Protein expression profiling of primary mammary epithelial cells derived from MMTV-neu mice revealed that HER2/NEU-driven changes in protein expression are functionally clustered

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.


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


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


Q9CXW4 60S ribosomal protein L11 Rpl11 14.537 6.078 1.26

P84099 60S ribosomal protein L19 Rpl19 3.7706 1.1426 1.72



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



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


Q9D7S7 Ribosomal protein L22-like 1 Rpl22l1 8.7002 2.6747 1.70



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


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


6B (MIP224)

Psmc4 7.0708 1.8142 1.96


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


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


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.

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50 PARK ET AL.

Documents

Protein expression profiling of primary mammary epithelial cells derived from MMTV-neu mice revealed that HER2/NEU-driven changes in protein expression are functionally clustered