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J Biol Chem. 2008 September 26; 283(39): 26518–26527. doi: 10.1074/jbc.M803337200. | PMCID: PMC2546538 |
Copyright © 2008, The American Society for Biochemistry and
Molecular Biology, Inc. Cell Surface Proteomics Identifies Molecules Functionally Linked to Tumor
Cell
Intravasation *![[S with combining enclosing square]](/corehtml/pmc/pmcents/x0053x20DE.gif) Erin M. Conn,‡ Mark A. Madsen,‡ Benjamin F. Cravatt,‡ Wolfram Ruf,§ Elena I. Deryugina,‡ and James P. Quigley‡1 Departments of ‡Cell Biology and §Immunology, The Scripps Research Institute, La Jolla, California 92037 Received May 1, 2008; Revised July 11, 2008. In order to better understand the molecular and cellular determinants of
tumor cell intravasation, our laboratory has generated a pair of congenic
human HT-1080 fibrosarcoma variants (i.e. HT-hi/diss and HT-lo/diss)
differing 50–100-fold in their ability to intravasate and disseminate.
To investigate the molecular differences underlying the distinct dissemination
capacities of these HT-1080 variants, we performed a comparative analysis of
the cell surface proteomes of HT-hi/diss and HT-lo/diss. Cell membrane
proteins were enriched by biotinylation and avidin precipitation and analyzed
by tandem mass spectrometry employing multidimensional protein identification
technology. By this approach, 47 cell surface-associated molecules were
identified as differentially expressed between the HT-1080 intravasation
variants. From these candidates, four targets (i.e. TIMP-2, NCAM-1,
JAM-C, and tissue factor (TF)) were selected for further biochemical
validation and in vivo functional verification. Western blot analysis
of the cell surface enriched fractions confirmed the proteomic array data,
demonstrating that, in vitro, TIMP-2 protein was increased in the
HT-lo/diss variant, whereas NCAM-1, JAM-C, and TF levels were increased in the
HT-hi/diss variant. Corresponding in vivo differences in levels of
TIMP-2, JAM-C, and TF were demonstrated in primary tumors grown in the chick
embryo. Finally, functional inhibition of one selected protein (i.e.
TF) by small interfering RNA silencing or ligation with a function-blocking
antibody significantly reduced HT-hi/diss intravasation, thus clearly
implicating TF in the early steps of tumor cell dissemination. Overall, our
cell surface proteomic analysis provides a powerful tool for identification of
specific cell membrane molecules that contribute functionally to intravasation
and metastasis in vivo. One of the early and possibly rate-limiting steps in cancer progression
from a localized tumor to systemic metastatic disease is intravasation
( i.e. the entry of metastatic cells into the vasculature)
( 1– 5).
Molecules involved in intravasation represent attractive therapeutic targets,
since preventing or inhibiting this process would confine tumor cells to their
primary site and provide a more focused target for clinical intervention
( 6). To identify cellular
attributes that functionally contribute to tumor cell intravasation and
metastasis, including escape from the primary site, invasion of local stoma,
and entry into the vasculature, we have employed a pair of congenic human
fibrosarcoma HT-1080 cell variants, differing 50–100-fold in their
ability to intravasate and disseminate (HT-hi/diss and HT-lo/diss) while
having similar capacities to form primary tumors
( 7). These cell variants
display a distinct differential during spontaneous metastasis but behave
comparably in experimental metastasis models where cells are inoculated
intravenously and only the later steps of the metastatic cascade are
recapitulated. Therefore, comparative analysis of the HT-hi/diss and
HT-lo/diss variants can be useful for identification of molecules specifically
contributing to early metastatic events. Previously, we have employed activity-based protein profiling
( 8) to identify molecules that
might underlie the differential intravasation potential of the HT-1080 cell
variants. This proteomic approach implicated urokinase activation as a key
step in HT-hi/diss dissemination
( 9). Since many groups of
proteins functionally linked to cancer progression are cell surface molecules,
such as growth factor receptors, transmembrane signaling molecules, and
cell-cell or cell-matrix adhesion proteins, we suggested that HT-hi/diss and
HT-lo/diss might differentially express cell surface molecules that facilitate
tumor cell intravasation and contribute to early steps of cancer
dissemination. Membrane-tethered proteins are present in relatively low abundance and
therefore are often overlooked or not identified in broad spectrum, whole
cell, or tissue arrays. Cell surface biotinylation followed by avidin
precipitation is a widely used method to enrich membrane proteins
( 10– 14).
One major caveat of this approach is a high level of nonspecific intracellular
protein contamination in avidin pull-downs. Our initial attempt with a
commercially available cell surface labeling kit (Pierce) was disappointing,
since it yielded an overwhelming number of known intracellular proteins but
few cell surface molecules. Several previous studies involving gel-based
detection for protein identification have also been hampered by limited
sensitivity of the method
( 12– 14). To increase the specificity and sensitivity of the cell surface proteomic
approach, we have introduced essential modifications to standard cell labeling
procedures and used a non-gel mass spectrometry approach employing
multidimensional protein identification technology
(MudPIT) 2
( 15– 17).
This approach was used to identify proteins differentially expressed between
the tumor cell intravasation variants by comparing the cell surface proteomes
of HT-hi/diss and HT-lo/diss. To link the proteomic data to the process of
actual in vivo metastasis, we selected several candidate proteins
that were identified by the array as being enriched in one cell variant over
the other and verified the differential levels of the selected candidates in
cell lysates and primary tumors by Western blotting. Finally, we analyzed the
functional role of one of the identified proteins, tissue factor (TF), in
HT-1080 intravasation by employing the human tumor-chick embryo spontaneous
metastasis model. In this assay, the ability of human tumor cells to
intravasate is determined by quantifying the number of human cells arrested in
the chorioallantoic membrane (CAM), which serves as a repository of cells that
have escaped from primary tumors and entered the vasculature
( 18,
19). By down-regulating TF
function via siRNA silencing or ligation with a function-blocking antibody, we
have demonstrated that TF positively contributes to HT-hi/diss intravasation,
thereby validating our cell surface proteomic approach. Cell Surface Protein Labeling, Enrichment, and Digestion—A
schematic of the labeling and enrichment procedure is presented in
. HT-hi/diss and
HT-lo/diss cells, generated from the HT-1080 human fibrosarcoma cell line
( 7), were grown in Dulbecco's
modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum
(Hyclone, Logan, UT) and 10 μg/ml gentamicin (Invitrogen) (D-10). Cells
were harvested by trypsinization and seeded in D-10 at 4 ×
10 6 cells/150-mm dish to achieve 90–100% confluence 24 h
later. Cell layers were then washed three times with ice-cold PBS, 1
m m CaCl 2, 1 m m MgCl 2 (PBS-Ca/Mg)
and then incubated in 0.2 mg/ml EZ-Link Sulfo-NHS-LC-Biotin (Pierce) for 45
min at room temperature with gentle swirling every 10 min. Nonbiotinylated
control plates were incubated in PBS-Ca/Mg alone. After washing three times
with TBS-Ca/Mg (pH 7.4) to quench and remove remaining biotinylation reagent,
the cells were scraped in TBS, pelleted by centrifugation, and lysed in 50
m m octyl-β- d-glucopyranoside in TBS-Ca/Mg
supplemented with protease inhibitors (aprotinin, leupeptin, and pepstatin,
each at 10 μg/ml, and phenylmethylsulfonyl fluoride at 1 m m) for
30 min at 4 °C with rocking. Lysates were clarified by centrifugation at
1,000 × g for 2 min and mixed with 300 μl of avidin beads
(Sigma). After incubation for 1 h at room temperature while rotating, the
beads were pelleted by centrifugation at 200 × g for 3 min and
washed three times with TBS-Ca/Mg, three times with 4 m urea in
TBS, and finally three times with 50 m m Tris (pH 7.4). After
pelleting at 200 × g for 5 min, the beads were resuspended in
300 μl of freshly prepared 8 m urea in 50 m m Tris.
The eluted proteins were then reduced with 5 m m
Tris(2-carboxyethyl)phosphine for 30 min at room temperature and alkylated
with 10 m m iodoacetamide for 30 min at room temperature in the
dark. Next, the proteins were digested with 0.4% trypsin (sequencing grade) in
50 m m Tris supplemented with 10.5 m m CaCl 2 at
37 °C for 12 h. Samples were spun at 10,000 × g for 4 min,
the peptide-containing supernatant was removed, and formic acid was added to a
final concentration of 5%. Identification of Cell Surface Proteins by MudPIT
Analysis—To identify biotinylated cell surface proteins enriched by
the avidin precipitation, the digested peptide mixtures were loaded onto a
biphasic (strong cation exchange/reverse phase) capillary column and analyzed
by two-dimensional liquid chromatography in combination with tandem mass
spectrometry (MS) as described
( 15,
16). Briefly, a fused silica
capillary (100-μm inner diameter) was pulled using a CO 2-based
laser puller to make a fritless nanospray column. This column was packed with
10 cm of 5-μm C 18 reverse phase particles (Aqua 5 μ c18;
Phenomenex) followed with 3 cm of 5-μm strong cation exchange particles
(Partisphere 5 SCX; Whatman, Clifton, NJ). The peptide digest containing
100–500 μg of protein was loaded onto the SCX portion of the column,
displaced with a stepwise salt gradient of KCl in 5% acetonitrile (pH 3), and
reloaded onto the reverse phase portion of the column. The peptides were
eluted from the reverse phase portion of the column after each salt step and
analyzed in the mass spectrometer (Finnigan LTQ; Thermo Electron Corp.,
Waltham, MA) using a linear gradient of 80% acetonitrile in 0.5% acetic acid.
The mass spectrometer was set in data-dependent acquisition mode, where one
full MS scan is followed by MS/MS scans of the seven most abundant unique
peptides. The MS/MS data were then searched against the most recent version of
the human IPI data base using the SEQUEST search algorithm
( 20) to identify the proteins
present in the sample. The relative abundance of the peptides was estimated by
spectral counting, with the following criteria set for a peptide to be
included in the analysis: cross-correlation score greater than 1.8 (+1), 2.5
(+2), 3.5 (+3), and δ correlation greater than 0.08
( 21,
22). Additionally, to ensure
accuracy, only those proteins that were identified by two or more independent
peptides were included in the final comparison. Silver Staining—Proteins were separated by SDS-PAGE on
4–20% Tris-glycine gels under reducing conditions. The gels were fixed
for 2 h in a mixture of 50% methanol, 12% acetic acid, and 0.05% formaldehyde,
washed three times in 35% ethanol for 20 min each, and sensitized in 0.02%
sodium thiosulfate for 2 min. Gels were washed three times with water for 5
min each, stained for 20 min with 0.2% silver nitrate and 0.076% formaldehyde,
followed by two washes in water (1 min each), and developed with a solution of
6% sodium carbonate, 0.05% formaldehyde, 0.0004% sodium thiosulfate. Once the
gels were sufficiently stained, the developing reaction was stopped with a
solution of 50% methanol and 12% acetic acid. Finally, the gels were imaged
with an Alpha Imager 2000 (Alpha Innotech, San Leandro, CA). Quantitative Chick Embryo Spontaneous Metastasis
Assay—Fertilized White Leghorn eggs were incubated at 37 °C in
a rotary incubator with 60% humidity. On day 10 of incubation, a portion of
the eggshell was shaved off to expose the shell membrane. The CAM was
separated from the shell membrane using mild suction through a hole in the air
sac of the egg. A window was created in the eggshell, and 0.25–0.5
× 106 HT-hi/diss or HT-lo/diss cells were applied to the
dropped CAM. The windows were sealed with tape, and the embryos were returned
to a stationary incubator to allow for primary tumor development and
dissemination of cells from the primary tumors. After 5 days, the embryos were
sacrificed and primary tumors were excised, weighed, and frozen for
biochemical analysis. Distal portions of the CAM were also harvested and
frozen at -80 °C to determine the number of intravasated human cells by
Alu-qPCR (described below). Western and Avidin-Horseradish Peroxidase Blot
Analysis—HT-hi/diss and HT-lo/diss cells were lysed in
octyl-β- d-glucopyranoside buffer as described above. Primary
CAM tumors were lysed by mechanical dissociation with scissors in modified
radioimmune precipitation assay buffer containing protease inhibitors
(aprotinin, leupeptin, and pepstatin, each at 10 μg/ml, and
phenylmethylsulfonyl fluoride at 1 m m), followed by incubation
under agitation for 30 min at 4 °C. The lysates were clarified by
centrifugation at 6,000 × g for 15 min. Protein concentrations
of the cell and tumor lysates were determined using the BCA™
(bicinchoninic acid) protein assay kit (Pierce). Equal amounts of HT-hi/diss
or HT-lo/diss cell lysates, tumor lysates (10–40 μg), or cell surface
enriched eluates were resolved on 4–20% SDS-polyacrylamide gels and
transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore,
Billerca, MA). After transfer, the membranes were blocked with 5% nonfat milk
in PBS plus 0.05% Tween 20 (PBS-T) for 1 h. Membranes were probed overnight at
4 °C with the following primary antibodies: murine anti-human TIMP-2
(Oncogene, Cambridge, MA), goat anti-human TF (isolated in the laboratory of
Dr. W. Ruf), murine anti-human NCAM-1 (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA), and goat anti-human JAM-C (Santa Cruz Biotechnology). To control
for variability of human cell numbers within individual CAM tumors and to
ensure that similar quantities of human protein were loaded, lysates were
probed with murine mAb 29-7, which recognizes human CD44 on HT-hi/diss and
HT-lo/diss but does not cross-react with chick tissue
( 7). After incubation with
primary antibodies, the membranes were washed three times with PBS-T and then
probed with horseradish peroxidase-conjugated secondary antibodies (anti-goat
IgG from Vector Laboratories (Burlingame, CA) or anti-mouse IgG from Bio-Rad)
in PBS-T/nonfat milk for 2 h. After washing three times in PBS-T,
immunoreactive bands were visualized with SuperSignal West Pico
Chemiluminescent Substrate (Pierce) and quantified with a Molecular Imager Gel
Doc XR System (Bio-Rad). Down-regulation of TF—Down-regulation of TF was achieved by
siRNA silencing and by function-blocking antibody ligation. Two 19-mer siRNA
sequences were generated against TF mRNA (Qiagen, Valencia, CA): TF-si167
(sense, GCGCUUCAGGCACUACAAA; antisense, UUUGUAGUGCCUGAAGCGC)
( 23) and TF-si1086, which
recognizes the 3′-untranslated region (sense, GGAAACGCAAAUGAGUAUU;
antisense, AAUACUCAUUUGCGUUUCC). The nonsilencing scrambled sequence of
TF-si167 ( i.e. scr-si167) was used as a control (sense,
GCGUCUAGACGUCCACAAA; antisense, UUUGUGGACGUCUAGACGC). A BLAST-n search was
performed to confirm that siRNA sequences did not cross-react with other known
mRNA sequences. For siRNA transfections, 1.75 × 10 6
HT-hi/diss cells were plated in 10-cm dishes in D-10 without antibiotics.
After 24 h, the 70–85% confluent cultures were transfected with 20
n m TF-si167, TF-si1086, scr-si167, or no siRNA (mock) with 9 μl
of Lipofectamine 2000 (Invitrogen). Following overnight incubation, the cells
were detached with trypsin/EDTA, washed in D-10, and resuspended in serum-free
Dulbecco's modified Eagle's medium. A total of 0.25–0.4 ×
10 6 cells were grafted onto the CAM of day 10 chick embryos.
Down-regulation of TF was confirmed by Western blotting in cell lysates 24 h
after siRNA transfection and in primary tumors harvested 5 days after grafting
of tumor cells. The TF function-blocking antibody 5G9
( 24) was introduced either
mixed with the cells prior to CAM grafting or by daily topical application to
developing tumors at 25 or 50 μg in 0.1 ml of PBS plus 5% DMSO/embryo. Five
days after cell grafting, the embryos were sacrificed, primary tumors were
weighed, and distal portions of CAM were frozen for genomic DNA extraction and
Alu-qPCR analysis. Quantitative Analysis of Tumor Cell Intravasation by
Alu-qPCR—Genomic DNA was extracted from distal CAM samples using
the Gentra PureGene system (Qiagen). To determine actual numbers of human
cells contained in individual samples, 60 ng of genomic DNA was used for
quantitative PCR using primers recognizing primate-specific Alu
repeat sequences and SYBR green double-stranded DNA binding dye for
quantification, as previously described
( 19,
25). The qPCR was carried out
in a Bio-Rad MyIQ light cycler. The Ct values were converted into
numbers of human cells using a standard curve generated by spiking constant
numbers of chicken cells with serial dilutions of HT-1080 cells. Cell Surface Protein Enrichment and Identification by Mass
Spectrometry—To compare the cell surface protein profiles of the
highly disseminating (HT-hi/diss) and low disseminating (HT-lo/diss) variants
of the parental HT-1080 cell line, an enrichment procedure was necessary,
since cell surface proteins are present in relatively low abundance compared
with the proteins in the intracellular pool. Enrichment was accomplished by
biotinylation of cell monolayers using EZ-Link Sulfo-NHS-LC-Biotin and
precipitation of the biotin-labeled proteins with avidin beads. Cell
monolayers incubated with PBS instead of the biotinylation reagent underwent
the same avidin precipitation procedure to serve as nonlabeled controls. Since
nonspecific binding of intracellular proteins to avidin beads represents a
frequent problem associated with surface protein enrichment, we increased the
stringency of the washes after avidin precipitation by incubating the beads in
urea. To control for stringency of the wash step, we compared three different
conditions, employing 4 m urea, 6 m urea, or PBS.
Western blotting with avidin-horseradish peroxidase was performed to estimate
amounts of biotinylated material eluted after each wash condition. The
resulting blot showed no decrease in the yield or distribution of biotinylated
proteins after washing the beads with 4 or 6 m urea as compared
with PBS, thus indicating that the urea wash did not cause significant
displacement of biotinylated proteins from the avidin beads. However, the
stringent washes with urea substantially reduced the total amount of proteins
pulled down in the nonbiotinylated control (data not shown). Since washing
with 4 and 6 m urea resulted in a similar decrease of nonspecific
protein binding, the stringent wash step with 4 m urea was used
during sample preparation for mass spectrometry. To identify the most abundant proteins and compare the surface protein
expression profiles of the intravasation variants, the cell surface enriched
protein samples from HT-lo/diss and HT-hi/diss were analyzed by MudPIT. To
subtract highly abundant intracellular proteins precipitated nonspecifically,
we included a sample containing HT-hi/diss proteins that had undergone the
same enrichment procedures but were not biotinylated. Since this sample
included only those proteins that bound nonspecifically to the avidin beads,
these molecules were excluded from analysis, further narrowing the pool of
proteins identified as cell surface molecules. A list of the proteins
identified in the biotinylated samples in either one or both of the HT-1080
variants is presented in supplemental Table 1. Ultimately, by using this proteomic array, a total of 168 proteins were
identified as differentially expressed between the HT-1080 intravasation
variants. Despite our efforts to increase stringency and to exclude
nonspecifically bound proteins from analysis, approximately two-thirds of the
resulting proteins were intracellular or secreted molecules. However, among
the proteins identified as true cell surface molecules, 26 and 21 were more
abundant in HT-hi/diss and HT-lo/diss, respectively. A list of these proteins
and their UniProt accession numbers is presented in
Table 1. Known functions of
some of these proteins include cell-cell or cell-matrix adhesion, signaling in
response to cytokines or growth factors, induction of coagulation, and
proteolysis ( i.e. all processes linked to metastasis). | TABLE 1Cell surface proteins identified by LC-MS/MS with abundance differences
between HT-hi/diss and HT-lo/diss Percent coverage, UniProt accession
numbers, and summarized known functions are as indicated. Four molecules
(underlined) were selected to verify (more ...) |
Confirmation of Abundance Differences of Cell Surface Proteins in
Vitro—To validate the findings of the proteomic array, we analyzed
by Western blotting the abundance differences of selected cell surface
proteins between HT-lo/diss and HT-hi/diss cells cultured in vitro.
TIMP-2, one of the tissue inhibitors of matrix metalloproteinases, was
identified by LC-MS/MS as more abundant in HT-lo/diss
( Table 1) and was selected for
biochemical validation because of its known role in tumor cell invasion and
dissemination ( 26). Three
proteins that were demonstrated to be more abundant in HT-hi/diss
( Table 1) were also chosen for
further analysis: NCAM-1 (neural cell adhesion molecule-1), which modulates
cell to cell adhesion ( 27);
JAM-C (junctional adhesion molecule-C), known to be involved in leukocyte and
potentially tumor cell transendothelial migration
( 28– 30);
and TF, a surface receptor that initiates the coagulation cascade by binding
the secreted coagulation factor VII at the cell surface
( 31– 34).
The levels of these molecules in cell surface protein enriched eluates and in
whole cell lysates were further assayed by comparative Western blot
analysis. To compare the relative total protein content between the surface enriched
eluates from HT-hi/diss and HT-lo/diss and to analyze the protein content of
the nonbiotinylated control samples pulled down nonspecifically with avidin
beads, an equal volume of each fraction was resolved on an SDS-polyacrylamide
gel and silver-stained to determine the relative protein content of these
fractions ().
The resulting gel allowed for visualization of surface enriched proteins from
HT-hi/diss and HT-lo/diss cells and indicated that the HT-lo/diss fraction had
a slightly higher protein content. The nonlabeled controls exposed to the same
enrichment procedure did not contain detectable levels by silver staining of
avidin-precipitated proteins, indicating high efficiency of the biotinylation,
avidin precipitation, and 4 m urea stripping procedures. Equal
amounts of the HT-hi/diss and HT-lo/diss biotinylated cell surface enriched
fractions, along with whole cell lysates from HT-lo/diss and HT-hi/diss, were
resolved on SDS-polyacrylamide gels for probing with antibodies to the
selected proteins (). We observed that TIMP-2 levels were increased in both the surface fraction
and total cell lysates of HT-lo/diss (), the latter being in agreement with our previous
findings ( 25). Consistent with the observed LC-MS/MS differential of NCAM-1 between the
HT-1080 variants ( Table 1),
Western blot analysis of the cell surface protein pool demonstrated that
NCAM-1 levels were ~2-fold greater on the surface of HT-hi/diss compared
with HT-lo/diss cells, although the total cell content of NCAM-1 was similar
between the two variants (). Surface enriched protein fractions also manifested 1.7-fold higher levels
of JAM-C in HT-hi/diss than in HT-lo/diss
(), thus
confirming the LC-MS/MS data ( Table
1). Levels of JAM-C in the total cell lysates also differed
(1.3-fold) between the two cell lines, albeit not as dramatically as in the
surface fraction. There was a slight difference in the apparent molecular
weight of this protein between the cell surface pool and the whole cell
lysate, possibly reflecting a different glycosolation pattern in cell surface
JAM-C, as this protein has two potential N-linked glycosolation sites
( 35). Among the selected proteins, TF represented a molecule exhibiting the
highest protein expression differential between the HT-1080 intravasation
variants. In the cell surface fractions, TF protein levels were dramatically,
10.8-fold, higher in HT-hi/diss as compared with HT-lo/diss and also 2-fold
higher in the HT-hi/diss total cell lysates over HT-lo/diss
(). Confirmation of Protein Abundance Differences in Primary
Tumors—Relative in vivo protein levels of TIMP-2, NCAM-1,
JAM-C, and TF were next analyzed in primary tumor xenografts. HT-hi/diss and
HT-lo/diss cells were grafted on the CAM of day 10 embryos and allowed to form
primary tumors for 5 days, at which time the tumors were harvested and lysed.
Lysates from individual CAM tumors were resolved by SDS-PAGE and transblotted,
and membranes were immunoprobed for TIMP-2, NCAM-1, JAM-C, and TF. TIMP-2
levels were higher in the HT-lo/diss tumors as compared with the HT-hi/diss
tumors (), thus
confirming the expression differential of this molecule demonstrated in the
cells cultured in vitro. The HT-hi/diss and HT-lo/diss primary tumor
lysates contained comparable amounts of NCAM-1 protein
(), which was in
agreement with the similar levels of NCAM-1 between the two variants detected
in total cell lysates of in vitro cultures. JAM-C protein levels were
higher in HT-hi/diss CAM primary tumors compared with the HT-lo/diss
counterparts (),
consistent with the differential indicated by the proteomic array and Western
blot analyses of both whole cell lysates and cell surface enriched fractions
(). The most
dramatic in vivo observation, however, was the average 4.2-fold
difference in TF protein levels between HT-hi/diss and HT-lo/diss primary
tumors (), reaffirming
its pronounced differential determined by the proteomic approach in cultured
cells (10.8-fold differential between the cell surface eluates). This finding,
along with the documented role of TF in tumor progression (reviewed in Ref.
34) prompted us to modulate
the function of this protein in vivo to verify its contribution to
tumor cell intravasation. Down-regulation of Tissue Factor Protein Decreases
Intravasation—In order to functionally link the increased
expression of TF to enhanced intravasation and dissemination of HT-hi/diss, we
took two approaches to decrease TF function in vivo:(a)
down-regulation of TF expression by siRNA interference and (b)
inhibition of TF with a function-blocking antibody. One of the advantages of the chick embryo metastasis assay is that
intravasation can be quantified as early as 4–5 days after tumor cell
grafting (i.e. when transient siRNA transfections still continue to
repress gene expression). TF expression was down-regulated in HT-hi/diss using
two independent siRNA constructs, TF-si167 and TF-si1086. Control cells were
mock-transfected (Lipofectamine alone) or transfected with a nonsilencing
scrambled construct (scr-si167). Both TF-specific siRNA constructs decreased
TF protein expression to 36–53% of control levels 24 h after
transfection (), at which time the TF-si167-, TF-si1086-, scr-si167-,
or mock-transfected cells were inoculated onto the CAMs of day 10 chick
embryos. To confirm that down-regulation of TF persisted until the time of
tumor harvest, individual primary tumors were lysed to analyze in
vivo TF expression by Western blotting. As can be seen in
, protein
levels of TF were still reduced 5 days after cell grafting on the CAM in
TF-siRNA primary tumors compared with control tumors. Overall, the siRNA
treatment did not significantly affect primary tumor growth
(,
top). Whether intravasation of HT-hi/diss was affected by down-regulation of TF
was determined by analysis of distal portions of the CAM using
Alu-qPCR to detect and quantify intravasated tumor cells. The numbers
of human cells in the CAM vasculature was significantly reduced in TF-si167
and TF-si1086 transfectants to 63 and 50% of control levels, respectively,
whereas nonsilencing scr-si167 did not have a significant effect on
intravasation (, bottom). Thus, it appears that the increase
in TF protein expression in HT-hi/diss facilitates tumor cell
intravasation. To further analyze the involvement of TF in tumor cell dissemination and to
control for any potential off-target effects of the siRNA transfection, we
turned to another method of blocking protein function, namely the inhibition
by a specific function-blocking antibody. We employed mAb 5G9, which
efficiently prevents TF from initiating the coagulation cascade at the cell
surface interface ( 24). This
function-blocking antibody was applied topically to developing CAM tumors
daily or introduced with the cells at the time of grafting onto the CAM. Since
both antibody treatment strategies produced similar inhibitory effects, the
data from these experiments were pooled to calculate fold changes over a mouse
IgG control. The TF function-blocking mAb 5G9 decreased HT-hi/diss
intravasation to 51% of control levels
(,
bottom) without significantly affecting primary tumor growth
(,
top). Thus, both methods of down-regulating TF ( i.e. by
decreasing expression at the message level or blocking function by specific
antibody ligation) reduced HT-hi/diss intravasation. To study tumor cell intravasation and dissemination, our laboratory has
generated a pair of congenic variants of the human fibrosarcoma HT-1080 cell
line ( i.e. HT-hi/diss and HT-lo/diss), which exhibit a
50–100-fold differential in their ability to spontaneously metastasize.
Importantly, both cell types display similar abilities to form primary tumors
in spontaneous metastasis and to colonize secondary organs after intravenous
injection in experimental metastasis, suggesting that their substantial
difference in tumor dissemination must lie in their ability to successfully
complete early steps in the metastatic cascade
( 7). We have taken multiple
approaches to investigate the nature of this metastasis differential,
including activity-based protein profiling
( 9), MMP expression profiling
( 25), cDNA and cytokine arrays
(data not shown), and the cell surface proteomic approach described herein.
Alterations in expression and function of cell surface proteins have long been
associated with cancer progression which is not surprising, since many growth
factor and cytokine receptors, adhesion molecules, and proteases or protease
receptors are functionally active at the cell membrane
( 36). We therefore focused
this study on identifying in vitro surface proteins differentially
expressed between the HT-1080 dissemination variants and validating in
vivo the functional impact of selected protein abundance differences. In this study, several modifications were introduced to standard cell
surface protein enrichment procedures in order to increase specificity and
decrease the contamination with nonsurface proteins
(). Analysis of the
resulting surface proteins from HT-lo/diss and HT-hi/diss by LC-MS/MS provided
means to compare their surface proteomes and identify proteins with potential
abundance differences between the intravasation variants. Ultimately, we
identified 26 known cell surface proteins with higher abundance in HT-hi/diss
and 21 known cell surface-associated proteins with higher abundance in
HT-lo/diss. It should be kept in mind that the identification by this analysis
of a particular protein in only one cell variant does not indicate the absence
of that protein in the other variant but points to a difference in surface
expression levels of the protein between the two tumor cell
counterparts. Cell surface protein enrichment combined with MudPIT provided a valuable
tool for identifying proteins with differential expression between the cell
dissemination variants and, therefore, with potential roles in metastasis.
Specifically, we focused on one protein that was identified by LC-MS/MS as
more abundant in HT-lo/diss ( i.e. TIMP-2) and three proteins
identified as more abundant in HT-hi/diss ( i.e. JAM-C, NCAM-1, and
TF) and confirmed their differentials by Western blot analyses in both whole
cell lysates and surface protein enriched fractions. It has been documented
that tumor cells cultured in the presence of fetal calf serum, which contains
an abundance of growth factors and cytokines, often have protein expression
profiles different from those of primary tumors in vivo
( 37). Therefore, we sought to
confirm that the differentials in surface protein expression identified by
LC-MS/MS in cultured cells were also manifested within the tumor
microenvironment and would have functional significance during actual
intravasation and dissemination of the tumor cells in vivo. To this
end, we next validated in vivo that our method of in vitro
labeling and surface protein enrichment can be exploited to identify protein
differentials with functional relevance for metastasis. This was accomplished
by probing primary tumors for the selected proteins and by in vivo
modulating the expression and function of TF, our most attractive candidate
protein. By Western blot analysis, we confirmed that the MMP inhibitor TIMP-2,
identified by LC-MS/MS as more abundant in HT-lo/diss was present at higher
levels by Western blot analysis in HT-lo/diss surface fractions, whole cell
lysates, and primary tumors as compared with HT-hi/diss. Previously, high
levels of TIMP-2 in HT-lo/diss were linked to their reduced ability to
intravasate ( 25), affirming
the functional relevance of this protein identified by our cell surface
proteomic approach. Although TIMP-2 is a secreted protein, it can be detected
at the cell surface due to its binding with MT1-MMP
( 38,
39). Since both HT-lo/diss and
HT-hi/diss express MT1-MMP ( 7,
25), the presence and
detection of TIMP-2 was not unexpected and probably represented the
MT1-MMP-bound pool of TIMP-2. In accordance with this, MT1-MMP was detected by
the proteomic array in the surface fraction of both cell variants, but since
it was also detected in the nonbiotinylated control, MT1-MMP was not listed in
supplemental Table 1. The confirmatory finding that TIMP-2 was more abundant
in HT-lo/diss by Western blotting provided a validation of our approach to
cell surface proteomics by LC-MS/MS. However, for further investigation, we
chose to focus on proteins identified as more abundant in HT-hi/diss, since
these proteins would represent targets for in vivo down-regulation to
directly assess their functional involvement in intravasation. Western blot analysis confirmed the proteomic array data demonstrating that
JAM-C, NCAM-1, and TF were significantly more abundant in HT-hi/diss than in
HT-lo/diss. In each case, these proteins were immunologically detected in
greater quantities in the cell surface protein fractions of HT-hi/diss as
compared with HT-lo/diss. Interestingly, in the case of NCAM-1, protein levels
in the cell surface enriched eluate differed 2-fold between the two variants,
whereas total lysates of cells cultured in vitro and primary tumors
grown in vivo had similar levels of this protein. A difference
specifically in surface NCAM-1 might represent a difference in cellular
trafficking or turnover of the surface pool (e.g. by degradation or
shedding) between the variants. This result indicates that the described cell
surface proteomic approach can be used to identify differentials in cell
surface pools even when total cellular contents of a specific protein are
similar and also highlights the importance of identifying aberrant or
differential subcellular localization and not just total protein expression of
potentially important targets. Thus, the specific difference in
membrane-associated NCAM-1 identified by cell surface proteomics in this
study, for example, would be missed by the more global arrays analyzing total
cellular proteins. Another protein that has been identified by our array as enriched in
HT-hi/diss is a transmembrane adhesion molecule, JAM-C, known to interact with
the endothelium during leukocyte transendothelial migration
( 28,
29). JAM-C is an intriguing
target, since several parallels can be drawn between tumor cell intravasation
and leukocyte transendothelial migration in terms of interactions with the
endothelium. In this regard, a recent study has reported that down-regulation
of JAM-C by siRNA inhibited dissemination of HT-1080 fibrosarcoma cells in a
mouse experimental metastasis model
( 40), implicating JAM-C in the
late stages of metastasis. We observed a difference in JAM-C protein
expression between the HT-lo/diss and HT-hi/diss intravasation variants in
primary tumor samples as well as in cell surface enriched fractions,
suggesting that JAM-C might also have a functional role in the early steps of
metastasis. Importantly, the difference in JAM-C levels between the HT-lo/diss
and HT-hi/diss variants was most pronounced in the cell surface enriched
fraction. Since JAM-C functions at the cell surface where ligand binding
occurs, our findings highlight an important difference in the specific
functional pool of this cell surface protein, which again might be overlooked
if only the total cell lysates are analyzed. A direct role of JAM-C in the
early steps of tumor dissemination is currently under investigation. The most dramatic difference between HT-lo/diss and HT-hi/diss observed in
this study was the up to 10.8-fold differential in TF levels in the surface
enriched protein fractions, whole cell lysates, and primary tumors. Since TF
is the major initiator of the coagulation cascade, increased TF expression has
long been associated with a hypercoagulable state frequently observed in
advanced, metastatic cancer
( 41). This association has
proved to be functionally relevant both for primary tumor growth and late
stages of metastasis as cells overexpressing TF become more angiogenic and
metastatic through TF-mediated thrombin generation, fibrin deposition, and
platelet activation. Additionally, TF can induce signaling events mediated by
protease-activated receptor-2 and, thus, may contribute to cancer progression
independently of coagulation
( 34,
42,
43). Tumor growth appears to
be facilitated by TF expression
( 34) via mechanisms that may
involve PAR-mediated signaling events
( 42). In addition, recent
evidence has implicated TF-induced signaling in cell migration
( 50,
51), a process critical for
cancer cell dissemination. In experimental metastasis models, which bypass
intravasation and recapitulate late stages of the metastatic cascade,
overexpression of TF in tumor cells enhanced colonization, whereas inhibition
of TF function correspondingly decreased experimental metastasis
( 24,
44– 49). Despite the documented role of TF in tumor growth, angiogenesis, and late
stages of metastasis, its role in the early process of tumor cell
intravasation has not yet been elucidated. Therefore, we investigated the
functional role of TF in intravasation using an in vivo spontaneous
metastasis model. To this end, TF function was modulated in HT-hi/diss by
down-regulating TF expression with TF-specific siRNA and by inhibiting protein
function with a TF function-blocking antibody. Both approaches to
down-regulate TF function substantially reduced HT-hi/diss intravasation
without affecting primary tumor development, thus implicating TF as a possible
critical factor in early steps of tumor cell dissemination. These findings are
consistent with previous reports linking TF expression to tumor progression
and also validate our proteomic method for identifying functionally relevant,
metastasis-associated protein targets. In summary, our cell surface proteomic array generated a list of 47 surface
proteins with abundance differences between the two congenic HT-1080
intravasation variants. Confirmation of the LC-MS/MS data was accomplished by
analyzing levels of selected proteins in the two variants by a second
independent method, Western blotting, performed with cells cultured in
vitro and primary tumors grown in vivo. Differential expression
at the cell surface was demonstrated between the variants for all selected
proteins, whereas differences in total protein contents were more restricted.
Importantly, selected proteins indicated by cell surface proteomics have been
implicated in vivo in spontaneous metastasis, since TIMP-2, JAM-C,
and TF were each differentially expressed between the two phenotypically
distinct variants in primary tumors. Functional modulation of one of the
selected proteins (i.e. TF) substantially decreased intravasation of
the HT-hi/diss variant. Overall, our approach to enrich and purify cell
surface proteins allowed us to identify by LC-MS/MS several specific molecules
that are related to tumor cell intravasation and represent attractive targets
for inhibition of tumor cell dissemination and metastasis. We thank Chenxing Li, Lauren Hayden, and David Balser for excellent
technical assistance and Sherry Niessen for assistance with the MudPIT
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