From Oxford Glycosciences, The Forum, 86 Milton Park,
Abingdon, Oxford OX14 4RY, United Kingdom, the ¶ Cancer
Research United Kingdom Molecular Oncology Laboratories, Weatherall
Institute of Molecular Medicine, John Radcliffe Hospital, Oxford
OX3 9DS, United Kingdom, and
Hybrigenics, 3-5 Impasse
Reille, Paris 75014, France
Received for publication, October 4, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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Proteins associated with cancer cell
plasma membranes are rich in known drug and antibody targets as well as
other proteins known to play key roles in the abnormal signal
transduction processes required for carcinogenesis. We describe here a
proteomics process that comprehensively annotates the protein content
of breast tumor cell membranes and defines the clinical relevance of
such proteins. Tumor-derived cell lines were used to ensure an
enrichment for cancer cell-specific plasma membrane proteins because it
is difficult to purify cancer cells and then obtain good membrane
preparations from clinical material. Multiple cell lines with different
molecular pathologies were used to represent the clinical heterogeneity of breast cancer. Peptide tandem mass spectra were searched against a
comprehensive data base containing known and conceptual proteins derived from many public data bases including the draft human genome
sequences. This plasma membrane-enriched proteome analysis created a
data base of more than 500 breast cancer cell line proteins, 27% of
which were of unknown function. The value of our approach is
demonstrated by further detailed analyses of three previously uncharacterized proteins whose clinical relevance has been defined by
their unique cancer expression profiles and the identification of
protein-binding partners that elucidate potential functionality in cancer.
Breast cancer is the most frequently diagnosed cancer in women and
accounts for 30% of all cancers diagnosed in the United States (1).
The implementation of screening programs for the early detection of
breast cancer and the advent of anticancer treatments such as
chemotherapy, radiotherapy, and anti-estrogen therapies to augment
surgical resection have improved the survival of breast cancer
patients. However, some breast tumors become refractory to such
treatments as the cancer cells develop resistance to chemotherapy drugs
or lose their hormone sensitivity, leading to recurrent or metastatic
disease that is often incurable. Cancer membrane-associated proteins
form the basis of a number of new drug and antibody cancer therapeutics
such as Gleevec (abl-kinase) (2), herceptin (her2neu) (3), Panorex
(Ep-CAM) (4), and IRESSA
(EGF1 receptor) (5).
Furthermore many other membrane-associated proteins such as small
GTPases, kinases, and catenins are implicated in carcinogenesis. Thus a
comprehensive definition of cancer cell membrane-associated proteins
can reveal further proteins involved in cancer biology which may
themselves represent new therapeutic targets.
Tumor-specific proteins have been identified for a number of cancer
types using techniques such as differential screening of cDNAs (6)
and the purification of cell surface proteins that are recognized by
tumor-specific antibodies (7). More recently, DNA "chips"
containing up to 12,000 expressed sequence elements have been used to
characterize tumor cell gene expression (8). However, there are several
reasons why the numerous and extensive previous transcriptomic analyses
of breast cancer may not have revealed all tumor-associated proteins.
These include (i) a lack of correlation between transcript and
disease-associated protein levels; (ii) translocation of a protein in
the disease state rather than simply differential levels of the
transcript; (iii) novel/uncharacterized genes that are not highly
represented within the "closed system" of a cDNA array where
there are restrictions on the number of expressed sequence elements per
chip and the knowledge and availability of DNA clones.
There have been previous attempts to use plasma membrane-enriched
preparations from cancer cell lines to discover new proteins associated
with tumor biology (9). In this study we have improved the proportion
and total number of membrane proteins obtained from around 30% in
previous studies (9) to more than 50%, with 31% being trans-plasma
membrane or plasma membrane-associated. Furthermore, 27% of the
proteins identified are of unknown function. Indeed, the three
uncharacterized proteins described in this paper (named breast cancer
membrane protein BCMP11, BCMP84, and BCMP101) all show cancer cell
membrane association as well as protein expression levels and
localizations that are unique to cancer cells over normal breast
epithelial cells.
Preparation of Membrane Fractions and Protein
Separation--
The human breast carcinoma cell lines MDA-MB-468
(ATCC:HB-132), T-47D (ATCC:HB-133), BT-474 (ATCC:HTB-20), and MCF-7
(ATCC:HTB-22) were cultured in Dulbecco's modified Eagle's
medium/F12 medium containing 10% fetal calf serum, 2 mM
glutamine, and 1% penicillin/streptomycin. The cells were maintained
at 37 °C in a humidified atmosphere of 95% air and 5%
CO2. Adherent cells (2 × 108) were washed
three times with PBS and scraped using a plastic cell lifter. Cells
were centrifuged at 1,000 × g for 5 min at 4 °C,
and the cell pellet was resuspended in homogenization buffer (250 mM sucrose, 10 mM HEPES, 1 mM EDTA,
1 mM vanadate, and 0.02% azide protease inhibitors). Cells
were fractionated using a ball bearing homogenizer (8.002-mm ball, HGM
Laboratory Equipment) until approximately 95% of the cells were
broken. Membranes were fractionated using the method described by
Pasquali et al. (10). The fractionated cells were
centrifuged at 3,000 × g for 10 min at 4 °C, and
the postnuclear supernatant was layered onto a 60% sucrose cushion and
centrifuged at 100,000 × g for 45 min. The membranes
were collected using a Pasteur pipette, layered on a preformed 15-60%
sucrose gradient, and spun at 100,000 × g for 17 h. Proteins from the fractionated sucrose gradient were run on a
4-20% one-dimensional gel (Novex) and subjected to Western blotting.
Those fractions containing alkaline phosphatase and transferrin
immunoreactivity but not oxidoreductase II or calnexin immunoreactivity
were pooled and represented the plasma membrane fraction.
Proteolysis and MALDI-TOF Mass Spectrometry--
Proteins
excised from the one-dimensional gels were analyzed by MALDI-TOF mass
spectrometry (Voyager STR, Applied Biosystems, Framingham, MA) using a
337-nm wavelength laser for desorption and the reflectron mode of
analysis. Selected masses for BCMP11 ((M+H) = 1,226.604, 1,258.674, and 922.455), BCMP84 ((M+H) = 1,648.697), and BCMP101
((M+H) = 2,099, 1,649 and 1,170) were characterized further by
MS/MS using a quadrupole TOF mass spectrometer equipped with a
nanospray ion source (Micromass U. K. Ltd., Manchester). Prior to MALDI analysis the samples were desalted and concentrated using C18 Zip TipsTM (Millipore, Bedford, MA). Samples for MS/MS were
purified using a nano LC system (LC Packings, Amsterdam, The
Netherlands) incorporating C18 SPE material. Using the SEQUEST search
program (11), uninterpreted tandem mass spectra of tryptic digest
peptides were searched against a FASTA data base of public domain
proteins constructed of protein entries in the nonredundant data base
held by the National Center for Biotechnology Information (NCBI) and
Swiss-Prot which are accessible at www.ncbi.nlm.nih.gov/and www.expasy.com. Peptide matches identified by SEQUEST were filtered according to their cross-correlation score, normalized difference correlation score, compatibility with trypsin digestion, and number of
observations of proteins and peptides. Peptides were only used for
protein identification where the cross-correlation score was Cloning, AFP Tagging, and Transient Transfection
Analysis--
Total RNA was prepared from cultured MDA-MB-468 and
T-47D cells using Trizol reagent (Invitrogen) and resuspended in
RNase-free water at a concentration of 1 µg/µl. 5 µg of total RNA
was used for oligo(dT) primed first strand cDNA synthesis using
Superscript II reverse transcriptase (Invitrogen). The predicted
full-length BCMP11, BCMP84, and BCMP101 open reading frames (ORFs) were
amplified by PCR from T-47D and MDA-MB-468 cDNAs using the
following primers: BCMP11F,
5'-ggccaagtcagcttcttctg-3'; BCMP11R,
5'-gtatttgtcaatgtgccagagg-3'; BCMP84F,
5'-ataggacaacagaactctcacc-3'; BCMP84R,
5'-gcttcaacggaactttgcagag-3'; BCMP101F,
5'-tgtgcaaatgaccctggagttg-3'; BCMP101R,
5'-ggctgctactgcaaacagttcc-3'. Reactions contained 10 ng of
cDNA and reagents for PCR (Qiagen) and used 40 cycles at 94 °C
for 30 s and 60 °C for 30 s. PCR products were column
purified (Qiagen), cloned into T:A vector (Invitrogen) and the sequence
verified. For fluorescent tagging, the BCMP11, BCMP84, and BCMP101
full-length ORFs were PCR cloned into the pQBI25/50-fN1 vector
(Qbiogene) resulting in an in-frame addition of the SuperGloTM
autofluorescent protein (AFP) to the C terminus of each expressed
protein. Transient transfection of SuperGloTM AFP-tagged BCMP11,
BCMP84, and BCMP101 cDNAs into MDA-MB-468, T-47D, and normal human
mammary epithelial (Clonetics, BioWhittaker, Inc. International) cell
lines was achieved using SuperfectTM transfection reagent (Qiagen).
Immunohistochemistry and
Immunocytochemistry--
Immunohistochemical analysis was carried out
on formalin-fixed paraffin-embedded tissue microarrays containing 1-mm
sections of breast carcinoma tissue from 50 donors as well as 200 sections of various normal tissues (Clinomics Laboratories Inc.,
Pittsfield, MA). A variety of other ethically approved carcinoma
tissues sections were provided by the University of Oxford, U. K.
Formalin-fixed sections were deparaffinized by two 5-min washes in
xylene, then rehydrated through successive graded ethanol solutions and
washed for 5 min in PBS. Antigen retrieval was achieved in 0.01 M citrate buffer (pH 6) and microwaving for 10 min at full
power (950 W). In addition, detection with the BCMP84 antibody required
that the tissue be treated with 1 mg/ml pepsin for 1.5 min at room temperature. Thawed frozen sections were fixed in acetone for 10 min at
room temperature then washed twice in PBS. Endogenous hydrogen
peroxidase activity was quenched by treating the slides in 3% hydrogen
peroxidase and PBS for 10 min. The tissue was blocked in 10% donkey
serum and PBS for 1 h before the addition of 2 µg/ml primary
polyclonal antibody (in 2.5% donkey serum). The BCMP11, BCMP84, and
BCMP101 polyclonal antibodies were raised in rabbits immunized with two
specific peptides (ABCAM Ltd., Cambridge, U. K.). The BCMP11 peptides
used were CAQNEEIQEMAQNKFIMLNLMHET and CTYEPRDLPLLIENMKKALRLIQSEL, the BCMP84 peptides used were
CEGGKETLTPSELRDLV and CEAAKSVKLERPVRGH, and the BCMP101 peptides used
were SYKEVPTADPTGVDR and LTDASQGRRGRVVND. Western blot analysis
of T-47D, MDA-MB-468, and other negative cell line lysates was used to
confirm that each antibody cross-reacted with a single band of the
predicted size in the correct samples. After three washes in PBS the
tissue sections were incubated with biotin-conjugated secondary
antibodies (Biotin-SP-conjugated AffiniPure donkey anti-rabbit, Jackson
ImmunoResearch) diluted at 1:200 (2.5 µg/ml in 2.5% donkey serum and
PBS) for 1 h. Slides were washed three times in PBS and the tissue
incubated with streptavidin-horseradish peroxidase (Jackson
ImmunoResearch) diluted 1:100 (5 µg/ml in 2.5% donkey serum and
PBS). Immunostaining was detected using DAB substrate solution (Dako
Ltd.) according to the manufacturer's instructions. Immunocytochemical
analysis was carried out on MDA-MB-468 cells fixed in 4%
paraformaldehyde and PBS and permeabilized on 0.3% Triton X-100. After
a 1-h incubation in blocking buffer (10% donkey serum and PBS) BCMP84
antibodies (1:50 dilution in blocking buffer) were incubated on the
cells for a further 1 h. Cells were washed three times in PBS and
incubated for 1 h in biotinylated anti-rabbit secondary antibody
(Jackson Laboratories) followed by the addition of streptavidin-Alexa
488 (Molecular Probes).
In Situ RT-PCR--
Direct in situ RT-PCR detection
of BCMP101 mRNA expression was examined in formalin-fixed,
paraffin-embedded breast cancer tissues (provided by Human Research
Tissue Bank, Department of Cellular Pathology, Peterborough District
Hospital, Peterborough PE3 6DA, U. K.). Briefly, dewaxed and
rehydrated tissue was permeabilized in 0.01% Triton X-100 for 3 min
followed by treatment with proteinase K for 30 min at 37 °C. Direct
in situ RT-PCR was carried out in a GeneAmp in Situ PCR
System 1000 (PerkinElmer Life Sciences) using a GeneAmp Thermostable
rTth RT-PCR kit (PerkinElmer Life Sciences). The primers used to
amplify BCMP101 were: sense, 5'-ttcacctctccgcgggtagcct-3', antisense,
5'-ggaagttacccacatatacggc -3'. The thermal cycling parameters were 1 cycle of 94 °C for 2.5 min followed by 20 cycles of 94 °C for
40 s, 60 °C for 50 s, and 72 °C for 30 s.
Amplified product was detectable through the direct incorporation of
alkali-stable digoxigenin-11-dUTP (Roche Molecular Biosciences), which
was added to the reaction mix. After washing in PBS an
anti-digoxigenin-gold antibody (Roche) was incubated on the tissue
section for 30 min at room temperature. This was followed by a silver
enhancement step (Roche silver enhancement reagents) during which time
the amplified expression product became visible by light microscopy. The tissue was counterstained with hematoxylin (Dako Ltd.).
Yeast Two-hybrid Cloning Analysis--
Baits were PCR amplified
(Pfu, Stratagene) and then cloned in the pB6 plasmid derived
from the original pAS2
The mating protocol has been described elsewhere (12). Briefly, the
screening conditions were adapted for each bait (test screen) before
performing the full-size screening. The selectivity of the
HIS3 reporter gene was modulated with 3-aminotriazole to obtain a maximum of 384 histidine-positive clones. For all selected clones, LacZ activity was measured in a semiquantitative
5-bromo-4-chloro-3-indolyl- Selection of Protein Candidates for Further Analysis--
To
discover proteins with novel relevance to breast cancer, bioinformatic
filters were applied to exclude those expressed ubiquitously or at high
levels in vital organs (liver, lung, heart, central nervous system,
digestive system, kidney). Proteomic data bases were queried for
frequency, and tissue and subcellular distribution, of proteins
identified in the present study. Accumulated cDNA data were also
queried for absolute abundance in various tissue types. Proteins were
identified for further analysis on the basis of the highest expression
in breast cancer, coupled with low or absent expression in vital
organs. A further exclusion step was applied to remove proteins already
known to be involved in breast cancer or those obviously not associated
with plasma membranes. The resulting class of breast cancer-specific or
transcriptionally low level proteins were examined for further evidence
relevant to tumorigenic processes, such as homology to protein families known to be associated with breast cancer or involved in cancer biology. Other proteins in this class which were previously supported only by cDNA evidence were also included as potential novel breast cancer-specific membrane proteins. Of the 22 proteins selected using
the above methods, the three described here were particularly interesting with respect to mRNA distribution, breast
cancer-specific changes, and relevant interacting proteins.
MS/MS Analysis of Membrane-associated Proteins in Breast
Cancer-derived Cell Lines--
Purified cell membrane protein
preparations were isolated from the MDA-MB-468, T-47D, BT-474, and
MCF-7 breast cancer cell lines. The T-47D/MCF-7 estrogen
receptor-positive and MDA-MB-468/BT-474 EGF receptor-positive cell
lines were pooled, and each pool was independently separated by
one-dimensional-PAGE. Sequential 0.5-mm gel slices containing the
proteins were subjected to trypsinolysis, and the resulting peptide
fragments analyzed by MALDI-TOF and MS/MS. Proteins were identified by
mass- and fragment-based data base searching (11, 14). Selection of
peptides for MS/MS analysis was deliberately biased away from those
masses contained in highly abundant proteins so as to increase the
discovery potential and coverage of lower abundance proteins. In total,
501 distinct proteins were identified from both cell membrane pools
(see Table I in the supplemental material). This table provides an
accession to each protein sequence obtained by MS/MS and indicates in
which cell line pool each protein was identified. The tandem mass
peptides used to identify each protein have also been included in
supplemental Table I because accession numbers can frequently change,
particularly for the more recently identified hypothetical/functionally
uncharacterized proteins. The proteins are categorized into five
locational groups based on Swiss-Prot annotations, literature
citations, and homology searches. However, it is noteworthy that many
of the proteins identified can exist in multiple cell locations, but
where there is good literature evidence of membrane association we have
reflected this in the categorizations in supplemental Table I, which
determines the contribution of each locational group to Fig.
1A. The proportion of proteins
in each category is illustrated in Fig. 1A. Categories T,
PA, and IM, which represented 51% of the total, contained known trans-plasma membrane (T), plasma membrane-associated (PA), and integral membrane-associated (IM) proteins, respectively (Fig. 1A). Examples of proteins in each of these categories are
shown in Fig. 1B and include known breast cancer membrane
antigens such as erbB2/her2neu tyrosine kinase receptor
(accession P04626) and EGF receptor (O00688). Intracellular signaling
proteins, such as Ras-related proteins and nonreceptor tyrosine
kinases, which bind to plasma membrane-associated proteins, were
observed in category PA (Fig. 1B and supplemental Table I),
demonstrating that multiple components of cancer signal transduction
pathways can be extracted and identified by these proteomic
methods.
Of the proteins identified, 27% were either hypothetical (proteins
predicted from the transcriptome) or of unknown function and cell
localization (Fig. 1A). Based on the number of known membrane-associated proteins found, it is likely that ~50% of these
hypothetical/uncharacterized proteins represent trans-plasma membrane
or generally membrane-associated proteins. The two breast cancer cell
line pools, one representing estrogen receptor-positive cell lines and
the other EGF receptor-positive cell lines, were deliberately chosen to
reflect some of the different molecular pathologies known in breast
cancer. Although failure to identify a protein is not evidence of its
absence in a membrane preparation, many proteins were apparently
restricted to, or denoted in, either the EGF receptor pool or the
estrogen receptor-positive pool (supplemental Table I). From the pool
of hypothetical/uncharacterized proteins we describe more detailed
analyses on three, BCMP11, BCMP84, and BCMP101. A representative MS/MS
result used to identify protein BCMP84 is shown in Fig.
2. All three unique proteins were
represented by a low number of expressed sequence tags (ESTs) that did
not represent a complete expression profile. In addition, BCMP11 was chosen because of its similarity to the Xenopus
laevis XAG embryonic differentiation proteins. BCMP84 was an
uncharacterized member of the S100 protein family, several of which are
associated with tumor biology. BCMP101 was chosen because, with no
known functionally characterized homologs, it represented a completely
unique breast cancer membrane-associated protein.
Identification and Cloning of BCMP11, BCMP84, and
BCMP101--
Four spectra from protein BCMP11, isolated from
T-47D/MCF-7 cell membranes, were found to match a translation of an EST
from a human lung carcinoma library (accession AI458391) (supplemental Table I) defining an ORF of 166 amino acids (Fig.
3A). A BLAST search of a human
EST data base (www.ncbi.nlm.nih.gov/blast) identified several
overlapping ESTs with matches to the sequence of AI458391, providing
additional 5'- and 3'-untranslated region sequences. Primers designated
to the 5'- and 3'-untranslated region sequences were used to amplify a
full-length clone from T-47D cDNA by PCR (Fig. 3A). The
predicted BCMP11 protein is highly homologous to hAG-2, a novel human
protein encoded by a cDNA cloned from the MCF-7 breast cancer cell
line (15) and localized to chromosomal band 7p21.3 (16). hAG-2 is the
human homolog of XAG-2, a secreted X. laevis protein that is
thought to be involved in the regulation of dorsoanterior ectodermal
cell fate during cement gland differentiation (17). BCMP11, like hAG-2,
is predicted to be an extracellular protein with an N-terminal signal
sequence (psort.nibb.ac.jp). In view of the high degree of sequence
identity between the two proteins, we have named BCMP11 "hAG-3"
(GenBank AY069977; Swiss-Prot Q8TD06).
Protein BCMP84 was isolated from the MDA-MB-468/BT-474 cell membrane
pool. Amino acid sequences from a single MS/MS were found to match a
translation of an EST from a human colon carcinoma cell line (accession
number AA315020) (Fig. 3B). Overlapping ESTs were identified
which established a complete ORF of 104 amino acids. A full-length
clone was amplified by PCR from MDA-MB-468 cDNA (Fig.
3B). The predicted BCMP84 protein shows similarity to the
S100 family of calcium-binding proteins and the predicted protein
product of a recently identified cDNA (AY007220), which is
identical to BCMP84. The protein encoded by AY007220 has been named
S100A14 and annotated as a novel member of the S100 family of calcium
binding proteins (Swiss-Prot Q9HCY8). The BCMP84 gene lies at
chromosomal position 1q21 within the S100 calcium binding protein gene
cluster (18).
Three spectra from protein BCMP101, isolated from MDA-MB-468/BT-474
cell membranes, were found to match a translation of an EST from a
human pooled library of testis, fetal lung, and B-cell cDNA
(GenBank accession AI827549; dbEST identification 2915502). Additional
overlapping ESTs were identified, and alignment with genomic clone
AC021396.2 established a complete ORF of 310 amino acids. Primers
designed to the 5'- and 3'-untranslated region sequences were used to
amplify a full-length clone from MDA-MB-468 cDNA by PCR (Fig.
3C). The predicted protein product of a recently identified,
uncharacterized cDNA clone, NSE-2 (GenBank AJ417849; Swiss-Prot
Q96KN1) is identical to BCMP101. The BCMP101 gene lies on chromosome 8 at position q24.21, and the ORF is encoded by a single exon. The
predicted BCMP101 protein has a homolog on chromosome 2 (GenBank
CAD10038); however, the functions of both proteins are unknown, and
analysis of the BCMP101 protein sequence identified no motifs that
might suggest a particular function or cellular location.
Cellular Localization and Proteins Interacting with BCMP11, BCMP84,
and BCMP101--
C-terminal tagging with green
SuperGloTM-AFP and immunocytochemistry were used to
determine the cellular localization of BCMP11, BCMP84, and BCMP101
proteins in MDA-MB-468 and T-47D cell lines. In addition, yeast
two-hybrid cloning analysis was used to identify tumor cell
line-derived proteins interacting with full-length BCMP84 and BCMP101
proteins and mature BCMP11 protein (signal sequence deleted).
Transient transfection analysis of AFP-tagged BCMP11 cDNA construct
demonstrated that the translated AFP fusion protein was localized in
secretory or endosome-like organelles in both T-47D and MDA-MB-468
cells (Fig. 4A). Indeed,
BCMP11 has a putative signal sequence and is predicted to be a secreted
extracellular protein. In addition, yeast two-hybrid cloning identified
the human homolog of a rat glycosylphosphatidylinositol-anchored
metastasis-associated protein, C4.4A, (GenBank NM_014400) (19)
and the extracellular domain of dystroglycan as BCMP11-interacting
proteins (20). An interaction between BCMP11 and C4.4A is particularly
interesting given that both proteins are expressed in carcinoma tissues
prone to metastasis.
Analysis of the cellular location of an AFP-tagged BCMP101 demonstrated
widespread intracellular localization but also significant expression
associated with the plasma membrane in both MDA-MB-468 and T-47D cell
lines (Fig. 4B). Indeed, we observed particularly high
levels of BCMP101 plasma membrane localization in areas of cell-cell
contact (Fig. 4B, ii). Consistent with this,
BCMP101 was found to interact specifically with
Transient transfection analysis of an AFP-tagged BCMP84 cDNA
construct demonstrated that the translated AFP fusion protein was
localized in the plasma membrane in both MDA-MB-468 and T-47D cancer
cells (Fig. 4C, i-ii). Because both our breast
cancer cell line membrane proteome data (supplemental Table I) and
Western blot analysis with an anti-BCMP84 antibody demonstrated the
absence of BCMP84 in the T-47D cell line, these data indicate that
T-47D cancer cells artificially expressing BCMP84 also target the
protein to the plasma membrane. In addition, immunocytochemical
analysis with an anti-BCMP84 antibody also showed clear plasma membrane localization of BCMP84 protein in MDA-MB-468 cells (Fig. 4C,
iii). In contrast to this tumor cell plasma membrane
localization, a primary culture of non-tumor-derived human mammary
epithelial cells transfected with the AFP-tagged BCMP84 construct
showed only cytosolic BCMP84 expression (Fig. 4C,
iv). Yeast two-hybrid cloning identified nucleobindin
(CALNUC) protein as a BCMP84-interacting protein. Nucleobindin is found
in both cytosolic and membrane fractions (see supplemental Table I,
plasma membrane-associated accession Q02818) where it is thought to act
as a major calcium-binding protein (21).
BCMP11, BCMP84, and BMCP101 Proteins Are Expressed in Clinical
Breast Cancer Tissues--
Immunohistochemical analysis was used to
determine the expression of BCMP11, BCMP84, and BCMP101 proteins in
sections of normal and breast cancer tissues. BCMP11 showed a
restricted expression profile in normal tissues, with only the
epithelial lining of the colonic mucosa showing significant levels of
staining with a specific antibody raised against BCMP11, entirely
consistent with the mRNA distribution of BCMP11 (data not shown).
In contrast, very high levels of BCMP11 staining were seen in 43 of 58 (74%) breast cancer donor tissues but not normal breast ductal
epithelial cells (Fig. 5A). In
each case the BCMP11 staining was restricted to the cancerous
epithelial cells of the tissue and was localized in the cytoplasm of
these cells (Fig. 5A, iv).
Real time quantitative RT-PCR, in situ RT-PCR, and
immunohistochemical analysis of BCMP101 expression demonstrated very
low levels in multiple normal tissues (data not shown). In contrast, high levels of BCMP101 mRNA and protein were detected in the
carcinoma cells of breast cancer tissues (Fig. 5C). Real
time RT-PCR analysis also demonstrated that BCMP101 mRNA was
up-regulated more than 2-fold in six of seven (85%) breast cancer
donor tissues relative to donor-matched adjacent normal breast tissue
(Fig. 5C).
The distribution of BCMP84 protein in normal tissues was restricted to
the stratified squamous epithelium of the skin, cervix, and tonsil
(data not shown). Strong immunoreactivity to the BCMP84 antibody was
observed in 10 of 58 (17%) breast cancer donor tissues with weak
staining seen in normal breast ductal tissue (Fig. 5B, v). In each case BCMP84 tumor staining was restricted to the
cancerous epithelial cells of the tissue and, as with the
immunocytochemical analysis in the MDA-MB-468 cell line (Fig.
4C, iii), showed a clear localization in the
plasma membrane (Fig. 5B, iv). In contrast, we
consistently observed weaker cytoplasmic staining in adjacent normal
ductal breast epithelial tissue (Fig. 6,
i). This is consistent with the cytosolic expression of
AFP-tagged BCMP84 in primary normal human mammary epithelial cells
(Fig. 4C, iv). Subsequent analysis of multiple
other tumor tissues demonstrated elevated BCMP84 protein expression in
squamous tonsil and bladder papillary transitional cell carcinoma
tissues (Fig. 6, ii-iii). For both of these tumor tissues
we observed that the less differentiated basal cancer cells showed
cytoplasmic BCMP84 staining that became distinctly plasma membrane
localized as the cells became more differentiated (Fig. 6).
Identification of tumor cell-specific plasma membrane-associated
proteins is an important first step in the development of antibody and
small molecule cancer therapies. In this paper we describe and list, to
our knowledge, the largest example of a tumor cell-derived membrane proteome.
The standard approach for resolving proteins involves two-dimensional
gel electrophoresis; however, two-dimensional gels are particularly
poor at separating relatively insoluble hydrophobic membrane proteins.
For this reason we chose to resolve our membrane preparations on
one-dimensional gels, which has allowed us to identify a spectrum of
membrane and associated proteins ranging from small membrane-associated
Ras signaling proteins to large multitransmembrane proteins (see
supplemental Table I). This comprehensive annotation of breast cancer
cell line proteins has been achieved by avoiding repetitive sequencing
of high abundance proteins and subjecting more than 16,000 peptides of
low relative intensity (<10% from MALDI) to MS/MS. Thus we observed a
wide dynamic range of breast cancer proteins, from the highly abundant
and amplified her2neu to rare unique proteins such as BCMP101 which
have never been sequenced at the protein level before and in this case
show only four matching ESTs from breast tissues. In addition to
one-dimensional gel electrophoresis, other exploratory peptide
separation technologies such as multidimensional protein identification
technology (22) or isotope-coded affinity tagging (23) as well as more
complex protein fractionation methods and new MS technologies such as anchor targets (www.brukerdaltonics.com) coupled to TOF-TOF instruments (www.appliedbiosystems.com) could enable the direct identification of
even lower abundance proteins. To ensure a representative and plasma
membrane-enriched breast cancer cell membrane preparation we have used
breast cancer-derived cell lines. The membrane preparations that can be
obtained from clinical breast tissue are far less pure and in many
instances are "contaminated" with non-tumor cell membranes.2 The four breast
cancer-derived cell lines used in this study, two representing estrogen
receptor-positive cell lines and the other two EGF receptor-positive
cell lines, were deliberately chosen to reflect some of the different
molecular pathologies known in breast cancer. Indeed, demonstrating
immunohistochemically that the three unique proteins we characterized
further are significantly expressed in clinical breast tumor tissues
has validated our use of tumor-derived cell lines in proteomics.
Of the three proteins we characterized, BCMP11/hAG3 shows the highest
prevalence in breast cancers (74%) with a strong positive correlation
with estrogen receptor status (20). We have observed no mRNA or
protein expression in normal breast epithelia or stromal tissue. The
finding that BCMP11 is potentially localized in secretory organelles is
consistent with it having a putative signal sequence and thus the
prediction that it is a secreted extracellular protein. Consistent with
this, yeast two-hybrid cloning identified the human homolog of a rat
glycosylphosphatidylinositol-anchored metastasis-associated protein,
C4.4A, as a BCMP11-interacting protein. C4.4A may therefore represent
an autocrine receptor for BCMP11. Further in vitro and in vivo studies will be required to determine the biological
role of BCMP11 in breast tumor cell growth and/or metastasis.
No clues as to the function of BCMP101 could be gained from its primary
sequence, but its mRNA expression was highly restricted to breast
cancer-derived cell lines and clinical breast cancer where it was
up-regulated more than 3-fold in six of seven breast cancer tissues
relative to the donor matched normal tissues. Fluorescently tagged
BCMP101 showed a localization in the plasma membrane, particularly in
areas of cell-cell contact, and a key finding with respect to this is
its interaction with BCMP84 is a member of the S100 family of proteins, and we have shown
here plasma membrane localization in both breast cancer-derived cell
lines and clinical breast cancer tissues but cytosolic expression in
non-tumor cells. The translocation of BCMP84 from cytosol to plasma
membrane in 17% of breast cancers is the lowest prevalence event of
the three proteins described here. However, BCMP84 shows this
proliferation and differentiation-associated event in other cancers
such as tonsil and bladder. The prevalence and significance of this
translocation to cancer remain to be established, but this 17% may
define molecularly a subset of breast cancers that have commonalities
with BCMP84-positive tumors derived from other tissues. Other members
of the S100 family of calcium-binding proteins are known to be
expressed in cancer tissues (25). In particular, S100A4 (p9Ka), whose
expression has been shown to induce metastasis in rodent models of
breast cancer (26, 27), is expressed in human breast cancers and
tightly correlates with poor prognosis (28, 29). Stradal and
Gimona (30) have demonstrated that S100A6 (calcyclin) exhibits a
calcium-dependent association with the plasma membrane and
nuclear envelope in porcine smooth muscle and human CaKi-2 cells. These
data suggest that the translocation of BCMP84 from the cytosol of
normal cells to the plasma membrane in tumor cells may be regulated in
a calcium-dependent manner. Indeed, further evidence for a
calcium-dependent localization of BCMP84 was discovered
through its interaction with nucleobindin. Nucleobindin is found in
both cytosolic and membrane fractions and has been strongly associated
with several different subfamilies of G In summary, we have demonstrated a process utilizing multiple proteomic
methodologies for the de novo discovery of cancer cell
membrane-associated proteins with potential clinical relevance to
breast cancer. This process also shows the primary discovery value of
cancer-derived cell lines where the ability to generate samples of high
purity and quality for proteome analysis far outweighs the perceived
problems of differences between cell lines and primary clinical
material. Indeed, our characterization of three of the novel proteins
identified in this study, BCMP11, BCMP84, and BCMP101, indicates their
potential as targets for breast cancer therapy and/or diagnostic
markers of the disease. Furthermore, the discovery potential of this
data set (supplemental Table I) extends well beyond these three
examples to include many uncharacterized proteins associated with the
membranes of breast cancer cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1.2 and the normalized difference correlation score
0.2. For conservative identification of abundant proteins two peptides with
these scores were required, but for some of the novel lower abundance
proteins and low molecular weight proteins identified we accepted a
single tandem peptide with these scores as long as there were mass
matches on the same protein sequence from the same gel slice.
(12). Random and oligo(dT)-primed cDNA
libraries from human placenta poly(A)+ RNA and pooled
breast cancer-derived cell line (T47-D, MDA-MB-468, MCF-7, BT-20)
poly(A)+ RNA were constructed into the pP6 plasmid derived
from the original pACT2 plasmid (13) and transformed in
Escherichia coli (DH10B; Invitrogen). The complexity of the
primary libraries was more than 50 million clones. The libraries were
then transformed into yeast, and 10 million independent yeast colonies
were collected, pooled, and stored at
80 °C as equivalent aliquot
fractions of the same library.
-D-galactopyranoside overlay
assay. The interacting "prey" fragments of the positive clones were
amplified by PCR, analyzed on an agarose gel, and sequenced at their
5'- and 3'-junctions on a PE3700 sequencer. The resulting sequences
were then used to identify the corresponding gene in the GenBank data
base (NCBI).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Classification of proteins identified
from breast cancer cell membrane preparations. A,
proteins were classified into the five groups illustrated using the
data in supplemental Table I. The numbers indicate the
percentage fraction of identified proteins represented by each group.
B, examples of proteins identified in each of the membrane
categories shown in A. The breast cancer cell line pools in
which each protein was identified are indicated (MDA 468/BT474,
T47D/MCF-7, or both).
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Fig. 2.
MS/MS showing the
m/z 1,648.697 ion used to identify a
tandem peptide (SANAEDAQEFSDVER) representing BCMP84. The y and b
ion trails are shown.
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[in a new window]
Fig. 3.
DNA and protein sequence of BCMP11
(A), BCMP84 (B), and BCMP101
(C). Peptides assigned to BCMP11, BCMP84, and
BCMP101 are in bold and underlined; tandem
spectra are in bold and italicized.
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Fig. 4.
Cellular localization of BCMP11, BCMP84, and
BCMP101 breast cancer cell lines. A, fluorescence
microscopy showing apparent endosomal expression of transiently
transfected AFP-tagged BCMP11 in MDA-MB-468 (i) and T-47D
(ii) cell lines. B, fluorescence microscopy
showing expression of AFP-tagged BCMP101 in MDA-MB-468 (i)
and T-47D (ii) cell lines. Membrane localization is
indicated by white arrowheads (magnification, ×60 using oil
immersion objective). C, fluorescence microscopy showing
plasma membrane-associated expression of BCMP84 with both transiently
transfected AFP-tagged BCMP84 and immunofluorescence using a
BCMP84-specific antibody. AFP-tagged BCMP84 expression in MDA-MB-468
(i) and T-47D (ii) cell lines is shown
(magnification, ×60 using oil immersion objective). iii,
immunofluorescence microscopy of BCMP84 in the MDA-MB-468 cell line
(magnification, ×40). iv, AFP-tagged BCMP84 expression in
human mammary epithelial cells (magnification, ×60) showing solely
cytosolic localization.
1-catenin
(Swiss-Prot accession P35221) by yeast two-hybrid analysis.
1-Catenin protein was also identified in our breast cancer cell
membrane preparations (see supplemental Table I; plasma
membrane-associated accession P35221).
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Fig. 5.
Expression of BCMP11, BCMP84, and BCMP101 in
breast cancer tissue sections. Immunohistochemical analysis of
BCMP11 (A) and BCMP84 (B) protein in three
separate 1-mm core sections of breast invasive ductal carcinoma
sections (i-iii) is shown. Panels labeled
iv show high power microscopy, demonstrating cytoplasmic
staining of BCMP11 and plasma membrane-associated staining of BCMP84 in
ductal carcinoma epithelial cells. Panels labeled
v show expression in normal breast ductal epithelial cells
(a representative hematoxylin and eosin stained section of this tissue
is shown in vi). C, in situ RT-PCR
(i) and immunohistochemical analysis (iii) of
BCMP101 in breast cancer tissue sections are shown. A nonspecific
primer in situ control RT-PCR is shown on a consecutive
section to indicate levels of background amplification (ii).
Expression of BCMP101 mRNA and protein in carcinoma cells is
indicated by arrowheads. Real time RT-PCR quantification of
BCMP101 mRNA levels in breast tumor and adjacent normal tissues
from seven donors is also shown (tumor tissue is represented by
red bars, normal tissue is represented by blue
bars).
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Fig. 6.
Immunohistochemical analysis demonstrating
subcellular translocation of BCMP84 protein in tumor tissues.
i, BCMP84 immunostaining in breast ductal carcinoma (grade
3) tissue (arrows) and adjacent normal tissue
(arrowheads). Compare the strong plasma membrane-associated
staining in the tumor cells with the granular cytoplasmic staining in
the normal tissue. BCMP84 immunostaining in squamous cell tonsil
carcinoma (ii) and bladder papillary transitional cell
carcinoma (iii) is shown. Note the predominantly cytoplasmic
staining in the less well differentiated carcinoma cells, which becomes
distinctly plasma membrane-associated as the cells become more
differentiated (arrows in ii). All tissues were
counterstained with hematoxylin. Magnification, ×40.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-catenin.
1-Catenin is known to associate
with the cytoplasmic domains of multiple plasma membrane-localized cadherins and as such are thought to play an important role in cell-cell adhesion. Interestingly,
1-catenin is mutated in the invasive human colon cancer cell family HCT-8 and is therefore an
invasion suppressor gene in human colon cancer (24). We can speculate
that BCMP101 may play a role in breast tumor development by binding to
1-catenin and blocking its tumor suppressor functions.
proteins on the luminal
surface of Golgi membranes where it is thought to act as a major
calcium-binding protein (21, 31). The finding that BCMP84 can interact
with nucleobindin which in turn can bind G proteins in a
calcium-dependent manner provides a possible mechanism by
which BCMP84 can associate with the plasma membrane in cancer cells and
suggests a role for BCMP84 in G protein-coupled signal transduction
events. In support of this we have identified both nucleobindin and
G
i3 (and multiple other G proteins) in our breast cancer
cell line membrane protein preparations (see supplemental Table I). The
translocation of BCMP84 to the plasma membrane in carcinoma cells
exemplifies the potential of our proteome-driven approach as
disease-associated cellular translocation of a protein would not be
detected at the level of the transcriptome.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Table I.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY069977 (BCMP11).
The nucleotide sequences reported in this paper have been submitted to the Swiss Protein Database under Swiss-Prot accession number(s) Q8TD06 (BCMP11).
§ Both authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 1235-207-687; Fax: 1235-207-670; E-mail: jon.terrett@ogs.co.uk.
Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M210184200
2 R. Boyd, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: EGF, epidermal growth factor; AFP, autofluorescent protein; BCMP, breast cancer membrane protein; EST, expressed sequence tag; IM, integral membrane-associated; MALDI, matrix-assisted laser desorption ionization; MS/MS, tandem mass spectrometry; ORF, open reading frame; PA, plasma membrane-associated; PBS, phosphate-buffered saline; RT, reverse transcription; T, trans-plasma membrane; TOF, time of flight.
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