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Advances in Brief |
Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Recent studies suggest that in addition to the COX-2 pathway, the invasiveness of colon cancer can be also influenced by growth factors such as HRG and serine proteases such as uPA. For example, HRG, which binds to the HER-3 and HER-4 receptor (13) , regulates the progression of cancer cells to a more invasive phenotype (14) . Recently, we confirmed that in the absence of HER-2 (also known as c-erbB2 or c-neu) overexpression, HRG activation of breast and colon cancer cells promotes the development of a more invasive phenotype in these cells (14 , 15) . The mechanisms and pathways by which HRG influences the biology of colon cancer cells remain elusive.
One of the important steps for tumor progression and invasion is the destruction of the ECM that separates the epithelial and stromal compartments by serine proteinases, thus facilitating the migration and intravasation of cancer cells (16) . The uPA system-mediated proteolysis contributes to the dissolution of connective tissues surrounding the cancer cells and of the perivascular basement membrane. Evidence suggests that the progression of colon cancer cells also involves the uPA, which has its own high-affinity cell surface receptor, uPAR, that greatly enhances the action of uPA on plasminogen (16, 17, 18) . uPAR localizes on cell-cell junctions and toward the leading edge of invading cells (19 , 20) . Regulated, spatially localized degradation of the ECM is required for tissue remodeling and invasiveness. Thus, uPA is spatially and metabolically positioned to play a pivotal role in the directed cascade of protease activity required for tissue invasion by the tumor cells.
The expression of uPA and uPAR have been demonstrated in essentially every solid tumor type examined to date (16) . Expression is not restricted to the tumor cells themselves; several tumor-associated cell types including macrophages, mast cells, endothelial cells, natural killer cells, and fibroblasts are all capable of uPA and uPAR expression (16) . The pattern of expression in these cells differs, depending on the type of tumor. For example, tumor cells that exclusively express uPAR (but not uPA), such as some forms of colon cancer, can recruit uPA made by surrounding stromal cells (20) . Tumor cell invasiveness and aggressiveness correlate strongly with the expression of uPAR. Expression of uPA and uPAR is often restricted to the leading edge of tumor progression and the tumor-host interface (21 , 22) .
Our previous studies showed that HRG stimulates the secretion of PGE2 and in vitro invasiveness of human colon cancer cells (15) . It is not known, however, whether uPA plays a role in the actions of HRG and PGE2 in colon cancer cells. The purpose of this study was to establish whether HRG-mediated stimulation of colon cancer invasiveness could be influenced by PGE2 and uPA. To demonstrate this, we used a three-dimensional model in which we could modulate the architectural remodeling of FET colon cells by blocking different levels in the proposed HRG-induced uPA activation pathway. Using a simplified in vitro model, this study demonstrated for the first time that both HRG and PGE2 augmentation involve the formation of distinct three-dimensional glandular structures. Because HRG is a paracrine growth factor, these findings raise the possibility that HRG- and PGE2-mediated up-regulation of uPA in tumor cells may enhance the ability of colon tumor cells to degrade the extracellular milieu and thereby invade normal tissue.
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Materials and Methods |
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Boyden Chamber Assay.
The effect of HRG on the invasiveness of FET colon cells was determined
by the Boyden chamber assay (14)
. Briefly, the bottom of
the porous 8 µm filter was coated with a thick layer of
Matrigel/DMEM:F12 (1:2) that serves as a chemoattractant for the cells
that are plated on the upper side of the filter. After 12 h, the
cells that pass through the pores and reattach on the lower surface of
the filter were quantitated. The filters were fixed, stained
with propidium iodide (0.1%, w/v), mounted on a slide, and analyzed by
confocal microscopy. The cells that invaded the Matrigel were
considered invasive, and results were expressed as the percentage of
total cells/microscopic field (x40 magnification) from both sides of
filter plus those en passage that were inside the pores.
Formation of Lumen-containing Three-dimensional Structures.
Cells were allowed to grow on a thick layer of Matrigel. Briefly, 100
µl of Matrigel/DMEM:F12 (diluted 1:2) were added to each well of a
Lab-Tek eight-chamber slide (Molecular Probes) and allowed to
gel for 30 min at 37°C. FET cells were trypsinized, washed
sequentially in DMEM:F12 supplemented with 10% serum and DMEM:F12
without serum, and resuspended into single cells, and 10,000 cells were
resuspended in 400 µl serum-free DMEM:F12/well. After 10 min, HRG
(10–100 ng/ml) or PGE2 (10–100 ng/ml) was
added, and slides were monitored for several days for morphological
changes. A concentration of 30 ng/ml HRG and 50 ng/ml
PGE2 was able to induce formation of cellular
clusters after 48 h for HRG and after 8 h for
PGE2. In some cases, we used Matrigel/DMEM:F12
(diluted 1:4)-coated Lab-Tek chambers to analyze changes in cellular
shapes reminiscent of a motile phenotype. All morphological changes
were analyzed after immunostaining and confocal microscopy analysis.
Northern Analysis.
Total cytoplasmic RNA was analyzed by Northern blot analysis using cDNA
probes against uPA and uPAR (American Type Culture Collection,
Manassas, VA).
Growth Assay.
Cell proliferation was measured by the MTT dye (Sigma) uptake method,
as described previously (25)
. About 7000 cells/0.5 ml
culture medium were seeded into each well of a 24-well plate. After
24 h, appropriate cultures were supplemented with HRG or
PGE2. For each time point, the medium was removed
from triplicate wells, MTT (5 mg/ml PBS) was added, and the plate was
wrapped in aluminum foil and kept at 37°C for 4 h. The dye
solution was aspirated, and the dye taken up by the cells was extracted
in 1 ml of isopropyl alcohol:1N HCl (96:4) for determination of
absorbance at 570 nm.
Immunostaining and Confocal Analysis.
For immunofluorescence staining, cells were grown on uncoated or
Matrigel-coated Lab-Tek chamber slides. After appropriate treatments,
cells were fixed for 10 min in a 1:2 mixture of cold methanol and
ethanol and incubated with the indicated primary antibodies followed by
the respective FITC- or rhodamine-conjugated secondary antibodies
(Molecular Probes). Confocal imaging, three-dimensional analysis, and
intensity quantification were performed using a Zeiss LSM and
personal computer-based LSM510 Version 2.01 software (14
, 15)
.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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; these modifications were reminiscent of those in
cells acquiring a more motile phenotype. HRG and
PGE2 treatments induced membrane ruffling
(arrows) and changes in cell shape. In contrast to the
uncoated slide, when cells were grown on Matrigel, the effect of
PGE2 was different from that of HRG, as assessed
by staining with ß-tubulin antibody (Fig. 1, D–F)
. HRG
treatment induced the formation of oblong cell clusters (Fig. 1E)
; the PGE2 treatment induced the
formation of intracellular pseudoglandular structures containing large
empty spaces (Fig. 1F)
. Consistent with the invasive nature
of PGE2-treated cells grown in Matrigel,
PGE2 promoted the invasion of FET cells using
Boyden chamber assays (Fig. 1G)
but did not have any effect
on cell growth as measured by the MTT assay after 48 h of
treatments (Fig. 1H)
. In contrast, HRG induced cell invasion
as well as cell proliferation (Fig. 1, G and H
,
respectively).
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. The results of phase-contrast microscopy studies
suggested that the structures induced by PGE2
were morphologically different from those induced by HRG and
characterized by different time kinetics. Using specific markers such
as ß-catenin for intercellular junctions or propidium iodide for
nuclear DNA, we analyzed these structures by confocal scanning
microscopy followed by three-dimensional reconstruction technique. Our
results showed that after 48 h of HRG treatment, the cells
displayed intracellular multiluminal three-dimensional structures that
appeared to be interconnected at different levels (Fig. 2
B,
yellow arrows). Two horizontal sections showing the apical
(red arrow) and basal (blue arrow) levels of the
structure are also shown (Fig. 2
B, right panels). In
contrast to HRG treatment, the three-dimensional structures in the
PGE2-treated cells were monoluminal (Fig. 2C)
, oriented perpendicularly on the slide surface, often
twice as long as the HRG-induced structures, and resembled colonic
glandular-like structures (Fig. 2C)
. Three different
sections were virtually cut at the basal level (yellow arrow
pointing to both sides), at the apical level (yellow
arrow pointing to the left), and vertically in the
Y axis (red arrow; Fig. 2C
). These
structures in PGE2-treated cells were in a state
of continuous remodeling, as indicated by the nuclear DNA staining,
which showed mitotic cells (white arrow) at the basal level
and apoptotic cells at the apical level (white arrowhead;
Fig. 2C
). Interestingly, no lumen-containing
three-dimensional structures were induced within the cells when
transforming growth factor 
was used as a signal (Fig. 2D)
. These findings suggest that the formation of
three-dimensional invasive structures requires ligand-triggered signals
(Fig. 2D)
.
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. Both HRG and PGE2 up-regulated the
levels of uPA mRNA, although they did so to different extents (Fig. 3A)
.
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. Both HRG and PGE2 treatments
substantially altered the cell shapes compared with no treatment in the
control cells. These morphological modifications were accompanied by a
dramatic redistribution of uPA, both at the level of focal adhesion
points and, in the case of PGE2 treatment, at the
site of dome-like structures (Fig. 3B
, lower and
upper, respectively). More interestingly, the uPA
redistribution occurred most frequently at the leading edges of the
cells, followed in incidence by redistribution at the focal
adhesion-like complexes in the case of cells treated with HRG. A low,
uniform, peripheral localization of uPA could be observed in the case
of the untreated FET cells (Fig. 3B)
. The epidermal growth
factor receptor was costained to identify the cell membranes, including
the free edges of FET cells, because costaining with ß-catenin
would identify mainly the intercellular junctions.
To evaluate the status of uPA distribution in the three-dimensional
clusters induced after 48 h of PGE2 and HRG
treatment, cells were costained for ß-catenin and uPA proteins and
analyzed using confocal microscopy and three-dimensional reconstruction
(Fig. 4)
. We also quantified the intensities of the pixels in each channel on
the predefined horizontal sections, where the values are plotted on
(a) a red histogram for the Alexa546 fluorochrome channel
that corresponds to ß-catenin and (b) a green histogram
for the FITC channel that corresponds to uPA. Superimposition of the
red and green peaks suggests the colocalization
of proteins ß-catenin and uPA (Fig. 4)
. In the structures induced by
HRG, uPA was distributed mainly on the internal membrane facing the
empty space of the structure and occasionally on cell membrane
projections, which, as an example, are represented by the middle
peak showing high pixel intensities in both the red and
green channels. This was different from the uPA
expression pattern in the PGE2-induced
structures, in which uPA was located on the cell membrane facing the
outside surface of the cellular walls (Fig. 4)
. The levels of
membrane-bound uPA were also different. Thus, the staining intensity of
uPA in HRG-treated cells was two to three times higher than that in the
PGE2-treated cells. However,
PGE2 treatment was accompanied by an increase in
the level of uPA staining compared with that found in the untreated
control cells. In brief, these results suggested that the temporal uPA
distribution and intensities in three-dimensional structures were
differentially influenced by HRG versus
PGE2. However, both HRG and
PGE2 increased the level of membrane-bound uPA
above the levels found in untreated control cells.
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. Thus, we could identify the appearance
of "hot spots" (arrows) on the cell-cluster surface that
are not in direct contact with the Matrigel; these hot spots are
characterized by uPA-bound and ß-catenin-positive membrane
projections. They represent sites where the cellular junctions are
disconnected, and the resulting free edges of the cells are flipped to
allow the initiation of lumen formation (Fig. 5A
,
arrows). The accumulation of membrane-bound uPA (hot spots)
appeared to be an early event that correlated with the presence of
luminal three-dimensional structures.
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, arrows), we
examined the effects of a specific COX-2 inhibitor, NS398
(15)
, and a specific uPAR antagonist, Å36, which
acts as a competitive inhibitor (IC50 = 5 nM) of uPAR on colon cancer RKO cells
(27)
. Interestingly, cotreatment of cultures with both HRG
and Å36 blocked the formation of lumen containing three-dimensional
clusters (Fig. 5C)
. Cotreatment of cells with HRG and COX-2
inhibitor NS398 also blocked the formation of a lumen inside the
three-dimensional clusters as well as the HRG effect on uPA
distribution, despite a slight increase in uPA staining at the level of
the focal adhesion complexes (Fig. 5C)
. Treatment of the
cells with NS398 did not affect the formation of
PGE2-induced three-dimensional structures (Fig. 5C)
. Treatment of the cells with NS398 blocked the
HRG-triggered increase in the invasiveness of FET cells (Fig. 5D)
. Similarly, treatment with Å36 inhibited the HRG- and
PGE2-triggered invasiveness of FET cells (Fig. 5D)
. These results suggest that PGE2
synthesis may be necessary for the formation of HRG-induced cell
structures on Matrigel and that both PGE2 and HRG
may use the uPA pathway for the formation of luminal three-dimensional
structures. These events may be closely linked to the invasiveness of
colon cancer cells. Colon cell lines are known to develop a distinct
crypt-like architecture when cocultured with fetal rat mesenchyme cells
(28)
or implanted under the kidney capsules of Swiss
nu/nu (nude) mice and allowed to grow for 10 days
(29)
. These models, although very laborious, emphasized
the need for a very active cellular stroma that originates from
the animal host and leads to different types of glandular structures.
Substitution of the stroma with exogenously added HRG in our model
enabled us to study, in a more accessible way, distinct multicell
architectural modifications, as well as dynamics of different
constituents such as cytoskeletal proteins or proteolytic enzymes. In summary, the results presented here suggest that HRG affects colonic tissue remodeling, at least, in part, by PGE2 up-regulation. In this model, HRG can induce a more invasive phenotype, probably due to its effects on the uPA/uPAR pathway as well as up-regulation of PGE2, which can, in turn, also feed into the uPA pathway. Although both HRG and PGE2 can trigger a series of distinct morphogenetic alterations, PGE2 was more potent that HRG. It is possible that over a period of time, the HRG-induced PGE2 may attain a significantly higher level in the conditioned medium. In addition, there may be other unidentified effects of HRG that could partially attenuate PGE2-stimulated remodeling, and HRG-treated cells may have a greater amount of bound uPA than PGE2-treated cells. Additional studies are required to address these and other possibilities. There is a regulatory interplay between the stroma-secreted ECM proteins and the epithelial cells that harbor various surface receptors. This interplay may be critical for a dynamic control of cellular movement and cytoskeleton organization. HRG and PGE2 heavily modulate these events in the colon system. The results of our study suggest a model of colon cancer progression in which the binding of HRG to its HER-3 and HER-4 receptors on adjacent tumor epithelial cells induces a series of events, including secretion of PGE2 and up-regulation of uPA. This new pathway may be integrated into a broad model of how cells function, die, migrate, or differentiate under the influence of HRG and PGE2 signals. In addition, our three-dimensional model system presented here may potentially be used to screen the effects of therapeutic compounds and biomolecules on different spatial aspects of colon biology, including cell growth, motility, invasion, survival, and apoptosis.
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FOOTNOTES |
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1 Supported in part by NIH Grant CA80066, American
Institute for Cancer Research Grant 96A077, and Cancer Center
Core Grant CA16672. 
2 Present address: Angstrom Pharmaceuticals, Inc.,
11585 Sorrento Valley Road, San Diego, CA 92121.
3 To whom requests for reprints should be
addressed, at Department of Molecular and Cellular Oncology, The
University of Texas M. D. Anderson Cancer Center 108, 1515 Holcombe
Boulevard, Houston, TX 77030. Fax: (713) 745-3792; E-mail: rkumar{at}notes.mdacc.tmc.edu
4 The abbreviations used are: COX-2,
cyclooxygenase-2; PG, prostaglandin; HRG, heregulin-ß1; HER, human
epidermal growth factor receptor; uPA, urokinase plasminogen activator;
uPAR, uPA receptor; ECM, extracellular matrix; DMEM:F12, DMEM:Ham’s
F-12; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide.
5 A. Mazumdar, L. Adam, A. Mazar, D. Boyd, and R.
Kumar. Heregulin regulation of plasminogen activator and its receptor:
human breast epithelial cell invasion, submitted for publication.
Received 3/15/00. Accepted 11/ 8/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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interferon-mediated growth inhibition of A431 cells. Cancer Res., 49: 3482-3488, 1989.This article has been cited by other articles:
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