Research Communication
Protein Expression Profiling of Primary Mammary Epithelial CellsDerived from MMTV-neu Mice Revealed that HER2/NEU-DrivenChanges in Protein Expression Are Functionally Clustered
Sungwoo Park1*, Kyung-min Lee1*, Ji-hyun Ju1, Jaeyoon Kim2, Dong-Young Noh3,
Taehoon Lee2,4 and Incheol Shin11Department of Life Science, Hanyang University, Seoul, Korea2Division of Molecular Life Science, POSTECH, Pohang, Korea3Department of Surgery, Seoul National University College of Medicine, Seoul, Korea4NOVA Cell Technology, Inc. Pohang, Korea
Summary
MMTV-neu transgenic mice overexpressing NEU in theirmammary glands develop tumor after 6 months of age. To finda novel protein biomarker using this mouse model, we identifiedand characterized the proteins that were differently expressedbetween primary mammary epithelial cells from 2 months oldMMTV-neu heterozygote mice and wild type (WT) littermatesusing two-dimensional digest (ChemDigestTM/Trypsin)-LC-MS/MS. The differentially expressed proteins were selected and an-alyzed using DAVID Bioinformatics resource. The proteinsinvolved in anti-apoptosis, purine metabolism, ribosome andproteasome functions were upregulated, whereas cell adhesion-related proteins were downregulated in PMECs from MMTV-neu mice when compared with WT PMECs. The results indi-cate that several functional units are coregulated by HER2/NEU. We hypothesize that these changes in the cellular pro-teome may be responsible for early onset of HER2/NEU-driventumorigenesis. � 2009 IUBMB
IUBMB Life, 62(1): 41–50, 2010
Keywords proteonomics; signal transduction; HER2/NEU.
Abbreviations AK, adenylate kinase; CAM, cell-adhesion mol-ecule; EGFR, epidermal growth factor receptor;
HPLC, high-pressure liquid chromatography;IKK, IjB kinase; MMTV, mouse mammarytumor virus; MS, mass spectrometry; NCAM,neural cell adhesion molecule; PMECs, primarymammary epithelial cells; PAPS, 30-phosphoa-denosine 50-phosphosulfate; PAPSS, 30-phos-phoadenosine 50-phosphosulfate synthetase;RPA, replication protein A; RRM2, ribonucleo-tide reductase M2 subunit; RNAP, RNA poly-merase; XDH, xanthine dehydrogenase; XO,xanthine oxidase.
INTRODUCTION
HER2/NEU (also known as ERBB2) receptor kinase is the
protein product of the her2 proto-oncogene and a member of
the transmembrane receptor tyrosine kinases comprised of epi-
dermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2/
HER2/NEU, ERBB3/HER3, and ERBB4/HER4. NEU is the rat
homologue of HER2 that was originally identified from chemi-
cally-induced neuroblastomas and homologous with retroviral
oncogene product (ERBB) closely related to EGFR (1). Her2
gene is located on human chromosome 17q12 (2), and its
expression has been implicated in the induction of many human
cancer (3) caused by increased proliferation and enhanced sur-
vival through formation of phosphorylated heterodimeric com-
plex with ligand-bound HER1/ERBB1, HER3/ERBB3, and
HER4/ERBB4 (4, 5). HER2 is responsible for transforming nor-
mal mammary epithelial cells, overexpressed in 20–30% of
women with breast cancer and correlated with more aggressive
tumor behavior and poor patient outcome (6, 7). Patients with
tumors overexpressing HER2 have lower response rates to anti-
estrogen therapy, facilitating the drug discovery targeting HER2
*These authors contributed equally to this study.
Address correspondence to: Incheol Shin, Department of Life Sci-
ence, Hanyang University, Seoul 133-791, Korea. Tel: 182 2 2220
2562. E-mail: [email protected] or Taehoon Lee, NOVA Cell
Technology, Inc., Division of Molecular and Life Science, POSTECH,
Pohang 790-784, Korea. Tel: 182 54 223 2475. E-mail: taehoon@postech.
ac.kr.
Additional Supporting Information may be found in the online ver-
sion of this article.
Received 6 March 2009; accepted 29 September 2009
ISSN 1521-6543 print/ISSN 1521-6551 online
DOI: 10.1002/iub.276
IUBMB Life, 62(1): 41–50, January 2010
including trastuzumab (Herceptin), a neutralizing monoclonal
HER2 antibody that has been shown to prolong survival (8, 9).
To understand the molecular basis for potent transforming
activity of HER2/NEU, many neu transgenic mouse models
have been generated over the past 20 years (3), and the study
using this model showed the first direct evidence that the acti-
vated neu allele in mammary gland is capable of inducing a
mammary tumor phenotype (10, 11). Mouse mammary tumor
virus (MMTV)-neu transgenic mice that overexpress NEU
protein with strong viral promoter/enhancer develop focal mam-
mary tumors with a latency period of 7–8 months (12). Because
of overexpression rather than point mutation of neu is compara-
ble to human her2-driven breast cancer (13), MMTV-neu mouse
model carrying unactivated neu under the transcriptional control
of MMTV promoter/enhancer was used in this study to find pro-
tein biomarker(s) that could be applied to prognosis of human
breast cancer.
Proteomic approaches are now being used to identify and
validate novel proteins especially as disease biomarkers. Many
tools were developed to explore the proteome such as high-pres-
sure liquid chromatography (HPLC), two-dimensional electro-
phoresis, and mass spectrometry (MS). In this report, we per-
formed the differential proteome analysis and functional classifi-
cation of identified proteins using primary mammary epithelial
cells (PMECs) derived from young (2 months) wild type (WT)
and MMTV-neu transgenic (TG) mice using a novel shotgun
method, Two Dimensional Digest (ChemDigestTM/Trypsin)-LC-
MS/MS. We tried to gather the proteome information exclu-
sively in breast epithelial cells, avoiding contamination from
surrounding connective tissue by using primary cultured cells
from mammary gland. Subsequent western blot experiments
were performed to confirm the differential expression patterns
of biomarker proteins. Functional classification of differential
proteins based on DAVID Bioinformatics Resources 2007
server (http://david.abcc.ncifcrf.gov) was conducted to analyze
the differential modulation of signaling pathways in both WT
and MMTV-neu PMECs.
MATERIALS AND METHODS
Mice and Genotyping
MMTV-neu (Fvb/N background) mice were purchased from
the Jackson Laboratory (http://www.jax.org) and maintained
under standard light (12 h)/dark (12 h) condition and at constant
temperature in animal facilities (Hanyang University). Mice
were housed in plastic mouse cage and fed with standard pellet
food (Daejong Instrument Industry, http://www.daejonglab.
co.kr) and tap water. MMTV-neu1/2 male transgenic mice were
crossed with the WT females to generate both MMTV-neu1/2
female mice as experimental group and WT females as control
group. Pups were ear-punched at 3 weeks, and the ear tissues
were digested in a digestion buffer (500 mM KCl, 0.1 mg/mL
gelatin, 0.45% Igepal CA-630, 0.45% Tween-20, 100 mM Tris,
pH 8.3 supplemented with 0.05 mg/mL of protease K). The tis-
sue sample was incubated for overnight at 568C and 10 min at
908C and was used as a template in the genotyping PCR reac-
tion with the neu-promoter specific primer: forward: 50-CGGAACCCACATCAGGCC-30 and reverse: 50-TTTCCTGCAGCAGCCTACGC-30.
Preparation and Culture of PMECs
Two months old mice were subjected to vaginal smearing to
select the mice in the diestrous stage. Selected mice were killed
by asphyxiation in the CO2 chamber, and the #4 mammary
glands were removed by surgery under aseptic condition. The
mammary glands were chopped in digestion medium [DMEM/
F12 (Gibco) containing 450 U collagenase (Sigma–Aldrich), 100
U hyaluronidase, 50 U penicillin, and 50 lg/mL streptomycin
(Gibco), filtered through 0.22 lm filter (Millipore)] with scalpel
to less than 1 mm3. The homogenates were incubated for 3–4 h
at 378C and the cells were spun down at 500g for 5 min. The cell
pellets resuspended in PBS (Gibco) containing 5% FBS (Gibco)
were centrifuged until the g value reached 350g and the centrifu-
gation was immediately halted. The same centrifugation steps
were repeated twice to collect pure epithelial cells free from
fibroblast contamination. The PMECs pellets were resuspended
in prewarmed DMEM/F12 (Gibco) containing 10 ng/mL epider-
mal growth factor (Invitrogen), 10 lg/mL insulin (Gibco), 0.5
lg/mL hydrocortisone (Sigma–Aldrich), 100 ng/mL choleratoxin
(Sigma–Aldrich), 5% Horse serum (Gibco), and 1% penicillin/
streptomycin (Gibco) and seeded on culture plates. The cells
were incubated at 378C in a humidified atmosphere containing
5% CO2. PMECs grown in typical epithelial cell colonies (Fig.
1A) were collected by cell scrapper at 70–80% confluency,
washed twice with PBS, and frozen until subjected to proteome
analysis by mass spectrometry or western blots.
Two Dimensional Digest (ChemDigestTM/Trypsin)-LC-MS/MS and Data Analyses
Here, we used a novel approach of mass spectrometer-based
proteomics; two dimensional (ChemDigest/Trypsin) digest-LC-
MS/MS for differential proteomic analysis of PMECs proteome
(Illustrated in Fig. 1C). Whole PMECs proteome was first
hydrolyzed at aspartic acid residue using ChemDigestTM (http://
www.sigmol.com) that denatured and alkylated the extracted
proteins, reduced disulfide bond, and cleaved proteins at aspar-
tic acid residues. The resulting hydrolysate was digested with
trypsin and subjected to chromatographic separation by off-line
C18 HPLC and analyzed by LC-MS/MS. LC-MS/MS data were
searched against the UniProt v14.6 using in-house MASCOT
software (ver 2.2.04). Enzyme cleavage rule was made by com-
bining rules of trypsin and ChemDigestTM. The following pa-
rameters were used: six maximum missed cleavages; N-acetyl
(Protein), oxidation (M), pyroglutamylation (N-term EQ) as
variable modification; and charge states 12, 13, and 14. Win-
dows of mass accuracy of 50 ppm and 0.25 Da were used for
precursor ions and MS/MS data, respectively. Using this
method, we identified 2,454 distinct proteins from the Uniprot
42 PARK ET AL.
database by accommodating a target-decoy search strategy (14)
that yielded 2% estimated false discovery rate.
To estimate fold-changes of identified proteins between exper-
imental groups, we applied label free quantitative analysis based
on comparison of ion intensity (15). We got the total ion intensity
(TII) that is the sum of all ion intensities in a MS/MS spectrum.
The values of Protein TII in each group (TIIWT, TIIMMTV-neu)
could be calculated by summation of TIIs of all spectra identified
from each condition. Log2 ratio of TIIMMTV-neu and TIIWT was
calculated and used for quantitative index. To avoid taking
logarithm on zero’s, we set the TIIWT or TIIMMTV-neu as 0.185
that is the 5 percentile value of TII if no peptide was identified in
an experimental group.
The proteins of which Log2 ratio of TIIMMTV-neu and TIIWT
were more than 1(two folds) were selected and analyzed using
DAVID Bioinformatics Resources 2007 (http://david.abcc.
ncifcrf.gov). Total mice proteins indicated in UniProt accession
number was submitted as background and specific genes that
were highly expressed in WT or MMTV-neu PMECs were used
as ‘gene list’ in this program. The results of analyses were
interpreted in KEGG (http://www.genome.ad.jp/kegg) to obtain
list of proteins related to cell signaling and metabolic pathways.
Western Blots
Whole cell lysates were obtained from PMECs in a cell lysis
buffer [20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.1 mM EDTA,
20 mM NaF, 1 mM Na3VO4, 13 protease inhibitor (Roche,
Indianapolis, IN), 1% NP-40, 0.1% triton X-100, 0.1% SDS],
and protein concentrations were determined with the BCA pro-
tein assay kit (Pierce, Rockford, IL). Equal amount of proteins
(30 lg) were boiled in 13 SDS sample buffer, subjected to
SDS-PAGE, and transferred onto 0.2 lm nitrocellulose mem-
brane (Whatman GmbH, Dassel Germany). After blocking of
the membrane with 5% nonfat dry milk or bovine serum albu-
min in TBST (20 mM Tris-Cl, pH 7.6, 140 mM NaCl, 0.05%
Tween-20), the membrane was incubated with appropriate dilu-
tions of specific primary antibodies: gamma-glutamyltransferase
1, Inositol polyphosphate-5-phosphatase B, IjB kinase(IKK)-c,BCL2, Caspase-3, NF-j-B, syntaxin-6 antibodies were from
Santa Cruz (Santa Cruz Biotechnology, http://www.scbt.com).
Proteasome subunit a type 5, IKK-b antibodies were obtained
from Cell Signaling Technology (http://www.cellsignal.com),
and HER2/NEU antibody was from NeoMarkers (http://
www.labvision.com). After incubation with primary antibody
solution, the membrane was washed with TBST and incubated
with horseradish peroxidase-conjugated anti mouse or anti
rabbit antibodies (Amersham Biosciences, Little Chalfont,
Buckinghamshire UK). After subsequent washing with TBST,
Immunoreactive protein bands were detected with West-zol Plus
system (Intron Biotechnology, http://www.intronbio.com).
RESULTS
Establishment of PMECs
PMECs were prepared as described in Materials and Meth-
ods. To confirm that our PMECs are epithelial and there was no
Figure 1. Photomicrographs of PMECs used in this study (A) and confirmation of epithelial cell phenotype by western blot with
epithelial markers (B). Schematic flow chart showing the experimental strategies (C).
43PROTEIN EXPRESSION PROFILING OF PMECs
contamination by fibroblasts, series of western blot analyses
were conducted (Fig. 1B). The epithelial markers, E-cadherin
(16) and cytokeratin 19 (17) were expressed in PMECs and pos-
itive control MCF-7 human mammary epithelial cancer cells.
The mesenchymal cell marker fibronectin (18) was not detected
in PMECs, but strongly expressed in MDA-MB-231 mesenchy-
mal breast cancer cells. These data strongly indicate that the
PMECS we used in this study were of epithelial origin.
Proteins Involved in Anti-apoptosis, Purine Metabolism,Ribosomal and Proteasome Proteins Were Upregulatedin PMECs From MMTV-neu Mice
Defects in apoptosis (programmed cell death) mechanisms
play pivotal roles in tumor pathogenesis allowing cancer cells
to survive and provide protection from various stresses (19).
Subsequent accumulation of genetic alterations allows deregula-
tion of cell proliferation, interference of differentiation, promo-
tion of angiogenesis, and increase cell motility and invasiveness
during tumor progression (19). Transformed epithelial cells
allowed to survive in a suspended state without attachment to
extracellular matrix by defects in apoptosis resulted in increased
metastasis (20). In our experiment, expression of anti-apoptotic
proteins BCL-XL, NFjB, and IKK-b were increased in PMECs
from MMTV-neu mice (Table 1, supporting information figure
S1A for KEGG pathway). BCL-XL belongs to BCL-2 family
that protects the integrity of mitochondria, inhibiting cyto-
chrome c release, and the subsequent activation of caspase-9.
Consistent with our results, overexpression of BCL-XL and
BCL-2 enhanced the metastatic characters in breast cancer cells
(21, 22), and overexpression of HER2 in breast carcinoma cell
lines (MCF-7) was accompanied by up-regulation of BCL-XL
and BCL-2 (23).
Activation of NF-jB typically involves the phosphorylation
of IjB by the IKK complex that subsequently induces IjB deg-
radation and these events release NF-jB, allowing its transloca-
tion to the nucleus (24) and resulting in regulation of apoptosis
and proliferation (25). Our results (Table 1) may suggest that
upregulation of these apoptosis-related proteins blocked the nor-
mal apoptotic pathways of mammary epithelial cells in MMTV-
neu mice. Quantitative confirmation of the protein levels was
conducted by western blot analyses (Fig. 2). The western blot
analyses revealed upregulation of both IKK-b and IKK-c subu-
nits. The level of effector caspase (caspase-3) was also exam-
ined to monitor activation of apoptotic signaling (Fig. 2).
Downregulation of cleaved 19 kDa caspase 3 fragment and up-
regulation of 35 kDa caspase 3 were observed, indicating down-
regulation of apoptotic pathways in MMTV-neu PMECs.
Transformed cancer cells divide rapidly than normal cells,
hence they need more DNA synthesis. Different key enzymes
of nucleotide metabolism and DNA biosynthesis are signifi-
cantly upregulated in certain tumor cells (26). Because the max-
imal proliferative potential of a cell is limited by the activities
and amount of the different rate-limiting enzymes of the essen-
tial purine and pyrimidine nucleotide biosynthesis, the biochem-
ical procedure of transformed cells has been reprogrammed to
maintain continued proliferation by enzymatic imbalance, which
induces cell cycle progression that leads to more malignant phe-
notype. This imbalanced metabolism supported by increased
activities of rate-limiting enzymes of the anabolic pathways,
concomitant with decreased activities of the opposing enzymes
involved in catabolic pathways implies a shift to the anabolic
direction (26).
Adenylosuccinate lyase, DNA-directed RNA polymerase,
ribonucleotide reductase, adenylate kinase 2, xanthine dehydro-
genase/oxidase, and bifunctional 30-phosphoadenosine 50-phos-phosulfate (PAPS) synthetase 1/PAPSS1/sulfurylase kinase 1
were upregulated in PMECs from MMTV-neu mice (Table 2,
supporting information figure S1B). Adenylosuccinate lyase (EC
4.3.2.2, Adenylosuccinase/ASL/ADSL) is a bifunctional enzyme
that catalyzes conversion of succinylaminoimidazole carboxa-
mide ribotide to aminoimidazole carboxamide ribotide and the
conversion of adenylosuccinate to AMP. Fumarate is a by-prod-
Table 1
Apoptosis-related proteins upregulated in MMTV-neu PMECs
UniProt
accession
number Protein name Gene name TIIMMTV-neu TIIWT Log2(TIIMMTV-neu/TIIWT)
Q64373 Apoptosis regulator Bcl-X
(Bcl-2-like 1 protein)
Bcl2l1 3.7852 1.5363 1.30
O88351 Inhibitor of nuclear factor kappa-B
kinase subunit beta
Ikbkb 1.8423 0.8236 1.16
Q9WTK5 Nuclear factor NF-kappa-B p100
subunit (DNA-binding factor KBF2)
Nfkb2 4.0209 1.1853 1.76
Q64373 Apoptosis regulator Bcl-X
(Bcl-2-like 1 protein)
Bcl2l1 3.7852 1.5363 1.30
O08529 Calpain-2 catalytic subunit
precursor (Calpain-2 large subunit)
Capn2 2.2874 1.1358 1.01
44 PARK ET AL.
uct from both reactions. The deficiency of adenylosuccinate
lyase affects human diseases such as psychomotor retardation,
autistic features, hypotonia, and seizures (27). DNA-directed
RNA polymerase I subunit replication protein A (RPA)1 (EC
2.7.7.6) and DNA-directed RNA polymerase II subunit RPB2
(EC 2.7.7.6) were also upregulated in PMECs from MMTV-neu
mice. RPA1, RPA2, and RPA3 are subunits that compose a het-
erotrimeric single-stranded DNA binding complex, RPA. RPA1
is the largest subunit conserved in eukaryotes and has essential
roles in DNA replication, recombination, and repair (28). Muta-
tion in Rpa1 could result in defective DNA double-strand break
repair, chromosomal instability, and cancer in mice (29). RNA
polymerase II (RNAP II) comprises 12 subunits from RPB1 to
RPB12 in Saccharomyces cerevisiae (30), and RPB1 and RPB2
are two largest subunits. RPB2 is not only structural protein of
RNAP II but also interacts with a general transcription factor
TFIIB and serve as start site selection when transcription ini-
tiates (31).
Ribonucleoside reductase (EC 1.17.4.1, RR) catalyzes the
de novo biosynthesis of deoxyribonucleotides (dNTPs) for DNA
synthesis by reduction of corresponding ribonucleotides (32).
Recent inhibition study using siRNA reveals that M2 subunit
(RRM2) of ribonucleoside reductase reduces the growth poten-
tial of cancer cells both in vitro and in vivo (33) and enhances
cellular invasiveness (34), suggesting that RRM2 could be a
candidate for cancer therapeutics. Adenylate kinase (EC 2.7.4.3,
AK/ATP-AMP transphosphorylase) is a ubiquitous enzyme that
contributes to the regulation of the homeostasis of the cellular
adenine and guanine nucleotide pools (35). Among three iso-
forms, AK1 is located mainly in the cytosol, AK2 is present in
the intermembrane space of mitochondia, and AK3 is located in
the mitochondrial matrix (36). AK2 is essential for maintenance
and cell growth but, in contrast, may play a role in apoptosis
through formation of a complex with Fas-associating protein
with death domain and caspase-10 (37). Xanthine dehydrogen-
ase (EC 1.17.1.4, XDH) /oxidase (EC 1.17.3.2, XO) is a mem-
ber of the molybdenum hydroxylase family that catalyzes the
oxidation of hypoxanthine and xanthine to uric acid. Mamma-
lian xanthine dehydrogenase can be converted to xanthine oxi-
dase by modification of cysteine residues or by proteolysis of
the polypeptide chain (38). Decreased activity and expression of
XO in breast cancer was previously reported (39). PAPSS(30-phosphoadenosine 50-phosphosulfate synthetase)1/sulfurylase ki-
nase 1 catalyzes the formation of PAPS, the activated sulfate car-
rier in a two-step that is mediated by two distinct domains of
PAPSS. PAPSS1 is an isoform of PAPSS highly expressed in
brain and skin and have been associated with mice and human dis-
ease with mutation such as murine brachymorphism (40), human
spondyloepimetaphyseal dysplasia (41), and osteoarthritis (42).
The ribosome, the protein-synthesizing machinery in cells, is
composed of �80 ribosomal proteins and 4 rRNAs in mammals.
Ribosomal proteins are extremely ancient molecules and can be
divided into two groups belonging to two subunits, a large sub-
unit binds tRNA molecules and contribute to peptidyl transfer
and a small subunit mediates mRNA binding, decoding, and fi-
delity. These ribosomal proteins are not only supporting archi-
tecture of the structural basis but also have extra-ribosomal
functions such as biological roles on gastrointestinal (43), pros-
tate (44), gastric (45), and breast (46) cancer. Indeed, various ri-
bosomal proteins in PMECs from MMTV-neu mice were
increased when compared with WT PMECs including 60S ribo-
somal proteins (large subunits); L3, L4, L5, L6, L7, L10, L11,
L12, L13a, L14, L17, L18, L18a, L19, L21, L22, L22-like 1,
L23, L24, L27, L28, L31, L35a, L37a, L38, 60S acidic
ribosomal proteins; P0, P1, P2, 40S ribosomal proteins (small
Figure 2. Validation of proteomics data by western blots.
PMECs were prepared as described in Materials and Methods
and the cells were subjected to western blot analyses with spe-
cific antibodies. Gamma-glutamyltransferase 1 in selenoamino
acid metabolism pathways, L28 of ribosomal protein, protea-
some subunit alpha type 5, NF-j-B and IKK-b in apoptosis-
related pathways were upregulated in MMTV-neu PMECs.
IKK-c, BCL2, which were involved in apoptotic pathways,
were also upregulated. The decreased apoptotic cell death in
MMTV-neu PMECs was demonstrated by the increase in
cleaved caspase-3. Upregulation of inositol polyphosphate-5-
phosphatase B related in phosphatidylinositol signaling and syn-
taxin-6 of SNARE interactions in WT PMECs were also con-
firmed.
45PROTEIN EXPRESSION PROFILING OF PMECs
subunits); S2, S3, S4, S5, S6, S7, S8, S11, S12, S13, S14, S15,
S15a, S19, S21, S23, S26, SA (p40), 39S mitochondrial ribo-
somal proteins (large subunits); L9, L10, L19, L24, L44, L46,
L49, and 28S mitochondrial ribosomal proteins (small subunits);
S5, S7, S9, S22, S29, S35 (Supporting Information data–Micro-
soft Excel Sheet). Among these, some proteins indicated in Ta-
ble 3 and supporting information figure S1C were upregulated
more than twice. These data suggests that the level of ribosomal
proteins may affect the activity of ribosome that should be
accelerated to maintain rapid cell proliferation. Consistently,
60S ribosomal protein L19 was increased in human breast
tumors overexpressing HER2 in previous report (47). Represen-
tatively, the protein level of 60S ribosomal protein L28 (Table
3) was confirmed by western blot (Fig. 2).
Table 2
Purine metabolism-related proteins upregulated in MMTV-neu PMECs
UniProt accession
number Protein name Gene name TIIMMTV-neu TIIWT Log2(TIIMMTV-neu/TIIWT)
P54822 Adenylosuccinate lyase
(Adenylosuccinase)
Adsl 3.751 0.1850 4.34
O35134 DNA-directed RNA
polymerase I subunit RPA1
Polr1a 2.5074 0.1850 3.76
Q8CFI7 DNA-directed RNA
polymerase II subunit RPB2
Polr2b 10.1832 3.4539 1.56
P11157 Ribonucleoside-diphosphate
reductase M2 subunit
Rrm2 3.6038 0.3403 3.40
Q9WTP6 Adenylate kinase isoenzyme
2, mitochondrial
Ak2 8.237 1.1276 2.87
Q00519 Xanthine dehydrogenase/oxidase Xdh 4.2327 0.1850 4.52
Q60967 Bifunctional 30-phosphoadenosine50-phosphosulfate synthetase
1 (PAPSS 1)
Papss1 1.8584 0.8210 1.18
Table 3
Ribosomal subunits upregulated in MMTV-neu PMECs
UniProt accession
number Protein name Gene name TIIMMTV-neu TIIWT Log2(TIIMMTV-neu/TIIWT)
Q9CXW4 60S ribosomal protein L11 Rpl11 14.537 6.078 1.26
P84099 60S ribosomal protein L19 Rpl19 3.7706 1.1426 1.72
P47962 60S ribosomal protein L5 Rpl5 7.2146 1.0367 2.80
P14148 60S ribosomal protein L7 Rpl7 268.8271 101.8727 1.40
P62264 40S ribosomal protein S14 Rps14 3.9127 1.9373 1.01
P19253 60S ribosomal protein L13a
(Transplantation antigen
P198) (Tum-P198 antigen)
Rps13a 35.7955 10.5136 1.77
P97461 40S ribosomal protein S5 Rps5 68.1632 18.3630 1.89
P62267 40S ribosomal protein S23 Rps23 76.5245 27.1019 1.50
Q6ZWV3 60S ribosomal protein L10 Rpl10 335.8045 104.4224 1.69
O09167 60S ribosomal protein L21 Rpl21 24.3708 8.0816 1.59
O55142 60S ribosomal protein L35a Rpl35a 1.9094 0.1850 3.37
P67984 60S ribosomal protein L22 Rpl22 76.3200 32.9543 1.22
Q9D7S7 Ribosomal protein L22-like 1 Rpl22l1 8.7002 2.6747 1.70
P61358 60S ribosomal protein L27 Rpl27 15.5347 3.8269 2.02
P41105 60S ribosomal protein L28 Rpl28 117.1344 57.1334 1.04
Q9CQR2 40S ribosomal protein S21 Rps21 1.8749 0.6619 1.50
46 PARK ET AL.
The proteasome is an abundantly expressed multicatalytic
protease that is responsible for the turnover of many proteins
including cell cycle, regulatory proteins, and tumor suppressor
proteins. Appropriate intracellular protein levels were main-
tained by this major machinery for protein degradation, ubiqui-
tin-proteasome pathway. Hence, the malfunction of proteasomal
degradation could either increase the level of oncoproteins or
reduce the amount of tumor suppressor proteins, resulting in the
onset of tumorigenesis (48). Recent study showed that 26S pro-
teasome regulatory subunit 4 (S4) was upregulated in primary
human breast tumors by heregulin-B1-related mechanism (49).
In our experiments, we found that many proteasomal subunits
were upregulated in PMECs from MMTV-neu mice (Table 4,
supporting information figure S1D), and proteasome subunit atype 5 was tested by western blot to confirm difference in protein
level (Fig. 2). These data suggest that overexpression of these
structural proteins of proteasome may play a pivotal role in tu-
morigenesis via causing imbalance of proteosome structure that
could lead to a dysfunction of proteasome that controls the bal-
anced level of oncogenic proteins and tumor suppression proteins.
Cell Adhesion Molecules Were Downregulated inPMECs From MMTV-neu Mice
Cell adhesion molecules (CAMs) are required for cell adhe-
sion, a fundamental process for the correct functioning of multi-
cellular organisms. Many CAMs have been reported to be
involved in a broad range of processes including basic cell to
cell and cell to matrix interactions, cell migration, cell cycle,
and intracellular cell signaling. We found that some CAMs are
downregulated in PMECs from MMTV-neu mice than PMECs
from WT mice (Table 5, supporting information figure S2).
Poliovirus receptor-related protein 2 precursor (CD112) is iden-
tified as a tumor cell antigen recognized by CD226 in natural
killer cells (50). Neural cell adhesion molecule 1 (NCAM,
CD56)-transfected human breast cancer cells in nude mice show
longer latency periods and slower growth rates (51). Taken to-
gether with this previous report, our data suggest that decreased
expression of NCAM and other CAMs in MMTV-neu mice may
be responsible for the onset of tumorigenesis in MMTV-neu mice.
In addition to the changes in functionally clustered proteins
mentioned in previous sections, some other functionally clus-
tered proteins that were differentially expressed between WT
PMECs and MMTV-neu PMECs were also identified. Proteins
involved in selenoamino acid metabolism (Supporting Informa-
tion Table S1 and figure S3), glutathione metabolism (Sup-
porting Information Table S2 and Figure S4), and prostaglan-
din and leukotriene metabolism (Supporting Information Table
S3 and Figure S5) were upregulated in MMTV-neu PMECs.
SNARE interactions in vesicular transport (Supporting Infor-
mation Table S4 and Figure S6), glycerophospholipid metabo-
lism (Supporting Information Table S5 and Figure S7), and
Table 4
Proteasomal subunits proteins upregulated in MMTV-neu PMECs
UniProt accession
number Protein name Gene name TIIMMTV-neu TIIWT Log2(TIIMMTV-neu/TIIWT)
Q9WVJ2 26S proteasome non-ATPase
regulatory subunit 13
Psmd13 40.9298 11.1428 1.88
O35226 26S proteasome non-ATPase
regulatory subunit 4
Psmd4 11.7724 3.0320 1.96
P46471 26S protease regulatory subunit
7(Protein MSS1)
Psmc2 1.9945 0.1850 3.43
P54775 26S protease regulatory subunit
6B (MIP224)
Psmc4 7.0708 1.8142 1.96
P62196 26S protease regulatory subunit
8 (Proteasome 26S subunit
ATPase 5)
Psmc5 47.9979 12.2908 1.97
Q9Z2U0 Proteasome subunit alpha type
7 (EC 3.4.25.1) (Proteasome
subunit RC6-1)
Psma7 11.2066 5.2048 1.11
Q9Z2U1 Proteasome subunit alpha type
5 (Proteasome zeta chain)
Psma5 16.6242 5.6790 1.55
O70435 Proteasome subunit alpha type
3 (Proteasome component C8)
Psma3 2.8390 0.7472 1.93
Q9R1P1 Proteasome subunit beta type
3 (Proteasome theta chain)
Psmb3 28.0754 10.9157 1.36
47PROTEIN EXPRESSION PROFILING OF PMECs
phosphatidylinositol signaling system (Supporting Information
Table S6 and Figure S8) were upregulated in WT PMECs.
The importance of these changes between WT and MMTV-
neu PMECs warrants further investigations.
Despite we could not test all of the proteins shown in the
KEGG pathways, some of representative upregulated proteins in
proteome profiles were virtually increased when confirmed by
western blot analyses (Fig. 2).
DISCUSSION
Our results, herein, revealed that at least a part of HER2/
NEU-driven changes in mouse mammary gland epithelial cell
proteome are functionally clustered into several units. Although
we could not verify all the changes in protein expression by
conventional methods like western blots and immunohisto-
chemstry mainly due to a lack of sufficient high-quality anti-
bodies, the results of the functional annotations themselves vali-
dated that these changes are likely to reflect real in vivo situa-
tions since the concomitant changes in the expression profiles
of the proteins in the same functional cluster could indicate that
their expressions are coregulated in that particular proteome for
a certain biological function.
A few studies were recently conducted to identify HER2/
NEU induced changes in protein expression profiles using laser
capture microdissected samples and flow-sorted human breast
luminal epithelial cells (52, 53). Zhang et al. (52) showed that
nine proteins involved in glycolysis and detoxification pathways
were upregulated in HER2-positive breast tumors, and Gharbi
et al. (53) identified a list of proteins differentially expressed
between HER2-negative and positive cell lines. In addition to
these reports, Landis et al. (54) reported that TGF-b-induciblegenes were downregulated in tumors from MMTV-neu trans-
genic mice when compared with WT mammary gland tissue
using Affymetrix microarrays. However, the authors failed to
identify the concomitant changes in the canonical TGF-b signal-
ing molecules by HER2/NEU expression. Some of the TGF-b-inducible genes, for example, serine protease 11 was also down-
regulated in preneoplastic mammary tissue as well as in overt
tumors. On the contrary to this report, we could not detect
changes in TGF-b-inducible protein between WT PMECs and
MMTV-neu PMECs. A study using cluster analysis of tissue
microarray sections of a large series of human invasive breast
cancer also revealed that breast tumors could be classified to 5
groups of distinct protein expression (55). In their report,
HER2/NEU is also shown to be a strong classifying marker in 2
groups. Group 3 is characterized by HER2/NEU and MUC1
overexpression, reduced E-cadherin expression, and overall nu-
clear receptor negativity. Another HER2/NEU-driven group 6
consists of tumors with strong expression of HER2/NEU, weak/
negative expression of MUC1, and strong expression of E-cad-
herin. These differences in HER2/NEU-driven groups may indi-
cate the molecular heterogeneity of HER2/NEU-induced
tumors.
In addition to these previous reports, by using two dimen-
sional digest-LC-MS/MS, a novel shotgun method for proteome
analyses, we were able to find more than 50 differentially
expressed proteins that are clustered into several functional
units in PMECS from WT and MMTV-neu mice. Furthermore,
by using Babelomics (http://babelomics.bioinfo.cipf.es/), we
found that these changes in mice proteome could be translated
into the similar changes in corresponding human proteome (data
not shown).
In conclusion, we report here that two-dimensional digest
(ChemDigestTM/Trypsin)-LC-MS/MS could be a method of
choice for differential proteome analysis, as it could help us
identify adequate number of differentially expressed proteins
and that the upregulated proteins may be a potential early prog-
nostic marker for HER2-positive breast cancer.
ACKNOWLEDGEMENTS
This work is supported by FPR08A2-080 of 21st Century Fron-
tier Functional Proteomics Project from Korean Ministry of Sci-
ence and Technology and by a grant of the Korea Healthcare
technology R&D Project, Ministry of Welfare and Family
Affairs, Republic of Korea, (A084466).
Table 5
Cell adhesion-related proteins upregulated in WT PMECs
UniProt accession
number Protein name Gene name TIIMMTV-neu TIIWT Log2(TIIMMTV-neu/TIIWT)
P13597 Intercellular adhesion molecule
1 precursor (ICAM-1)
Icam1 0.9002 23.6059 24.71
P32507 Poliovirus receptor-related protein
2 precursor (mHveB)
Pvrl2 1.5981 3.4750 21.12
P13595 Neural cell adhesion molecule
1, 180 kDa isoform precursor
(NCAM-180)
Ncam1 0.1850 4.3242 24.59
P18828 Syndecan-1 precursor (SYND1) Sdc1 0.1850 3.4365 24.22
48 PARK ET AL.
REFERENCES1. Schechter, A. L., Stern, D. F., Vaidyanathan, L., Decker, S. J., Drebin,
J. A., Greene, M. I., and Weinberg, R. A. (1984) The neu oncogene: an
erb-B-related gene encoding a 185,000-Mr tumour antigen. Nature 312,
513–516.
2. Yamamoto, T., Ikawa, S., Akiyama, T., Semba, K., Nomura, N., Miya-
jima, N., Saito, T., and Toyoshima, K. (1986) Similarity of protein
encoded by the human c-erb-B-2 gene to epidermal growth factor recep-
tor. Nature 319, 230–234.
3. Ursini-Siegel, J., Schade, B., Cardiff, R. D., and Muller, W. J. (2007)
Insights from transgenic mouse models of ERBB2-induced breast can-
cer. Nat. Rev. Cancer 7, 389–397
4. Olayioye, M. A., Neve, R. M., Lane, H. A., and Hynes, N. E. (2000)
The ErbB signaling network: receptor heterodimerization in develop-
ment and cancer. EMBO J. 19, 3159–3167.
5. Yarden, Y. and Sliwkowski, M. X. (2001) Untangling the ErbB signal-
ling network. Nat. Rev. Mol. Cell. Biol. 2, 127–137.
6. Ross, J. S. and Fletcher, J. A. (1998) The HER-2/neu oncogene in
breast cancer: prognostic facto, predictive facto, and target for therapy.
Stem Cells 16, 413–428.
7. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., and
McGuire, W. L. (1987) Human breast cancer: correlation of relapse and
survival with amplification of the HER-2/neu oncogene. Science 235,
177–182.
8. Piccart-Gebhart, M. J. and Loi, S. M. (2005) Fulvestrant—ready to start
its journey in the breast cancer adjuvant endocrine world? Eur. J. Cancer
41, 341–343.
9. Romond, E. H., Perez, E. A., Bryant, J., Suman, V. J., Geyer, C. E. Jr.,
Davidson, N. E., Tan-Chiu, E., Martino, S., Paik, S., Kaufman, P. A.,
Swain, S. M., Pisansky, T. M., Fehrenbacher, L., Kutteh, L. A., Vogel,
V. G., Visscher, D. W., Yothers, G., Jenkins, R. B., Brown, A. M.,
Dakhil, S. R., Mamounas, E. P., Lingle, W. L., Klein, P. M., Ingle, J.
N., and Wolmark, N. (2005) Trastuzumab plus adjuvant chemotherapy
for operable HER2-positive breast cancer. N. Engl. J. Med. 353, 1673–
1684.
10. Bouchard, L., Lamarre, L., Tremblay, P. J., and Jolicoeur, P. (1989)
Stochastic appearance of mammary tumors in transgenic mice carrying
the MMTV/c-neu oncogene. Cell 57, 931–936.
11. Muller, W. J., Sinn, E., Pattengale, P. K., Wallace, R., and Leder, P.
(1988) Single-step induction of mammary adenocarcinoma in transgenic
mice bearing the activated c-neu oncogene. Cell 54, 105–115.
12. Guy, C. T., Webster, M. A., Schaller, M., Parsons, T. J., Cardiff, R. D.,
and Muller, W. J. (1992) Expression of the neu protooncogene in the
mammary epithelium of transgenic mice induces metastatic disease.
Proc. Natl. Acad. Sci. USA 89, 10578–10582.
13. Lemoine, N. R., Staddon, S., Dickson, C., Barnes, D. M., and Gullick,
W. J. (1990) Absence of activating transmembrane mutations in the c-
erbB-2 proto-oncogene in human breast cancer. Oncogene 5, 237–239.
14. Elias, J. E. and Gygi, S. P. (2007) Target-decoy search strategy for
increased confidence in large-scale protein identifications by mass spec-
trometry. Nat. Methods 4, 207–214.
15. Nakamura, T. and Oda, Y. (2007) Mass spectrometry-based quantitative
proteomics. Biotechnol. Genet. Eng. Rev. 24, 147–163.
16. Frixen, U. H., Behrens, J., Sachs, M., Eberle, G., Voss, B., Warda, A.,
Lochner, D., and Birchmeier, W. (1991) E-cadherin-mediated cell-cell
adhesion prevents invasiveness of human carcinoma cells. J. Cell Biol.113, 173–185.
17. Pujol, J. L., Grenier, J., Daures, J. P., Daver, A., Pujol, H., and Michel,
F. B. (1993) Serum fragment of cytokeratin subunit 19 measured by
CYFRA 21–1 immunoradiometric assay as a marker of lung cancer.
Cancer Res. 53, 61–66.
18. Kasai, H., Allen, J. T., Mason, R. M., Kamimura, T., and Zhang, Z.
(2005) TGF-beta1 induces human alveolar epithelial to mesenchymal
cell transition (EMT). Respir. Res. 6, 56.
19. Reed, J. C. (2003) Apoptosis-targeted therapies for cancer. Cancer Cell3, 17–22.
20. Frisch, S. M. and Screaton, R. A. (2001) Anoikis mechanisms. Curr.
Opin. Cell Biol. 13, 555–562.
21. Espana, L., Fernandez, Y., Rubio, N., Torregrosa, A., Blanco, J., and Si-
erra, A. (2004) Overexpression of Bcl-xL in human breast cancer cells
enhances organ-selective lymph node metastasis. Breast Cancer Res.
Treat. 87, 33–44.
22. Del Bufalo, D., Biroccio, A., Leonetti, C., and Zupi, G. (1997) Bcl-2
overexpression enhances the metastatic potential of a human breast can-
cer line. FASEB J. 11, 947–953.
23. Kumar, R., Mandal, M., Lipton, A., Harvey, H., and Thompson, C. B.
(1996) Overexpression of HER2 modulates bcl-, bcl-X, and tamoxifen-
induced apoptosis in human MCF-7 breast cancer cells. Clin. Cancer
Res. 2, 1215–1219.
24. Hayden, M. S. and Ghosh, S. (2004) Signaling to NF-kappaB. Genes
Dev. 18, 2195–2224.
25. Perkins, N. D. (2007) Integrating cell-signalling pathways with NF-kap-
paB and IKK function. Nature Rev. 8, 49–62.
26. Hatse, S., De Clercq, E., and Balzarini, J. (1999) Role of antimetabo-
lites of purine and pyrimidine nucleotide metabolism in tumor cell dif-
ferentiation. Biochem. pharmacol. 58, 539–555.
27. Spiegel, E. K., Colman, R. F., and Patterson, D. (2006) Adenylosucci-
nate lyase deficiency. Mol. Genet. Metab. 89, 19–31.
28. Wold, M. S. (1997) Replication protein A: a heterotrimeri, single-
stranded DNA-binding protein required for eukaryotic DNA metabo-
lism. Annu. Rev. Biochem. 66, 61–92.
29. Wang, Y., Putnam, C. D., Kane, M. F., Zhang, W., Edelmann, L., Rus-
sell, R., Carrion, D. V., Chin, L., Kucherlapati, R., Kolodner, R. D., and
Edelmann, W. (2005) Mutation in Rpa1 results in defective DNA dou-
ble-strand break repai, chromosomal instability and cancer in mice. Nat.
Genet. 37, 750–755.
30. Woychik, N. A. and Hampsey, M. (2002) The RNA polymerase II ma-
chinery: structure illuminates function. Cell 108, 453–463.
31. Chen, B. S. and Hampsey, M. (2004) Functional interaction between
TFIIB and the Rpb2 subunit of RNA polymerase II: implications for the
mechanism of transcription initiation. Mol. Cell Biol. 24, 3983–3991.
32. Tsimberidou, A. M., Alvarado, Y., and Giles, F. J. (2002) Evolving role
of ribonucleoside reductase inhibitors in hematologic malignancies.
Expert Rev. Anticancer Ther. 2, 437–448.
33. Heidel, J. D., Liu, J. Y., Yen, Y., Zhou, B., Heale, B. S., Rossi, J. J.,
Bartlett, D. W., and Davis, M. E. (2007) Potent siRNA inhibitors of
ribonucleotide reductase subunit RRM2 reduce cell proliferation in vitro
and in vivo. Clin. Cancer Res. 13, 2207–2215.
34. Duxbury, M. S. and Whang, E. E. (2007) RRM2 induces NF-kappaB-
dependent MMP-9 activation and enhances cellular invasiveness. Bio-chem. Biophys. Res. Commun. 354, 190–196.
35. Kohler, C., Gahm, A., Noma, T., Nakazawa, A., Orrenius, S., and Zhi-
votovsky, B. (1999) Release of adenylate kinase 2 from the mitochon-
drial intermembrane space during apoptosis. FEBS Lett. 447, 10–12.
36. Nobumoto, M., Yamada, M., Song, S., Inouye, S., and Nakazawa, A.
(1998) Mechanism of mitochondrial import of adenylate kinase iso-
zymes. J. Biochem. (Tokyo) 123, 128–135.
37. Lee, H. J., Pyo, J. O., Oh, Y., Kim, H. J., Hong, S. H., Jeon, Y. J.,
Kim, H., Cho, D. H., Woo, H. N., Song, S., Nam, J. H., Kim, H. J.,
Kim, K. S., and Jung, Y. K. (2007) AK2 activates a novel apoptotic
pathway through formation of a complex with FADD and caspase-10.
Nat. Cell Biol. 9, 1303–1310.
38. Nishino, T., Okamoto, K., Kawaguchi, Y., Hori, H., Matsumura, T.,
Eger, B. T., Pai, E. F., and Nishino, T. (2005) Mechanism of the con-
version of xanthine dehydrogenase to xanthine oxidase: identification of
the two cysteine disulfide bonds and crystal structure of a non-converti-
ble rat liver xanthine dehydrogenase mutant. J. Biol. Chem. 280,
24888–24894.
49PROTEIN EXPRESSION PROFILING OF PMECs
39. Linder, N., Lundin, J., Isola, J., Lundin, M., Raivio, K. O., and Joensuu,
H. (2005) Down-regulated xanthine oxidoreductase is a feature of
aggressive breast cancer. Clin. Cancer Res. 11, 4372–4381.
40. Kurima, K., Warman, M. L., Krishnan, S., Domowicz, M., Krueger, R.
C. Jr., Deyrup, A., and Schwartz, N. B. (1998) A member of a family
of sulfate-activating enzymes causes murine brachymorphism. Proc.
Natl. Acad. Sci. USA 95, 8681–8685.
41. ul Haque, M. F., King, L. M., Krakow, D., Cantor, R. M., Rusiniak, M.
E., Swank, R. T., Superti-Furga, A., Haque, S., Abbas, H., Ahmad, W.,
Ahmad, M., and Cohn, D. H. (1998) Mutations in orthologous genes in
human spondyloepimetaphyseal dysplasia and the brachymorphic
mouse. Nat. Genet. 20, 157–162.42. Ikeda, T., Mabuchi, A., Fukuda, A., Hiraoka, H., Kawakami, A.,
Yamamoto, S., Machida, H., Takatori, Y., Kawaguchi, H., Naka-
mura, K., and Ikegawa, S. (2001) Identification of sequence poly-
morphisms in two sulfation-related gene, PAPSS2 and SLC26A, and
an association analysis with knee osteoarthritis. J. Hum. Genet. 46,
538–543.
43. Kobayashi, T., Sasaki, Y., Oshima, Y., Yamamoto, H., Mita, H.,
Suzuki, H., Toyota, M., Tokino, T., Itoh, F., Imai, K., and Shinomura,
Y. (2006) Activation of the ribosomal protein L13 gene in human gas-
trointestinal cancer. Int. J. Mol. Med. 18, 161–170.
44. Bee, A., Ke, Y., Forootan, S., Lin, K., Beesley, C., Forrest, S. E., and
Foster, C. S. (2006) Ribosomal protein l19 is a prognostic marker for
human prostate cancer. Clin. Cancer Res. 12, 2061–2065.
45. Wang, H., Zhao, L. N., Li, K. Z., Ling, R., Li, X. J., and Wang, L.
(2006) Overexpression of ribosomal protein L15 is associated with cell
proliferation in gastric cancer. BMC Cancer 6, 91.
46. Zhu, Y., Lin, H., Li, Z., Wang, M., and Luo, J. (2001) Modulation of
expression of ribosomal protein L7a (rpL7a) by ethanol in human breast
cancer cells. Breast Cancer Res. Treat. 69, 29–38.
47. Henry, J. L., Coggin, D. L., and King, C. R. (1993) High-level expres-
sion of the ribosomal protein L19 in human breast tumors that overex-
press erbB-2. Cancer Res. 53, 1403–1408.
48. Mani, A. and Gelmann, E. P. (2005) The ubiquitin-proteasome pathway
and its role in cancer. J. Clin. Oncol. 23, 4776–4789.
49. Barnes, C. J., Li, F., Talukder, A. H., and Kumar, R. (2005) Growth
factor regulation of a 26S proteasomal subunit in breast cancer. Clin.Cancer Res. 11, 2868–2874.
50. Pende, D., Bottino, C., Castriconi, R., Cantoni, C., Marcenaro, S., Riv-
era, P., Spaggiari, G. M., Dondero, A., Carnemolla, B., Reymond, N.,
Mingari, M. C., Lopez, M., Moretta, L., and Moretta, A. (2005) PVR
(CD155) and Nectin-2 (CD112) as ligands of the human DNAM-1
(CD226) activating receptor: involvement in tumor cell lysis. Molecular
immunology 42, 463–469.
51. Edvardsen, K., Bock, E., Jirus, S., Frandsen, T. L., Holst-Hansen, C.,
Moser, C., Spang-Thomsen, M., Pedersen, N., Walsh, F. S., Vindelov,
L. L., and Brunner, N. (1997) Effect of NCAM-transfection on growth
and invasion of a human cancer cell line. APMIS 105, 919–930.
52. Zhang, D., Tai, L. K., Wong, L. L., Chiu, L. L., Sethi, S. K., and Koay,
E. S. (2005) Proteomic study reveals that proteins involved in metabolic
and detoxification pathways are highly expressed in HER-2/neu-positive
breast cancer. Mol. Cell Proteomics 4, 1686–1696.53. Gharbi, S., Gaffney, P., Yang, A., Zvelebil, M. J., Cramer, R., Water-
field, M. D., and Timms, J. F. (2002) Evaluation of two-dimensional
differential gel electrophoresis for proteomic expression analysis of a
model breast cancer cell system. Mol. Cell Proteomics 1, 91–98.54. Landis, M. D., Seachrist, D. D., Montanez-Wiscovich, M. E., Daniel-
pour, D., and Keri, R. A. (2005) Gene expression profiling of cancer
progression reveals intrinsic regulation of transforming growth factor-
beta signaling in ErbB2/Neu-induced tumors from transgenic mice.
Oncogene 24, 5173–5190.
55. Abd El-Rehim, D. M., Ball, G., Pinder, S. E., Rakha, E., Paish, C.,
Robertson, J. F., Macmillan, D., Blamey, R. W., and Ellis, I. O. (2005)
High-throughput protein expression analysis using tissue microarray
technology of a large well-characterised series identifies biologically
distinct classes of breast cancer confirming recent cDNA expression
analyses. Int. J. Cancer 116, 340–350.
50 PARK ET AL.