Full text of DOI:10.1155/2001/657605

295
A proteomic approach to the identification of
lung cancer markers
Samir Hanasha,∗, Franck Brichorya and
David Beerb
lamide gels for protein separations, the 2-D gel ap-
proach is being increasingly complemented with addi-
tional analyses using liquid based protein separations
and protein microarrays. Proteomic analysis of tis-
sues and cell populations uniquely contributes an un-
derstanding of protein post-translational modifications
and of the distribution of protein gene products in sub-
cellular compartments. An important objective of our
lung cancer effort is the identification of novel markers
for early detection.
A large numberof studies involvinglung cancer have
been independently performed in the laboratory. At
the protein level, these studies have resulted in over
1000 samples related to lung cancer, that have been
processed using 2-D gels and for which information
has been recorded in the Lung Protein Database. This
number represents a fraction of over 30,000 2-D Gels
produced by our group for different studies, including
studies of other cancer types. While lung adenocar-
cinomas represent a major portion of the lung cancer
database, other lung tumor types including squamous
cell carcinomas and small cell lung cancers are repre-
sented, as are control lung tissues. Other 2-D patterns
were produced from studies of cell lines that have been
manipulated by transfection or by treatment with spe-
cific agents, as well as patterns produced after different
cell fractionation schemes.
Mass Spectrometry and/or N-terminal sequencing of
protein spots from 2-D gels of lung tumor samples or
cell lines has led to the identification of a large num-
ber of proteins expressed in lung cancer. Also, most
identifications made for proteins from a sample type
can often be confidently transferred to matching pro-
tein spots on master images from lung studies. Fig-
ure 1 exhibits some of the progress we have made in
identifying proteins in 2-D gels of lung samples.
An important first step in mining lung cancer pro-
teomic data for various applications is to determine the
ability of proteomic profiling to distinguish between
known types of lung cancer. Specific protein differ-
ences between differenttypes of cancer have been iden-
tified by other groups. In a recent study of breast, ovary
aDepartment of Pediatrics, University of Michigan
Medical Center, Ann Arbor, MI 48109, USA
bDepartment of Surgery, University of Michigan
Medical Center, Ann Arbor, MI 48109, USA
We have developed a comprehensive approach to the identi-
fication of protein markers in lung cancer that includes pro-
filing of tumor tissue and cell lines as well as the analysis
of serum for autoantibodies to lung tumor antigens. A large
number of proteins that are differentially expressed in the
major subtypes of lung cancer have been identified by mass
spectrometry. A database of protein expression in lung can-
cer and other types of cancer has been constructed that inte-
grates two-dimensional gel profiles, mass spectrometry data,
quantitative protein data and gene expression data at the RNA
level, that serves as a resource for biomarker identification.
Analysis of the serological response in lung cancer has led
to the identification of novel markers detectable in serum of
lung cancer patients at the time of diagnosis. The proteomic
approach is likely to yield novel classification schemes and
novel markers for early diagnosis of lung cancer.
Keywords: Lung, cancer, proteomics, markers
1. Introduction
We have implemented a comprehensive strategy for
the molecular analysis of lung cancer that includes pro-
teomic analysis of lung tumor tissue and cell lines, as
well as serum analysis for the identification of lung
tumor proteins that induce a serological response in
the form of autoantibodies. Although we have relied
to date primarily on two-dimensional (2-D) polyacry-
∗Address for correspondence: Samir Hanash, University of Michi-
gan Medical Center, 1150 W. Medical Center Drive, A520 Medi-
cal Science Research Building I, Ann Arbor, MI 48109-0656, USA.
Tel.: +1 734 763 9311; Fax: +1 734 647 8148; E-mail: shanash@
umich.edu.
Disease Markers 17 (2001) 295–300
ISSN 0278-0240 / $8.00  2001, IOS Press. All rights reserved

296
S. Hanash et al. / A proteomic approach to the identification of lung cancer markers
and lung tumors, 20 differentially expressed proteins
were identified [1] and in a prior study, 16 polypeptides
were found to be associated with different histopatho-
logical features of lung cancer [2,3]. In a study of
25 adenocarcinomas of the lung, 12 small cell lung
cancers, and 16 squamous cell tumors, by our group
(manuscript submitted) an initial analysis of protein
2-D patterns uncovered a group of 52 protein spots
that differed in average integrated intensity between the
three groups in a statistically significant manner. We
have identified 39 of this set of 52 spots by either N-
terminal sequencing and/or mass spectrometry of spot
digests.
the detection of tumor antigens that induce an immune
response. A number of antigens have been detected by
screening expression libraries with patient sera [4–6,
20–22]. The merits of our proteomic approach is that it
allows proteins, in their modification states as they oc-
cur in cells, to be analyzed for their antigenicity. Given
that proteins are subject to post-translational modifica-
tions, antibodies to epitopes that result from such post-
translational modifications can be detected. Addition-
ally, the 2-D approach allows for serial serum samples
to be analyzed much more readily than the screening
of expression libraries.
We have implemented a proteomic approach for the
identification of tumor antigens that elicit a humoral
response [23–25]. To this end, we have utilized 2-D
PAGE to simultaneously separate several thousand in-
dividual cellular proteins from tumor tissue or tumor
cell lines. Separatedproteins are transferredonto mem-
branes. Sera from cancer patients are screened individ-
ually, for antibodies that react against separated pro-
teins, by Western blot analysis. Proteins that specif-
ically react with sera from cancer patients are identi-
fied by mass spectrometric analysis and/or amino acid
sequencing and further evaluated with respect to their
specificity.
2. Serological approaches for the identification of
lung cancer markers
There is increasing evidence for an immuneresponse
to cancer in humans, demonstratedin part by the identi-
fication of autoantibodies against a number of intracel-
lular and surface antigens detectable in sera from pa-
tients with different cancer types [4–7]. The majority
of tumor derived antigens that have been identified as
eliciting a humoral response in lung cancer, as in other
tumor types, are not the products of mutated genes.
They includedifferentiationantigens and otherproteins
that are overexpressed in tumors [8]. The oncogenic
proteins L-Myc and C-Myc have been found to elicit
autoantibodies in a small percentage of patients [5,9].
There is some evidence that occurrence of autoantibod-
ies in lung cancer is of prognostic relevance [10–15].
Remarkably, tumor regression has been demonstrated
in some patients with small-cell lung carcinoma and
autoantibodies to onconeural antigens [15,16]. It is not
clear why only a subset of patients with a tumor type
develop a humoral response to a particular antigen. Im-
munogenicity may depend on the level of expression,
post-translational modification or other types of pro-
cessing of a protein, the extent of which may be vari-
able among tumors of a similar type. Other factors that
influence the immune response may include variability
among individuals and tumors in major histocompati-
bility complex molecules. Cytokines are also known
to affect the immune response and may vary in concen-
tration between tumors or in circulation [17–19].
3. A combined serological and proteomic-based
approach to the identification of novel lung
cancer markers
We have identified using our proteomic approach a
battery of proteins that induce autoantibodies that are
specific for different types of cancer including some
that show specificity for lung cancer. The availability
of a database of protein expression in lung cancer has
facilitated the identification of proteins that induce au-
toantibodies, in addition to providing valuable infor-
mation regarding the expression pattern of such protein
antigens in different tumor types and cell lines. One
such antigenwe have identified in lungcancer is protein
PGP 9.5 (Fig. 2) [24]. PGP 9.5 was identified as a pro-
tein in lung cancer that induces autoantibodies as part of
a study in which sera from 64 newly diagnosed patients
with lung cancer, from 99 patients with other types of
cancer and from 71 non-cancer controls were analyzed
for antibody-based reactivity against lung adenocarci-
noma proteins resolved by 2-D PAGE. Gels containing
separated proteins were blotted and subsequently hy-
bridized with individual sera from patients or controls.
Unlike controls, autoantibodies against a protein iden-
The identification of panels of tumor antigens that
elicit an antibody response may have utility in can-
cer screening, diagnosis or in establishing prognosis.
Such antigens may also have utility in immunotherapy
against the disease. There are several approaches for

S. Hanash et al. / A proteomic approach to the identification of lung cancer markers
297
Fig. 1. A small cell lung cancer master image with identified proteins.
tified by mass spectrometry as protein gene product 9.5
(PGP 9.5) were detected in sera from 9 of 64 patients
with lung cancer.
Circulating PGP 9.5 antigen was detected in sera
from two additional patients with lung cancer, without
detectable PGP 9.5 autoantibodies. PGP 9.5 was first
identified as a specific marker for neurons and neu-
roendocrine cells [26]. PGP 9.5 belongs to a family
of ubiquitin C-terminal hydrolase (UCH) isoenzymes
that play a regulatory role in the ubiquitin system [27].
It has been implicated in the mechanism to remove
ubiquitin from ubiquitinated proteins and thus prevent-
ing their degradation by proteasomes [28]. Ubiquiti-
nation of cellular proteins and their targeting for sub-
sequent degradation via ubiquitin-mediatedproteolysis
is an important mechanism that regulates the activity
of a variety of genes, notably cell cycle genes [27,29].
In lung tumors, increased de-ubiquitination of cyclins
by PGP 9.5 may contribute to uncontrolled prolifera-
tion [28]. In our study we demonstrated by 2-D PAGE

298
S. Hanash et al. / A proteomic approach to the identification of lung cancer markers
4.0 5.0 6.0
7.0
8.0 8.3
pI
160 kDa
70
55
A
43
MW
A5
P1 P2 P3
A2
A
27
18
14
Fig. 2. A 2-D image of a lung adenocarcinoma. Boxed areas containing annexins I and II and PGP 9.5 proteins are presented in Fig. 3.
and Western blot analyses that 80% of the lung tumors
we have studied contained detectable levels of PGP 9.5.
In previous analyses by immunohistochemistry, PGP
9.5 was detected in 40–82.5% of NSCLC and 50–90%
of SCLC [30–33]. Hibi et al reported ectopic expres-
sion of PGP 9.5 in lung cancers by SAGE analysis and
by immunochemistry [31,34]. In primary NSCLCs,
54% of the cases had positive PGP 9.5 staining, and
protein expression was associated with pathological
stage (44% of stage I and 75% of stages II and IIIA).
PGP 9.5 was observed in both SCLC and NSCLC cell
lines, independent of neuronal differentiation. Using
A549 lung adenocarcinoma cell line, we have demon-
strated that PGP 9.5 was present at the cell surface, as
well as secreted. Thus, the findings of PGP 9.5 antigen
and/or antibodies in serum of patients with lung cancer
suggest that PGP 9.5 may have utility in lung cancer
screening and diagnosis, as part of a panel of such pro-
teins or their corresponding antibodies, which we have
identified.
and/or II. Immunohistochemical analysis showed that
annexin I was diffusely expressed in neoplastic cells in
lung tumor tissues, whereas annexin II was predomi-
nant at the cell surface.
Annexin I is a 37 kDa protein which has been im-
plicated in glucocorticoid induced inhibition of cell
growth [35,36]. Annexin II is a 36 kDa protein that
occurs in a monomericform or as a tetramer, associated
with the annexinII light chain (p11), which is a member
of the S100 family [37,38]. Annexin II has been im-
plicated in cell-cell adhesion and in plasminogen acti-
vation and may function as a cell surface receptor [39].
Annexin II tetramers have been shown to interact with
procathepsinB on the surface of tumorcells and may be
involved in extracellular proteolysis, facilitating tumor
invasion and metastasis [40]. Interestingly, annexin I is
a target of autoantibodies in autoimmune diseases such
as systemic lupus erythematosus [41,42] and rheuma-
toid arthritis [43]. AnnexinII, specifically, has not been
previously implicated as a target of autoantibodies in
any disorders.
Annexins are known to undergo post-translational
modification including glycosylation [44]. Annexin I
and annexin II are both phosphorylated by various ki-
nases [45]. In our study, immunoreactivity against an-
nexin I was found to be dependent on N-glycosylation.
A potential N-linked glycosylation site is present at po-
sitions 42 and 61 from the N-terminus of annexins I and
In another study, sera from 54 newly diagnosed pa-
tients with lung cancer and 60 patients with other can-
cers and from 61 non-cancer controls were analyzed
for autoantibodies to lung tumor proteins. Sera from
60% of patients with lung adenocarcinoma, and 33% of
patients with squamous cell lung carcinoma but none of
the non-cancer controls exhibited IgG based reactivity
against proteins identified as glycosylated annexins I

S. Hanash et al. / A proteomic approach to the identification of lung cancer markers
299
for cancer diagnosis. The approach is currently being
P1
P1
P2
P3
expanded in several ways. First, proteomic analysis
is increasingly focused on the analysis of individual
sub-cellular compartments such as surface membranes,
nuclear proteins etc. Second the 2-D gel based ap-
proach is increasingly complemented with other sepa-
ration modes such as the use of multi-dimensional liq-
uid chromatography that is particularly suited for au-
tomation and for the analysis of small molecular weight
proteins and peptides. Other emerging technologies in-
clude the use of protein microarraysin which antigen or
antibody, representing probes, is deposited onto glass
surfaces and interrogated with targets represented by
reagents, sera, biological fluids or cell or tissue lysates.
One would envisage the development of specialized
microarrays to interrogate targets relevant to lung can-
cer. Proteomics is likely to contribute substantially to
our understanding of the pathophysiology of lung can-
cer and to the development of novel diagnostics and
therapeutics.
A
B
P3
P2
A1
C
D
E
A2
A1
A2
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