I. Introduction
Breast cancer is the most frequently
diagnosed cancer and the second leading cause of cancer deaths in American
women today. It is estimated that there will be 212,920 new cases of breast
cancer and 48,970 breast cancer deaths in American women in the year 2006
(Jemal et al, 2006). Metastasis, the spread of tumor cells from the primary
site to a distant organ to form a secondary tumor, is a major cause of deaths
of breast cancer patients (Marshall, 1993). The prevention of tumor metastasis
is one of the most important challenges in the design of therapies for patients
with malignancies. The metastatic spread of a primary tumor to distant organs
involves a series of predictable processes that include tumor cell detachment
from the primary tumor, migration and invasion through the basement membranes
of blood and lymph vessels, embolization, arrest and binding to vascular
endothelium at secondary organs, extravasation, and invasion of the secondary
organ. In these processes, cell motility is one of the essential cellular
functions that plays an important role in tumor metastasis (Miyake and
Hakomori, 1991).
Motility related protein-1 (MRP-1) was
originally recognized, and thus isolated by a monoclonal antibody that inhibits
cell motility and tumor cell metastasis (Miyake and Hakomori, 1991; Miyake et
al, 1991). The sequence of MRP-1 protein and
cDNA revealed that it is an identical gene to CD9, a cell surface antigen
initially discovered on B lineage leukemic cells and cloned by two different
groups independently (Boucheix et al, 1991; Lanza
et al, 1991). The MRP-1/CD9 (also named as CD9
or p24/CD9) is a tetraspanin transmembrane glycoprotein (Boucheix et al, 1991; Lanza et al, 1991)
that is located at the cell surface in a specific membrane microdomain called a
lipid raft (Ishii et al, 2006). CD9 has been
implicated in playing roles in cell adhesion, spreading (Cook et al, 1999), and motility (Ikeyama et al, 1993; Garcia-Lopez et al,
2005). CD9 is highly expressed in macrophages (Kaji et al, 2001) and forms a complex with integrin aIIbb3 and CD63, another member of the
tetraspanin superfamily, in activated platelets (Israels et al, 2001). In addition, CD9 has been shown to play
critical roles in sperm and egg fusion (Komorowski et al, 2006; Rubinstein et al,
2006). Recent data also suggest that extracellular domains of
tetraspanin CD9 protein can inhibit infection of macrophage by HIV virus, probably by
blocking viral entry via modulation of the activity of viral receptors that
form complexes with endogenous tetraspanins (Ho et al, 2006).
Among all these CD9-associated phenotypes
and functions, cumulative data have demonstrated one prominent CD9 feature,
i.e., suppression of tumor progression and metastasis. Downregulation of CD9
expression appears to be an acquired event in the development of malignant
tumors. There is an inverse relationship between CD9 expression and metastatic
potential in several cancers. CD9 protein levels in the metastatic lymph nodes
were found to be lower than those in the respective primary breast cancers in
half of the cases analyzed (Miyake et al, 1995).
A similar inverse relationship was also observed in colon cancer (Mori et al, 1998), oesophageal cancer (Uchida et al, 1999), endometrical cancer (Miyamoto et al, 2001),
oral cancer (Kusukawa et al, 2001), and
melanoma (Si and Hersey 1993). By using differential display cloning technique
in matched primary and metastatic derived human colon carcinoma cell lines,
Cajot and colleagues confirmed in 1997 that CD9 gene was more highly expressed
in the primary tumor as compared to its metastatic counterpart. Moreover,
reduced CD9 expression was found to be associated with a poor prognosis in the
patients with breast cancer (Seymour et al, 1990; Miyake
et al, 1996), non-small cell lung cancer (Higashiyama
et al, 1995), and pancreatic cancer (Sho et al, 1998).
Transfection of human MRP-1/CD9 cDNA revealed that cell motility was altered in
the MRP-1/CD9-expressing cells (Ikeyama et al, 1993),
suggesting that MRP-1/CD9 regulates cell motility. Restored expression of CD9
in highly metastatic human small-cell lung cancer cells reduced liver
metastasis in SCID mice. Moreover, no detectable levels of CD9 were expressed
in metastatic tumor cells in mice bearing CD9-transfected SCLC cells (Zheng et
al, 2005). Taken together, these data suggest
that CD9 may be a tumor-metastasis suppressor gene which may reduce tumor
metastasis via the inhibition of cell proliferation and motility, and that
decreased expression of CD9 may contribute to the malignant progression of
tumors.
Gene therapy has been demonstrated to be a
novel and promising modality to combat malignant diseases (Hanania et al, 1995; Marchisone et al,
2000). Adenovirus vectors have multiple advantages over other viral vectors
to serve as a gene delivery vehicle. Adenovirus is able to infect a wide
variety of cell types, and is able to infect non-dividing cells, it has a high
transduction efficiency and a high level of expression of transgene. It can
accommodate a large piece of foreign DNA and can be concentrated at high titers
(Lu, 2001). In this report, we generated a recombinant adenovirus expressing
human wild-type CD9 (Ad-CD9), and analyzed the potential therapeutic effects of
CD9 on suppression of breast tumor metastasis. In addition, we also used Ad-CD9
as a gene transfer vehicle to analyze the mechanism of CD9-mediated biological
modulation.
Ad-CD9, the
replication-deficient recombinant adenovirus, containing human MRP-1/CD9 cDNA
under the control of a CMV promoter, was generated as following: a 846-bp
fragment containing the full-length human CD9 cDNA gene was released from
plasmid pRc/CMVp24/CD9 (Cook et al, 1999) by
EcoR I and Xba I double digestion. After EcoR I and Xba I digestion of an
E1-deleted adenoviral shuttle vector, pacAd5CMVKNpA (University of Iowa
Research Foundation, Iowa City, IA), it was ligated to the above-mentioned
846-bp CD9 cDNA to generate the resultant adenoviral shuttle vector pacAd5-CD9.
The pacAd5-CD9 and pacAd5 9.2-100, an adenoviral type 5 genome backbone plasmid
(Anderson et al, 2000), were cotransfected into 293 cells by FuGENE 6 reagent
(Roche, Indianapolis, IN) according to the manufacturerÕs protocol. The
individual plaques were screened by the direct plaque screening PCR method (Lu
et al, 1998; Steiner et al, 2000) using a pair of primers recognizing the
adenoviral genome and CD9 cDNA respectively.
E. Immunofluorescence staining
Cells were grown on
coverslips and either untreated or transduced with virus (Ad-CD9 or Ad-lacZ) at
moi=200 for 48 h. The cells were fixed with 3.5% formaldehyde in PBS for 30 min
at room temperature. After washing 3 times with PBS, the cells were processed
to immunofluorescence staining under non-permeabilization conditions as
described previously (Lu et al, 2000). The cells were incubated with 10% normal
goat serum (NGS) in PBS for 20 min for blocking, then with 4 mg/ml anti-CD9 antibody mAb7
in 10% NGS in PBS for 1 h at room temperature, followed by incubation with 5 mg/ml FITC-labeled goat
anti-mouse IgG for 1 h at room temperature. After washing and fixing, the
coverslips were mounted on the slides and photographed under fluorescence
microscopy.
F. Flow cytometry assay
The assay followed the
procedure described previously (Cook et al, 1999).
Briefly, untreated or virus transduced cells were harvested, the cells were
suspended in DMEM with 5% NGS for 30 min for blocking, then 5x105
cells were labeled with 4 mg/ml anti-CD9 antibody mAb7 for 1 h on ice, followed by incubation with
5 mg/ml
FITC-labeled goat anti-mouse IgG for 1 h on ice. After washing and
centrifugation, the cells were resuspended in PBS and subjected to antibody
binding assay using a FACS Calibur flow cytometer (Becton Dickinson
Immunocytometry Systems, San Jose, CA).
G. Growth inhibition assay
The growth inhibition assay
followed the protocol as previously described (Steiner et al, 2000). Briefly,
three groups of cells were used for each breast cancer cell line: (a) control
untreated, (b) control virus Ad-lacZ treated, and (c) Ad-CD9 treated. Cells were transduced at multiplicity
of infection (moi) of 200. Cellular proliferation was measured by cell counting
of the attached cells at day 5 post viral transduction. StudentÕs t-test was
used for statistical analysis throughout the entire project.
The cell migration/motility
was measured by a modified BoydenÕs chamber method using a Transwell plate
containing polycarbonate filters with a pore size of 8.0 mm (Nunc, Roskilde, Denmark).
The filters were precoated on the undersurface (between upper and lower
chambers) with 10 mg/ml fibronectin at 370C for 3 h. Cells were transduced with
Ad-CD9 at moi of 200 for 48 h, the cells were harvested and counted. Cell
suspensions (2 ml of 2.5x104 cells/ml in serum-free DMEM medium with
1% BSA per well) were then placed into the upper compartment of the chamber and
serum-free medium with 1% BSA was placed into the lower chamber. Chambers were
incubated at 370C with 5%CO2 for 3 h. The filters were
then removed and nonmigrating cells remaining on the upper side of the filter
were scraped off. The cells that had migrated to the lower side of the filter
were fixed in Giemsa staining solution (Sigma, St. Louis, MO). After extensive
washing with water, the migrated cells were counted in five different fields
under a microscope at x200 magnification. Migratory activity was expressed as
the mean number of cells that migrated to the lower side of the filter, and
results were represented as sum of total cell numbers in five randomly selected
fields of view. Untreated or Ad-lacZ transduced cells were used as controls.
I. Adhesion assay
To examine the CD9 effects on
ability of cells to adhere extracellular matrix (ECM) components including
laminin, fibronectin, and matrigel, we transduced cells with Ad-CD9 at moi of
200 for 48 h. The cells were harvested and replated at 4x105 per
well in the 24-well culture plate precoated with fibronectin (10 mg/ml) (GibcoBRL,
Gaithersburg, MD), laminin (10 mg/ml) (GibcoBRL), or matrigel (Collaborative Biomedical Inc., Bedford,
MA). After incubation for 4 h at 370C, the nonadherent cells were
removed by washing the plate with PBS. The adherent cells were fixed with 4% of
paraformaldehyde solution for 10 min at room temperature. The cells were
stained with 1% toluidine blue for 5 min and rinsed with water. Cells were then
solubilized by adding 1% SDS and quantified using a microtiter plate reader at
595 nm. Untreated or Ad-lacZ transduced cells were used as controls.
The in vitro cell invasion assay was performed with 6-well-plate
Biocoat matrigel invasion chambers (Becton Dickinson Labware, Bedford, MA)
according to the manufacturerÕs procedure. Briefly, the chamber was first
rehydrated with serum-free media (DMEM) for 2 hr at 370C. After
rehydration, the chambers were placed in the lower compartment previously
loaded with DMEM media containing 5% FBS. Meanwhile, the JygMC(A) mouse breast
cancer cells of untreated control, as well as control virus (Ad-lacZ) treated
and Ad-CD9 transduced cells (moi=200 for 48 hr) were harvested. The cell
suspensions were adjusted to 2.5x105 cells/ml with serum-free
medium. The cell suspension (2ml per well) was immediately added to the upper
compartment of the chamber. The cells were then allowed to invade through the
matrigel for 22 hrs at 370C, and the noninvading cells were removed
by scrubbing the upper surface with a wet cotton swab. The filters were stained
with Diff-Quick stain kit (Dade Behring Inc., Newark, DE), drained and counted.
Highly metastatic breast
cancer JygMC(A) cells were either untreated, or transduced with Ad-lacZ or
Ad-CD9 (both at moi of 200). Forty-eight hours after viral infection, the cells
were harvested and the viable cell numbers were counted in a hemocytometer using
trypan blue exclusion. Cells (1x107 cells per mouse) were injected
subcutaneously into the flanks of 6-week-old female nude mice (athymic nude
mice, Harlan, Indianapolis, IN). Three groups of mice, with five mice in each
group, were formed corresponding to the three groups of cells mentioned above.
Size of primary tumors were
measured every three days with calipers, and tumor volumes were calculated
using the standard formula: width2 x length x 0.5 (Steiner et al,
2000). All the nude mice were sacrificed at day 34 post inoculation when some
of them became moribund. At the time of sacrifice, the allograft primary tumors
were collected and weighed. To examine metastasis, lung, liver, spleen, and
kidney were removed and metastases were counted under the dissection
stereoscope after fixation in Bouin's solution (Sigma) (Lu et al, 1994). Other
organs, including heart, brain, pancreas, lymph nodes, bone, and lumbar spinal
muscle were also carefully checked under microscopy to detect potential
metastasis.
III.
Results
A. Ad-CD9 expresses high levels of
p16 protein in breast cancer cells
B. CD9 inhibits breast cancer cell growth in vitro
Cell
migration is an important aspect of the tumor metastatic process. To analyze
the effect of CD9 on breast cancer cell migration, JygMC(A) cells treated with
Ad-CD9 at moi of 20 for 48 h were harvested and plated in the upper compartment
of the chamber which has been precoated with fibronectin on the undersurface of
the chamber/filter. By comparison with the untreated control cells, Ad-CD9
transduced cells exhibited an almost two-fold increase on cell motility, as
evaluated by counting the cells that were on the undersurface of the filter.
The control virus (Ad-lacZ) transduced cells did not cause a significant change
on the cell motility compared with untreated control cells (Figure 5). This
result suggested that CD9 promoted breast cancer cell migration towards a
fibronectin-enriched environment, that is, a haptotactic cell motility to
fibronectin.
IV. Discussion
In summary, in this study we showed that
adenoviral-mediated CD9 expression increased cell migration of the breast
cancer line JygMC(A) cells in a fibronectin-coated transwell; CD9 significantly
increased JygMC(A) cell adhesion to laminin and fibronectin with a minor
increase in adhesion to matrigel; CD9 also suppressed JygMC(A) cell growth and in vitro invasion ability in terms of
penetrating the matrigel. Moreover, Ad-CD9 mediated expression significantly
decreased tumor metastasis of JygMC(A) cells in a spontaneous metastasis animal
model. These results suggest that CD9 may have a therapeutic potential for
clinical applications in suppressing malignant progression and metastasis of
cancer. One of the practical examples of clinical application of Ad-CD9 is the
adjuvant gene therapy for cancer patients: After surgical resection of the
primary tumor, Ad-CD9 can be injected into the surgical wound sites to prevent
the recurrence of tumor at the primary site and metastasis to other
organs. Of course, the optimal goal
of long-term is to develop a therapeutic gene therapy virus targeting
metastatic cancer cells that can be delivered to the patients via systemic
administration. In that case,
either a regulatory promoter or cancer (or cell-type)- specific promoter should
be employed to ensure the therapeutic gene expression can be regulated or the
transgene expression is within the desired target cells---the distant
metastatic lesions (Lu, 2001).
Our data showed that CD9
expression reduced invasion of JygMC(A) cells to penetrate matrigel (Figure 7). Consistently, CD9Õs
anti-invasion ability was shown in the in
vitro mouse embryo implantation: blocking CD9 function in the embryo by
either monoclonal antibody or antisense oligonucleotide against CD9 led to
significantly enhanced embryo-outgrowth ability on the monolayer of uterus
epithelial cells and stimulated matrix metalloproteinase 2 (MMP-2) production,
suggesting that CD9 was able to impair embryo invasion and inhibit production
of MMP-2 (Liu et al, 2006). To explore the
mechanism of CD9-mediated suppression of breast tumor metastasis, we analyzed
protein expression levels of several tumor metastasis-associated proteinases,
the enzymes which breakdown the extracellular matrix barrier to facilitate
tumor cell spreading, in our model. Our Western blot analysis showed that MMP-2
and MMP-9 expression, at least at the protein level, were not altered by
overexpression of CD9 in JygMC(A) tumors (data not shown), suggesting that CD9-mediated
suppression of tumor metastasis was not due to the suppression of
tumor-associated proteinases.
In our study, CD9 enhances cell mobility and adhesion,
whereas it inhibits invasion into matrigel. These two aspects are not contradictory
if we take a detailed look for how the experiments have been done. Both cell mobility and cell adhesion
assays were designed by measuring CD9Õs attractiveness towards its receptor
fibronectin, a component of ECM, precoated on the undersurface of the filter
(mobility assay) or directly on plates (adhesion assay) within 3-4 h. Because fibronectin is suggested to be a
receptor for CD9 (Cook et al., 2002), it is not surprising that
CD9-overexpressing cells would have enhanced ability to bind to (adhesion) or
migrate towards to (cell mobility) fibronectin-coated surface. On the other hand, in vitro invasion assay was done in 22 h by measuring cellsÕ
ability to penetrate the matrigel, which is composed of ECM including
fibronectin. Thus,
CD9-overexpressing cells would have more interaction (Òligand-receptorÓ
binding) with ECM proteins in the matrigel, thus they are inclined to be
ÒtrappedÓ or retarded inside matrigel more easily than the control cells,
reflecting as a reduced cell ability to penetrate the entire layer of the
matrigel in the in vitro invasion
assay. This reduced invasion
ability, together with CD9-induced apoptosis and growth inhibition, contribute
to reduced distant metastasis of CD9-overexpressing tumor cells.
Expression of CD9 is significantly inversely
associated with the stage of disease in human breast cancer (Miyake et al, 1995; Cajot et al, 1997).
cDNA microarray results showed that CD9 was downregulated in metastatic breast
carcinoma cells compared to primary breast carcinoma cells. Moreover, the relapse
free survival in 5 years is significantly higher in CD9 positive cases than in
negative cases (Mimori et al, 2005). CD9
downregulation has also been reported to be associated with tumor progression,
metastasis and clinical outcome in various other solid tumors (Seymour et al, 1990a, b; Si and Hersey 1993; Higashiyama et al,
1995; Cajot et al, 1997; Mori et al, 1998; Sho et al, 1998; Uchida
et al, 1999; Kusukawa et al, 2001; Miyamoto et al, 2001; Sauer et al, 2003; Mimori et al, 2005).
However, one study reported that CD9 is not a prognostic factor in human
osteosarcoma (Kubista et al, 2004). These
data, taken together, suggest that CD9 may be best used as a prognostic factor
for solid tumors.
While several studies, including this report, have
shown that expression of CD9 appears to suppress malignancy of tumor cells (Ikeyama et
al, 1993; Miyake et al,
2000), one study showed, however, that CD9 overexpression did not
affect in vivo tumorigenic or metastatic
properties of human prostate cancer cells (Zvieriev et al, 2005). One explanation is that other proteins, such
as CD9 partners, are needed for CD9 full anti-tumorigenic action in a
particular cell type environment. While the exact mechanism of how CD9
functions to suppress tumor metastasis remains unclear, some reports
demonstrate that CD9 cDNA transfection altered cell motility (Lanza et al, 1991; Ono et al, 1999)
and induced apoptosis (Ono et al, 1999). One
possibility is that CD9 may exert a malignant-suppressing effect via a
combination of cell motility alteration and apoptotic induction.
CD9Õs anti-metastasis function was also demonstrated
in a CD9-knockdown system by others: ovarian carcinoma cell line transfected
with small interfering RNA against CD9, showed a CD9-negative phenotype and
reduced adhesion to extracellular matrix and increased peritoneal dissemination
(Furuya et al, 2005). These results suggest
that downregulation of CD9 may be an acquired event in the process of tumor
dissemination. Consistently, our results indicate that acquisition of CD9
expression in JygMC(A) breast cancer cells increased adhesion to extracellular
matrix components (Figure 6).
Cumulative data have suggested that CD9 and other
tetraspanin family members, including KAI-1/CD82 (Ono et al, 1999) and CD63 (Israels et al, 2001), are involved in metastasis suppression, and
this effect may be related to their association with integrins (Israels et al, 2001; Miyamoto et al, 2001). During tumor
progression, a reduction of CD9 gene expression results in tumor cells with
high metastatic potential. However, the mechanism of action of CD9 remains
unclear. Microarray and real-time PCR were used to analyze the changes of gene
expression in tumor cell lines and their CD9-transfectants. It has revealed
that the Wnt signaling pathway may be the downstream of the CD9 signal (Huang
et al, 2004). A recent study showed that CD9
regulates the actin cytoskeleton by downregulating the WAVE2, through the
Wnt-independent signaling pathway (Huang et al, 2006).
The mechanistic aspects of function of CD9 are currently under investigation in
our lab. Our ongoing studies have
indicated that cells overexpressing CD9 have a significantly higher level of
phosphorylated Akt than control transfected cells when plated on fibronectin,
and inhibitors of the PI 3-kinase pathway inhibited CD9-promoted haptotactic
motility (data not shown), suggesting that the PI 3-kinase pathway may be
involved in CD9-mediated migration.
In addition, our microarray results indicated that genes responsible for
deacetylation of histones were increased in CD9 overexpressing cells (our
unpublished data). We are in the
process to examine whether trichostatin A (TSA), a histone deacetylase
inhibitor, blocks the CD9-mediated actions. Moreover, our recent studies showed
that CD9 appeared to modulate p130Cas (data not shown), an adaptor protein that
is thought to be part of a Òmolecular switchÓ for induction of cell motility (Klemke
et al, 1998). Therefore, CD9-mediated motility and anti-tumor function may be
regulated in a multiple-platform manner, and may involve many components in a
complex network. Tetraspanins,
including CD9, can form protein complexes with integrins. To investigate whether alteration of CD9
expression may change contents of intergrins, thus modulating integrin-derived
signalling pathway to regulate the cancer cell ability to adhere and invade,
expression of several integrins at cell-surface (the functional integrins) of
JygMC(A) cells were examined for potential alteration by the CD9
overexpression. We found that Ad-CD9 had no significant effect on cell-surface
expression of integrin §1; but Ad-CD9 caused a significant reduction of a5 integrin on cell-surface (mean fluorescent intensity of 28.2 in
untransduced cells versus 5.11 in Ad-CD9 transduced cells). However, this alteration is probably not
due to the CD9 expression, rather, it is due to the adenoviral protein(s), as
the cells transduced by control adenovirus Ad-lacZ also showed a significant
reduction of §1 integrin on cell-surface (mean fluorescent intensity of
8.28). So we conclude that CD9 does
not appear to affect integrins a5 and §1 expression at
cell-surface. Whether CD9 alters
other integrinsÕ expression at cell-surface needs to be further investigated.
Acknowledgments
This work was supported
in part by National Institutes of Health grants DK65962 (Y.L.) and CA107162
(Y.L.), as well as by the Elsa U. Pardee Foundation (Y.L.), University of
Tennessee Vascular Biology Center of Excellence Pilot and Feasibility grant
(Y.L.) and the Cancer Research and Prevention Foundation (Y.L.).
We thank Dr. Syamal Bhattacharya and Ms. Patti Johnson
for reviewing this manuscript. We
also thank Dr. Qiusha Guo for assembling the flow cytometry plots.
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Yi Lu
antibody
(anti-CD9). The membrane was then incubated for 1 hr at room temperature with
the second antibody coupled to peroxidase (ECL Kit, Amersham, Buckinghamshire,
England), and enhanced chemiluminescent (ECL) staining was performed according
to the manufacturer's protocol. The antibody against CD9, mAb7, a mouse anti-human
CD9 monoclonal antibody, was purified from this laboratory (Lanza et al, 1991; Cook et al, 2002).
