGFBP7
Q53YE6
+, #
#
+, #
CYR61, IGFBP10
Q6FI18
+, #
+, #
+, #
RTN4, ASY
Q7L7Q6
+, #
+, #
#
KLK10
KLK6
CSTB
CSTC
TIMP1
TIMP2
PLK
Q53YL3
Q6H301
Q76LA1
Q6FGW9
Q5H9B5
P16035
Q99459
+, #
+, #
+
+
+, #
+, #
+
+, #
+, #
+
+
+, #
+, #
+
+
+
#
+, #
+
Secreted endopeptidase inhibitor
Secreted complement lysis inhibitor
Secrerted growth factor
Secreted cell adhesion molecule
Secreted growth factor binding
protein
Secreted growth factor binding
protein
Secreted growth factor binding
protein
Secreted growth factor binding
protein
Secreted growth factor binding
protein
Secreted growth factor binding
protein
Endoplasmic reticulum membrane
protein
Secreted serine protease
Secreted serine protease
Secreted cysteine protease inhibitor
Secreted cysteine protease inhibitor
Secreted metalloproteinase inhibitor
Secreted metalloproteinase inhibitor
Serine/threonine kinase
SPARC
Q6IBK4
+, #
+, #
#
Secreted calcium binding protein
OPN
Q4W597
+, #
+, #
#
Secreted cell adhesion molecule
#
#
+, #
#
+, #
+
#
+
+, #
#
+, #
Secreted endopeptidase inhibitor
Calcium ion binding, regulation of
cell proliferation
Growth factor, immunosuppressant
Secreted/cytoplasmic cytokine
Phosphotransferase
references
for proteins
found in
blood
29
30, 31
31, 32
21
30-34
31, 32
32
31, 32
33
30-32
32
31, 32
32
32
32, 34
+ ) identified with Mascot; # ) identified with ProteinPilot.
peptides identified are given in Supplementary Table 5s in
Supporting Information.
Identification of Proteins Known To Be Associated with
Cancers. A number of studies have shown that extracellular
chaperonin 10 is associated with various cancers.18-20,35-37
Evidence suggests that the extracellular form of chaperonin 10,
homologous to the intramitochondrial heat-shock protein, is
produced by a separate gene.38,39 This extracellular protein was
first discovered as “early pregnancy factor” and was found to
be released from the placenta into the blood stream within 6
h of conception.40,41 Our analyses show that extracellular
chaperonin 10 carries two post-translational modifications at
the N-terminus.35,42 Extracellular chaperonin 10 probably functions as an immunosuppressant41 and a growth factor43 in
neoplastic cell proliferation. PK-M2 is expressed selectively in
metastatic cells,44-47 primarily in its dimeric form. The only
expression in healthy tissues is the lung in which PK-M2 is
present in the active tetrameric form.48,49 The dimer, but not
the tetramer, shows strong immunochemical staining with
monoclonal antibodies against PK-M2.50 In tumor cells, PKM2 oscillates from the inactive dimer to the active tetramer
based on allosteric activation by fructose-1,6-diphospate.48,49
PK-M2 plays a key role in the survival of cancers in hypoxic
environments and in the provision of metabolites for rapid cell
division.46,47 PK-M2 was found to be overexpressed in endometrial cancer tissues18-20 and in the plasma of patients of
gastrointestinal cancer.51 Calgizzarin is a calcium-binding
protein that has been observed to be significantly upregulated
in colorectal and lung carcinoma cell lines.52 It was also
identified as a tumor marker in mouse colon53 and human
endometrial cancers.18 AAT has been found to be associated
with a number of cancers, including lymphoma and cancers
of the liver, lung, stomach, bladder, gall-bladder,54,55 and
endometrium.18-20 MIF has been found to be overexpressed
in a number of cancers, including endometrial,18 hepatocellular,56 non-small-cell lung,57 and brain.58,59
In addition to the above, a number of proteins that have
been linked to other cancers have also been identified in this
study. Kallikrein 10 is a predictive biomarker for ovarian60 and
breast cancers.61 We identified kallikrein 10 and 6 in the two
endometrial cancer cell lines. Another protein of potential
interest is mesothelin, a glycosyl-phosphatidylinositol-anchored
glycoprotein present on cell surface. This protein is highly
expressed in mesothelioma, ovarian cancer, and pancreatic
cancer, and appears to be a target for immune-based therapies.62,63 Twenty percent of the identified proteins are in a
subgroup with functions affiliated with “signal transduction”,
“cell growth regulator”, and/or “immune response”. These
proteins include growth factors and their regulators, protein
kinases, calcium-binding proteins, and receptors. Insulin-like
growth factor-binding proteins (IGFBPs) 2, 3, 4, 6, 7, and 10
were all observed. Insulin-like growth factors (IGFs) are multifunctional regulatory peptides of cell growth and survival,
attributes that are important for tumorigenesis. Functions of
these IGF binding proteins have been investigated extensively,
and the levels of these proteins in the circulation have been
examined as indications of prostate, breast, and bladder
cancers.64,65 Our results are consistent with literature data
indicative of the secretory nature of IGFBPs and show that these
proteins constitute a major protein group released by endomeJournal of Proteome Research • Vol. 6, No. 7, 2007 2619
research articles
trial cancer cells. Prostate differentiation factor (also named
as GDF15 or MIC-1) was previously detected in the secretomes
of breast cancer cell line MA11 and a malignant melanoma cell
line WM266-4.27 This transforming growth factor-β (TGF-β)
superfamily protein is highly expressed in the placenta and
plays a pivotal role in human placental development. It may
have a similar role in promoting angiogenesis in tumor
development, as it is overexpressed in many tumors, including
metastatic prostate, breast, and colon cancers.14 Clusterin, a
known secreted glycoprotein and one of the potential endometrial cancer markers discovered in our recent tissue homogenate study,19 was detected in all three cell lines. Overexpression
of this antiapotopic protein has been implicated in various
tumor progression, including prostate, breast, lung, bladder,
and colon cancers. Furthermore, it has been proposed to be a
potential diagnostic and prognostic marker for colon carcinoma
aggressiveness.66 WFDC2 is known to be overexpressed in a
number of cell lines, including ovarian, renal, lung, colon, and
breast. The bulk of initial studies on WFDC2 were focused on
using it as a biomarker for ovarian carcinoma.67 A recent review
suggested that the overexpression of WFDC2 is a good, early
marker for ovarian cancer, even better than CA125 for that
purpose.68 Cell division cycle 5-like protein kinase (also named
as polo-like kinase 1, PLK1) was also identified in all three cell
lines. This serine/threonine kinase is an important regulator
of mitotic cell division. Its overexpression is significantly
associated with p53 accumulation in colorectal cancer, and it
was suggested that this kinase might have potential as a new
tumor marker for colorectal cancer.69,70 Reticulon 4 (RTN4 or
ASY) has been characterized as a human apoptosis-induced
protein without any known apoptosis-related motifs.71 This
protein induces apoptosis in various cancer cells when overexpressed, whereas normal cells are relatively resistant to ASYdependent apoptosis. A better understanding of the role played
by ASY in apoptosis may lead to development of novel cancer
treatment strategies.
Another major group of identified proteins are matrixassociated proteins, secreted extracellular glycoproteins, and
adhesion molecules. These include osteopontin (OPN), secreted
protein acidic and rich in cysteine (SPARC)/osteonectin, calumenin, cofilin, and agrin. OPN is a secreted extracellular matrix
glycophosphoprotein. It is a key molecule in neoplastic transformation and cancer development in a variety of tumors,
including breast, skin, and ovary.72 OPN levels in plasma and
cerebrospinal fluid were found to be elevated in patients with
atypical teratoid/rhabdoid tumors.73 Tumor progression is
facilitated by degradation or remodeling of the extracellular
matrix. Matrix metalloproteinases and their inhibitors play a
crucial role in this process. Not surprisingly, a large number
of serine proteases and their regulators/serpins have been
identified in this study, including tissue inhibitor of metalloproteinases (TIMPs) 1 and 2. TIMPs are implicated in many
cancers, and their expressions are typically elevated in cancer
patients.74 Cystatin B and C are natural inhibitors of cathepsin
B. Both cystatins were found to be released by all three cell
lines. Elevated levels of these proteins in serum were found in
melanoma and colorectal cancer patients and correlated
significantly with a higher risk of adverse outcome.75,76
Conclusions
We have identified and analyzed proteins released by endometrial and cervical cancer cell lines. These proteins include
several differentially expressed proteins that we previously
2620
Journal of Proteome Research • Vol. 6, No. 7, 2007
Li et al.
identified as candidate EmCa biomarkers in comparisons
between homogenates of malignant and benign tissues. An
obvious next step would be to target detection of these
biomarkers in bodily fluids, including blood plasma and/or
serum, of EmCa patients. In addition, other released proteins
have been found to associate with other cancer types. It would
be of value to determine in the future the extents of differential
expression of these proteins in biopsied EmCa samples.
Acknowledgment. We are grateful to Ms. X’avia C. Y.
Chan and Dr. Jingzhong Guo for performing preliminary work
and helping to develop some of the experimental strategies.
We thank Professor John C. McDermott for helpful suggestions
and discussions. This work was supported by Canadian Cancer
Society Research Grant No. 016172 of the National Cancer
Institute of Canada. A salary support (to L.V.D.S.) from the
Ontario Genomics Institute and Genome Canada was graciously acknowledged. Infrastructural support was provided by
the Ontario Research and Development Challenge Fund, and
Applied Biosystems/MDS SCIEX.
Supporting Information Available: Proteins identified in serum-free culture media of KLE, Table 1s; HEC-1-A,
Table 2s; and HeLa, Table 3s. Summary of Mascot and
ProteinPilot results, Table 4s. Details of peptides identified for
proteins in Table 1, Table 5s. These materials are available free
of charge via the Internet at http://pubs.acs.org.
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