Protein Expression Profiling Identifies Macrophage Migration Inhibitory Factor

[CANCER RESEARCH 63, 1652–1656, April 1, 2003]
Protein Expression Profiling Identifies Macrophage Migration Inhibitory Factor
and Cyclophilin A as Potential Molecular Targets in Non-Small Cell
Lung Cancer
1
Michael J. Campa, Michael Z. Wang, Brandon Howard, Michael C. Fitzgerald, and Edward F. Patz, Jr.
2
Department of Radiology, Duke University Medical Center, Durham, North Carolina 27710 [M. J. C., B. H., E. F. P.], and Department of Chemistry, Duke University, Durham,
North Carolina 27708 [M. Z. W., M. C. F.]
ABSTRACT
Current diagnostic and therapeutic strategies for lung cancer have had
no significant impact on lung cancer mortality over the last several
decades. This study used a matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF MS) discovery platform
to generate protein expression profiles in search of overexpressed proteins
in lung tumors as potentially novel molecular targets. Two differentially
expressed protein peaks at m/z 12,338 and 17,882 in the MALDI-TOF
spectra were identified in lung tumor specimens as macrophage migration
inhibitory factor and cyclophilin A, respectively. Overexpression of both
proteins was confirmed by Western blotting, and cyclophilin A was local-
ized to the tumor cells by immunohistochemistry. These data demonstrate
the feasibility of using a MALDI-TOF platform to generate protein ex-
pression profiles and identify potential molecular targets for cancer diag-
nostics and therapeutics.
INTRODUCTION
Lung cancer continues to be a major public health issue with
⬎170,000 new cases diagnosed in the United States each year alone.
Despite extensive research efforts in genomics, drug discovery, and
lung cancer screening, the overall 5-year survival remains ⬃14% (1).
Thus, alternative diagnostic and therapeutic strategies are necessary
for changes in outcome to be realized. Recent efforts to improve
diagnostic capabilities, facilitate phenotypic stratification, and pro-
mote the discovery of new molecular targets for cancer therapies have
focused on the spectrum of genetic changes of cancer using gene
arrays (2–6). Given the disparity between gene transcription and
protein expression, however, and the absence of information on post-
translational modification, it is not apparent that genetic analysis alone
will be sufficient to improve clinical outcomes.
As another means of addressing this challenge, we sought to de-
termine whether protein expression profiling with a MALDI-TOF
MS
3
platform could be used as an alternative strategy in the search for
new diagnostic and therapeutic targets. Because the phenotype of a
disease is largely a reflection of its protein complement, we reasoned
that a display of the expressed proteins within a tissue could form a
pattern that could hold detailed information regarding the biology of
the disease. For this study, we developed a mass spectrometry-based
platform to generate protein expression profiles from lung tissue
lysates in an effort to discover and identify novel molecular targets for
lung cancer.
MATERIALS AND METHODS
Patients and Specimens. All patients at our institution who were to un-
dergo pulmonary resection for lung cancer or have a lung biopsy for unknown
lung disease were considered eligible for the study. After informed consent
approved by the Institutional Review Board, patients with lung cancer had a
sample of the tumor and adjacent normal lung removed from the gross
specimen at the time of resection. Those patients with unknown lung disease
and open biopsy had a sample of the abnormal lung tissue obtained for
MALDI-TOF MS analysis.
After gross examination by a staff pathologist, tissue specimens were frozen
in liquid nitrogen usually within 5 min after resection. Regions of specimens
used to make tissue lysates were sectioned, stained with H&E, and examined
to verify the histological diagnosis (i.e., tumor, normal lung, or other lung
disease).
Lysate Preparation. Tissue specimens were minced, and blood contami-
nation was reduced by end-over-end rotation for 30 min at 4°C in a conical
centrifuge tube containing 10 ml of PBS. A portion of the washed tissue (⬃10
mg) was then placed into a microcentrifuge tube containing 70
␮ l of mam-
malian protein extraction reagent (Pierce, Rockford, IL), crushed with a plastic
pestle, and shaken for 30 min at 4°C. Cellular debris was then removed by
centrifugation at 16,000 ⫻gfor 20 min at 4°C. Total protein content in the
supernatant fraction was estimated by a Bradford dye binding assay using BSA
as standard (7).
MALDI-TOF Analysis. Cell lysate samples were prepared for MALDI
analysis using a conventional dried droplet protocol. In this protocol, sinapinic
acid was used as the MALDI matrix. The sinapinic acid matrix was prepared
as a saturated, aqueous solution that contained 50% (volume for volume)
acetonitrile and 0.1% (volume for volume) trifluoroacetic acid. A 1-
␮ l aliquot
of each cell lysate was mixed with 30 ␮ l of the sinapinic acid matrix solution
before depositing 2 ␮ l of this mixture on the MALDI sample stage where the
solvent was evaporated under ambient conditions.
All MALDI-TOF mass spectra were acquired on a Voyager DE Biospec-
trometry Workstation (PerSeptive Biosystems, Inc., Framingham, MA) in the
linear mode using a nitrogen laser (337 nm). Mass spectra were collected in the
positive ion mode using an acceleration voltage of 25 kV, a grid voltage of 23
kV, a guide wire voltage of 75 V, and a delay time of 225 ns. Each spectrum
obtained was the sum of ⬃32 laser shots. The raw data in each mass spectrum
were smoothed using a Savitsky-Golay smoothing routine before mass cali-
bration using proteins of known mass as internal or external calibrants. S/Ns
were evaluated using the standard software provided by the instrument man-
ufacturer. All spectra used in this study were obtained from ⬃500 ng of total
lysate protein.
Data Analysis. MALDI-TOF mass spectra from each sample were ana-
lyzed using a T
2
test-based statistical pattern recognition approach to deter-
mine which parts of the spectra, or bins, were the most valuable in terms of
their ability to separate the two sets of data (i.e., tumor or normal). The test
yields a Pfor each bin, which is a measure of the probability that the means
of the two groups of data in that bin are equal. Consequently, a low Pindicates
that the two means are not close to each other; hence, that bin has a reasonable
capability of separating the sample sets. The lower the P, the more separable
the data are in that particular bin.
Protein Identification. Partial purification of the proteins responsible for
the ion signals at m/z 12,338 and 17,882 was accomplished by first performing
preparative liquid phase isoelectric focusing (Rotofor; Bio-Rad, Hercules, CA)
on seven pooled tumor lysates. The fractions from this separation technique
containing the m/z 12,338 and 17,882 peaks were identified by MALDI-TOF
MS, pooled, lyophilized, and subjected to further purification by reversed-
Received 10/30/02; accepted 2/3/03.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported in part by the National Cancer Institute: Early Detection Research Net-
work (1CFAMB-10057-A2).
2
To whom requests for reprints should be addressed, at Duke University Medical
Center, Department of Radiology, Box 3808, Durham, NC 27710. Phone: [phone];
Fax: [phone]; E-mail: [email protected].
3
The abbreviations used are: MALDI-TOF MS, matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry; MIF, macrophage migration inhibitory fac-
tor; CyP-A, cyclophilin A; S/N, signal:noise ratio; CT, computed tomography; PET,
positron emission tomography; FDG,
18
F-2-deoxy-D-glucose.
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phase high-performance liquid chromatography on a C4 column (Vydac,
Hesperia, CA). The high-performance liquid chromatography fractions con-
taining the peaks of interest were then pooled, lyophilized, and subjected to
two-dimensional gel electrophoresis using a pH 3–10 ReadyStrip immobilized
pH gradient strip (Bio-Rad) for the first dimension and an 8–16% polyacryl-
amide gel for the second dimension. After silver staining, protein spots
migrating between ⬃10 and 20 kDa were selected and subjected to peptide
mapping experiments at the Protein Core Facility at the University of North
Carolina at Chapel Hill using a Bruker Reflex III and an Applied Biosystems
4700 Proteomics Analyzer.
Immunoblotting. Tissue lysates were subjected to one-dimensional SDS-
PAGE (10 ␮ g of lysate protein/well) on a 15% polyacrylamide gel, and the
proteins were electroblotted to polyvinylidene difluoride using standard meth-
ods. Blots were probed with a rabbit polyclonal antihuman CyP-A antibody
(Upstate Biotechnology, Inc., Lake Placid, NY), and bound antibody was
detected using horseradish peroxidase-conjugated secondary antibody and a
chemiluminescent detection system (Pierce, Rockford, IL). The blots were then
stripped of bound antibodies (Restore stripping solution; Pierce) and reprobed
with a mouse monoclonal antihuman MIF antibody (R&D Systems, Minne-
apolis, MN). Before MIF immunoblot analysis, removal of anti-CyP-A anti-
bodies was verified by incubation with horseradish peroxidase-conjugated
antirabbit antibody and chemiluminescent detection.
Immunohistochemistry. Immunohistochemical analysis of acetone-fixed
(⫺70°C, 30 s), 5–8- ␮ m sections of lung tumor and normal lung tissue was
performed as described previously (8). Briefly, polyvalent anti-CyP-A rabbit
antibody (Upstate Biotechnology) and normal rabbit immunoglobulins were
used at 13 ␮ g/ml; incubation with biotinylated goat antirabbit IgG and horse-
radish peroxidase-conjugated Streptavidin (Zymed, South San Francisco, CA)
was performed following manufacturer’s recommendations. Slides were de-
veloped with diaminobenzidine (Immuno-Pure Ultra-Sensitive ABC Staining
Kit and Metal Enhanced 3,3⬘-diaminobenzidine substrate Kit; Pierce), coun-
terstained with hematoxylin, dehydrated, mounted, and scored by two inde-
pendent observers. Analysis was performed at the Immunohistochemistry
Shared Resource of the Duke Comprehensive Cancer Center.
RESULTS
MALDI-TOF Analysis of Tissue Lysates. For this study, we
developed a mass spectrometry-based platform to generate protein
expression profiles from lung tissue lysates in an effort to discover
and identify novel molecular targets. We initially obtained 10 lung
cancer specimens and the matched normal lung tissue specimens from
the same 10 patients, which were frozen immediately after surgical
resection. We then prepared tissue lysates from these specimens,
analyzed the lysates by MALDI-TOF MS, and compared all 10 tumor
lysate protein profiles to all 10 normal lung profiles using a statistical
pattern recognition approach with a T
2
test to elucidate the differences
in protein expression. The most statistically significant differentially
expressed peaks in the tumor as compared with the normal tissue
lysates were found at m/z 12,338 and 17,882 (⫾0.1% mass accuracy).
To validate the above results on a larger sample size, we prepared
lung tumor tissue/normal lung tissue lysate pairs from an additional 30
patients who had pulmonary resection. Twenty-four patients had
radiological findings suspicious for malignancy and were diagnosed
with non-small cell lung cancer, 4 patients had radiological findings
suspicious for malignancy but were diagnosed with benign lung
disease, and 2 patients had imaging findings suggestive of interstitial
lung disease which were confirmed by pathology. Figs. 1 and 2 show
2 different patients both with CT and PET findings suspicious for lung
cancer, although only the mass spectral data from patient 1 shows the
peaks in the tissue lysates at m/z 12,338 and 17,882 consistent with
malignancy (Fig. 1C).
Using a classification scheme based on our mass spectral analyses
of the original set of 10 tumor/normal lysate pairs, we classified the
blinded tissue lysates from these additional 30 patients (Fig. 3).
Overall, both of the distinguishing ion signals were seen in 27 of 34
Fig. 1. MALDI-TOF analysis of a 1.5-cm lung nodule suspicious for lung cancer by CT
and PET imaging. In A, axial CT image of a 69-year-old female demonstrated an irregular
1.8-cm right lower lobe nodule of uncertain etiology (arrow). In B, further evaluation with
PET and FDG was performed in this patient in an attempt to differentiate a benign from
malignant lesion. Coronal FDG-PET image showed increased FDG uptake in the nodule
(arrow), suggestive of malignancy. In C, because of the imaging findings and concern for lung
cancer, the patient was taken to surgery. Mass spectrum of the nodule lysate showed the peaks
at m/z 12,338 and 17,882, consistent with malignancy (top black spectrum). The lysate from
the patient’s normal lung at the time of resection did not show the tumor peaks (bottom gray
spectrum). Pathologic examination of the nodule confirmed adenocarcinoma.
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tumor lysates and in only 1 of 40 nonmalignant tissue lysates (79%
sensitivity, 98% specificity). Ion signals at either m/z 12,338 or 17,882
were found in 30 of 34 tumor lysates and 3 of 40 nonmalignant lysates
(88% sensitivity, 93% specificity). When examined individually, the
peak at m/z 12,338 was seen in 30 of 34 tumor lysates and 3 of 40
nonmalignant lysates (88% sensitivity, 93% specificity). The peak at
m/z 17,882 was found in 27 of 34 tumor lysates and 1 of 40 nonma-
lignant lysates (79% sensitivity, 98% specificity).
Protein Identification. A mass spectrometry-based peptide map-
ping strategy was used to identify the proteins producing the ion
signals at m/z 12,338 and 17,882. Ultimately, several proteins in spots
that had migration distances consistent with 12,338 and 17,882 kDa
proteins were subjected to an in-gel tryptic digestion. The resulting
peptides from each protein digest were sequenced by mass spectrom-
etry. The identification of CyP-A was substantiated by MS/MS data
on the following 7 tryptic peptides: (a) VSFELFADK; (b) FEDEN-
FILK; (c) EGMNIVEAMER; (d) VSFELFADKVPK; (e) VKEGM-
NIVEAMER; (f) SIYGEKFEDENFILK; and (g) HTGPGILSMAN-
AGPNTNGSQFFICTAK. These peptides spanned ⬃40% of CyP-A’s
linear amino acid sequence. MIF was identified based on MS/MS data
from one tryptic peptide with the sequence PMFIVNTNVPR and on
mapping the mass of an additional tryptic peptide to the following
linear sequence in MIF: ISPDRVYINYYDMNAANVGWNNSTFA.
The two proteins identified in this peptide mapping experiment,
MIF and CyP-A, had reported molecular masses of 12,345 and 17,881
kDa and were consistent with those of the protein ion signals observed
in our initial MALDI-TOF mass spectra of tumor (the error of our m/z
measurements was typically ⫾0.1%).
Fig. 2. MALDI-TOF analysis of a 1.8-cm lung nodule suspicious for lung cancer by CT
and PET imaging. In A, axial CT of a 46-year-old male demonstrated a 2.2-cm right lower
lobe nodule (arrow). In B, further evaluation of this patient with FDG-PET was per-
formed, and a coronal image showed increased FDG uptake (arrow), suggestive of a
malignancy. In C, because of the concern for lung cancer, the patient was taken to surgery.
Mass spectrum of the nodule lysate did not contain the peaks at m/z 12,338 and 17,882.
This pattern was suggestive of a benign abnormality, and pathologic examination of the
nodule showed that this was caused by a filarial infection, Dirofilarisis (dog heartworm). Fig. 3. Comparison of S/Ns for the MIF and CyP-A peaks in MALDI-TOF spectra of
lysates from all 40 tumor/normal pairs and six other lung diseases. This ratio was used to
determine whether overexpression in the sample was present. A, plot of specimen number
versus average S/N for the MIF peak in the 10 tumor/normal pairs from the training set
(F/Œ), 24 additional tumor/normal pairs (E/‚), and in six other lung diseases (䡺). B,
CyP-A peak data, symbol key same as in A. The Yaxis of each plot is displayed on a log
scale for clarity. The error bars represent the SD associated with the S/Ns extracted from
10 different spectra.
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Immunoblot Analysis and Immunohistochemistry. After pro-
tein identification, we verified by immunological techniques that MIF
and CyP-A were overexpressed in lung tumor cell lysates. We per-
formed immunoblot analyses on cell lysate proteins from a series of
tumor/normal lung matched pairs, as well as from a series of other
malignant and nonmalignant lung diseases. We found MIF to be more
abundant in lung tumor cell lysates from five of seven matched
tumor/normal pairs (Fig. 4A; Tumors 1, 4–6, 10) and three non-
matched tumor lysates (Fig. 4A; Tumors 7–9). Higher levels of CyP-A
expression were found in tumor lysates in six of seven matched
tumor/normal pairs (Fig. 4B; Tumors 1, 3–6, 10) and three non-
matched tumor lysates (Fig. 4B; Tumors 7–9). The level of MIF and
CyP-A in the other lung disease tissues was approximately equivalent
to the normal specimens, with the exception of the patient with
hypersensitivity pneumonitis (Fig. 4, Aand B; HP).
Tissue localization of CyP-A to the cytoplasm and nuclei of the
epithelial tumor cells was demonstrated by strongly positive immu-
nohistochemical staining of frozen lung tumor sections. These data
confirmed that the malignant cells were responsible for CyP-A over-
expression in the lysates and that this was not attributable to a
surrounding host inflammatory response. The matched normal lung
tissue was negative (Fig. 5). We did not similarly assess MIF by
immunohistochemistry because a previous study reported cytoplasmic
and nuclear overexpression of MIF in adenocarcinoma of the lung (9).
DISCUSSION
The discovery of new molecular targets for lung cancer diagnostics
and therapeutics has the potential to significantly change the clinical
approach and outcome of this disease. Current diagnostic techniques,
including noninvasive imaging followed by pathologic examination of
tissue, are still fundamentally based on anatomical and morphological
analysis. Therefore, we have designed an alternative strategy and ex-
plored the possibility of using a MALDI-TOF MS-based platform as a
means of generating protein expression profiles for the discovery of novel
molecular targets. In theory, this approach has several advantages over
other modalities used to identify differential expression of proteins (10,
11). MALDI-TOF analyses are capable of detecting small amounts of
protein material, hence, the potential to detect low abundance proteins.
MALDI-TOF exhibits excellent resolution of proteins of low molecular
mass, and it is able to detect both very acidic and basic proteins, which
can be particularly troublesome on two-dimensional gels. More impor-
tantly, this type of discovery technique makes absolutely no assumptions
about known or unknown proteins, allowing the process to be completely
independent of all presupposed hypotheses.
The current study demonstrates that it is possible to generate mass
spectrometry profiles from small human tissue samples and that lung
cancer tissue can be differentiated from normal lung and other lung
diseases based on a MALDI-TOF-generated protein expression profile.
Elucidation of MIF by this process helps validate our experimental
approach, because we were able to identify this protein, which has been
reported to be overexpressed in adenocarcinoma of the lung (9). Although
initially described as a cytokine causing T-cell activation, subsequent
reports have shown MIF to have a variety of cytokine activities in a
spectrum of diseases (12). Additional studies are needed to elucidate
MIF’s potential as a diagnostic or therapeutic target in cancer.
Fig. 5. Anti-CyP-A immunohistochemistry of a lung
tumor and the matched normal lung. In A, nuclear and
cytoplasmic staining are demonstrated in the lung
tumor. In B, normal lung shows no CyP-A staining.
Fig. 4. MIF and CyP-A immunoblot analyses of lysate protein from lung tumor,
matched normal lung, and other lung lesions. In A, MIF immunoblot of 10
␮ g of lysate
protein from tumor (T), matched normal (N), and various other lung lesions (Emphysema,
Pneumonia, Hypersensitivity Pneumonitis, Melanoma, Cystic Fibrosis, and Atelectasis)
was performed using an antihuman MIF antibody (R&D Systems). B, CyP-A immunoblot
of the same blot as in A, stripped and reprobed with a rabbit polyclonal anti-CyP-A
antibody (Upstate Biotechnology). We used actin immunoreactivity to verify equal protein
loading of gels before immunoblotting.
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More importantly, however, the discovery of CyP-A overexpres-
sion in lung cancer demonstrates the power of this technology to
elucidate novel molecular targets. CyP-A has never been reported in
lung cancer. CyP-A is a member of the immunophilin family of
proteins, typically studied for their binding of various immunosup-
pressive drugs, most notably Cyclosporin A, and their role in cellular
signaling pathways (13, 14). CyP-A has also been shown to possess
peptidyl prolyl cis-trans isomerase activity and thus may have a role
in protein folding (15). Although CyP-A has numerous known activ-
ities, its role in cellular growth and differentiation, transcriptional
control, cell signaling, and immunosuppression suggests that it could
be involved in an important aspect of oncogenesis.
Although this study focused on these two most statistically signif-
icant differentially expressed proteins, many other differentially ex-
pressed proteins have yet to be explored. The MALDI mass spectra
generated in this study were from unfractionated tissue cell lysates,
and thus, spectra contained ion signals for only a small fraction of the
thousands of different proteins that are expected to be present in such
a sample. The particular subset of proteins that we observed was
defined by the relative desorption/ionization efficiencies of each pro-
tein under the conditions of our MALDI analysis (16, 17). A number
of factors has been noted to contribute to the relative desorption/
ionization efficiency of proteins in complex mixtures, such as the
protein’s relative abundance, relative ionization efficiency, and spe-
cific interactions with the MALDI matrix. We expect that the sepa-
ration of the specimen proteins into less complex mixtures will result
in more extensive representation of the expressed proteins and in turn
yield spectra with higher information content.
Although there is a tremendous interest in proteomics, this is the
first study to fingerprint lung cancer using MALDI-TOF MS. We
identified two potential molecular targets, MIF, an overexpressed
protein in lung cancer reported previously, and CyP-A, a protein not
known previously to be overexpressed in non-small cell lung cancer.
The process of protein profiling has the potential to expand the current
repertoire of molecular targets, with the possibility for phenotypic
stratification. This type of information should lead to more rationally
designed diagnostic and treatment strategies, which will hopefully
translate into improved patient outcomes.
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