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Tumor Biology |
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 METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Localized breast cancer, before metastasis, can be cured with surgery. The high mortality rate associated with breast cancer, however, results from a propensity for the tumor to metastasize while the primary site is small and undetected. The process of metastasis requires, among other steps, changes in adhesion, increased migration, the production of proteases, and the invasion of the stroma. 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 such as uPA.
In addition to growth factors, the proteolytic enzyme uPA and its receptor (uPAR; Ref. 8 ) also regulate cancer invasion and metastasis. The production or expression of uPA/uPAR by either the tumor cells or the neighboring stromal cells is thought to initiate the degradation of ECM components around the cancer cells and the perivascular basement membrane, thus facilitating the migration and intravasation of cancer cells. Although several recent studies have established the prognostic value of uPA and uPAR in breast cancer patients (9, 10, 11, 12) , the issue of uPA/uPAR localization in tumor versus stromal cells or in both is not fully resolved. uPAR expression has been observed in breast cancer sections, predominantly in macrophages, and sometimes in tumor cells (10, 11, 12, 13) . However, using RNA in situ hybridization, the expression of uPA and uPAR has been detected in both tumor cells and stromal cells (14 , 15) , and Bastholm et al. (13) observed that seven of nine patients with confirmed lymph node metastasis demonstrated extensive uPAR-positive immunostaining in the primary tumor.
The uPA system consists of uPA serine protease, its cell surface-associated receptor (uPAR), plasminogen-activator inhibitors, and the proenzyme plasminogen that can be activated by uPA to form plasmin. Regulated, spatially localized degradation of the ECM is required for tissue remodeling, invasiveness, and angiogenesis. A lack of regulation in this process is the hallmark of malignancy and enables metastatic tumor cells to invade normal tissue and to obtain a blood supply. uPA is unique among serine proteases in that uPA has its own high-affinity cell-surface receptor, uPAR, which greatly enhances the action of uPA on plasminogen (16 , 17) . uPAR localizes on cell-cell junctions and to the leading edge of invading cells (18 , 19) . Thus, uPA is spatially and metabolically positioned to play a pivotal role in the directed cascade of protease activity required for tissue invasion. The binding of uPA to uPAR also initiates signaling cascades that require receptor occupancy but not uPA catalytic activity (20) . Recent studies have established that uPAR is an adhesion molecule capable of interacting with components of the ECM and integrins. These activities further support the idea that the uPA system plays a role in cell motility (21, 22, 23, 24) .
Expression of uPA and uPAR has been demonstrated in every solid tumor type examined to date (25 , 26) . Expression is not restricted to tumor cells themselves, because several tumor-associated cell types, including macrophages, mast cells, endothelial cells, natural killer cells, and fibroblasts, are all capable of uPA and uPAR expression in various tumor types. The pattern of expression in these cells differs depending upon the type of tumor involved. For example, tumor cells that exclusively express uPAR, but not uPA, can interact with uPA that is made by surrounding stromal cells. This is seen in colon cancer (27) , in which invasion and aggressiveness among cell lines correlate strongly with expression of uPAR. Expression of uPA and uPAR in breast cancer sections has been observed in tumor cells (10, 11, 12, 13) . A significant correlation was shown between advanced breast cancer and high expression levels of uPA, uPAR, or both. uPAR staining occurs only at the invasive edge (tumor-host interface) and is localized mostly in the endothelial cells in breast cancer (8 , 12) . Because the activation of uPA-dependent proteolysis and uPA-dependent signaling both depend on the binding of uPA to uPAR, inhibition of this interaction between uPA and uPAR has been proposed as a potential therapeutic modality to inhibit tumor progression. Preclinical tumor models using antibodies, high molecular weight inhibitors of uPA-uPAR interaction, and gene-expression-targeting approaches have demonstrated significant inhibitory effects on tumor growth, metastasis, and angiogenesis (28, 29, 30) .
Our previous studies showed that HRG stimulates the motility and invasion of human breast cancer cells (5, 6, 7) . However, it is not known whether uPA and uPAR play a role in HRG action in breast cancer cells. The purpose of this study was to investigate the regulation by HRG of the expression and function of uPA and uPAR in breast cancer cells. The present study is the first to show regulation of uPA and uPAR by HRG. Our results suggest that the uPA/uPAR system mediates, in part, the regulation by HRG of breast cancer cell invasion. Because HRG is a paracrine growth factor secreted from mesenchymal cells, our present findings raise the possibility that HRG-mediated up-regulation of uPA and uPAR in tumor cells enhances the ability of tumor cells to degrade the extracellular milieu, and thereby invade normal tissue.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Extracts, Immunoblotting, and Immunoprecipitation.
For preparation of cell extracts, cells were washed three times with
PBS and lysed in buffer [50 mM Tris-HCl (pH 7.5), 120
mM NaCl, 0.5% NP-40, 100 mM NaF, 200
mM NaVO5, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin) for 15 min on ice. The lysates were centrifuged in an
Eppendorf centrifuge at 4°C for 15 min. Cell lysates containing equal
amounts of protein were resolved on a 10% SDS-polyacrylamide gel,
transferred to nitrocellulose, and probed with the appropriate
antibodies, using an enhanced chemiluminescence (Amersham) method or
alkaline phosphatase-based color reaction method (5, 6, 7)
.
Zymography.
The proteinase activity of uPA was measured as described
(5)
. Briefly, equal amounts of proteins from serum-free
cell culture supernatants were resolved by nonreducing SDS-PAGE
containing 0.1% (w/v) casein (Sigma) and 10 µg/ml plasminogen
(Boehringer Mannheim). The gel was first incubated for 2.5 h at
22°C in 50 mM Tris-HCl (pH 7.5) containing 2.5% Triton
X-100 (Sigma) and 0.05% sodium azide (Sigma), and then for 16 h
at 37°C in 0.1 M Tris-HCl (pH 8.3) containing 0.5%
sodium azide and 0.15 M NaCl. The gels were stained with
Coomassie Brilliant Blue R250, and the enzymatic conversation of
plasminogen to plasmin by uPA was observed as cleared zones in the
blue-stained background. Supernatant from MDA-MB-231 cells were used as
a positive control in zymographic gels.
Northern Hybridization and Promoter-Luciferase/CAT Assays.
Total cytoplasmic RNA was analyzed by Northern hybridization using cDNA
probes for human uPA and uPAR mRNA. 28S and 18S RNA were used to assess
the integrity of the RNA; for RNA-loading control, the blots were
routinely reprobed with a GAPDH cDNA. uPA and uPAR-luciferase or -CAT
constructs used in the present study are well characterized in our
earlier publications (32, 33, 34)
. Briefly, the uPA-CAT
construct included 2345 bp of the 5'-flanking region of the urokinase
promoter fused with the CAT reporter, and the uPAR-CAT reporter system
consisted of a 449-bp sequence stretching from -398 to +51 (relative
to transcription start site) cloned into the XbaI site of
the basic pCAT vector (Promega, Madison, WI). MCF-7 cells (2 x 106) were serum-starved in low-serum
medium (DMEM containing 0.1% serum) for 24 h before transfection.
Serum-starved cells were transiently cotransfected with the desired
plasmids and control pSV b-Gal vector (Promega) using Lipofectamine
(Life Technologies, Inc.). Cotransfection studies were performed in the
presence or absence of dominant-negative mutants of MEK
(35)
, p38MAPK
(7)
, PI-3 kinase (5
, 36)
, NF-
B
(37)
, or control empty vector. The transfection
efficiency was normalized by cotransfection with pSV b-Gal vector
(Promega). Luciferase or CAT activity was measured at 24 h.
Where indicated, cells were treated with HRG (30 ng/ml) before lysis.
Each experiment was repeated with three independent transfections, and
transfection efficiency varied between 40–50%.
Immunofluorescence.
For indirect immunofluorescence, fixed cells were washed with PBS,
blocked by incubation with 10% normal goat serum (Sigma) in PBS for
1 h at ambient temperature. Cells then were allowed to react for
1 h at ambient temperature with rabbit polyclonal antibody
against ß-catenin and anti-uPA monoclonal antibody. For
controls, cells were treated with the secondary antibody only,
omitting the primary antibody, and no signals were detected in control
untreated cells. Cells were viewed with an inverted Zeiss fluorescence
microscope and analyzed by confocal microscopy using the PC-based
LSM-510 version 2.01 software (5
, 7)
.
Boyden Chamber Invasion Assays.
The cell migration assays were performed as described (5
, 7)
. Briefly, the bottom of the porous 8-µm filter was coated
with a thick layer of Matrigel/ DMEM:Ham’s F-12 (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 invade the Matrigel reattach on the lower surface of the
filter. 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) from both sides of the filter, plus those en passage that
were inside the pores. Results were expressed as the percentage of
migrating cells compared with the total number of cells (cells present
in all Z-sections) and represent the means ± SE
of quadruplicate wells from three or more experiments.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This uPA secretion was caused by up-regulation of uPA
mRNA expression in HRG-treated cells, demonstrated by Northern blot
analysis hybridization using a human uPA cDNA. HRG increased 3-
to 4-fold the steady-state levels of the 2.5-kb transcript of uPA
compared with untreated MCF-7 cells. The maximal induction occurred
1–4 h post-HRG treatment, and the uPA levels of uPA transcript were
maintained up to 24 h (Fig. 1B)
; this was
dose-dependent (Fig. 1C)
. The enhanced expression of uPA
mRNA observed in MCF-7 cells was a specific effect of HRG, because the
related transforming growth factor, TGF-
, did not influence the
expression of uPA mRNA (Fig. 1D)
. Since Grimaldi et
al. (38)
have shown a close correlation between the
proliferative state of cells and uPA transcription, we next examined
the expected mitogenic effects of HRG and TGF- in MCF-7 cells.
Results suggested that both HRG and TGF- stimulated the growth of
MCF-7 cells under serum-free culture conditions (Fig. 1E)
,
but only HRG was able to induce the expression of uPA (Fig. 1D)
. These observations suggested that the observed
up-regulation of uPA in HRG-treated cells may not be a consequence of
growth-promoting action of HRG. In addition, there was no change in the
rate of [3H]thymidine incorporation into DNA
between 4–12 h after HRG, a time point of maximum uPA expression (data
not shown).
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. To evaluate translational regulation, we used cycloheximide, a
translational inhibitor. Treatment of cells with cycloheximide
partially blocked the level of uPA expression compared with levels in
cells treated with HRG alone, and there was a stabilizing effect of
cycloheximide on the level of uPA mRNA. The results also suggest that
HRG regulates uPA expression at a pretranslational level.
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. The HRG up-regulated the expression of uPA in HC11 cells,
and this was in an Act-D-sensitive and cycloheximide-insensitive manner
(Fig. 3B)
. In brief, these results suggest that HRG
regulation of uPA may regulate motility and invasion in both normal and
pathological conditions in breast epithelial cells.
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Because MCF-7 cells express low levels of uPAR (40
, 41)
,
we next evaluated the potential regulation of uPAR mRNA expression in
MCF-7 cells, which are known to respond to HRG with increased cell
motility and invasion (5, 6, 7)
. Total RNA was isolated both
from control and from HRG-treated cells, and expression of uPAR mRNA
was analyzed by Northern blot analysis. Results in Fig. 4A
demonstrate the kinetics of uPAR mRNA expression in
HRG-treated MCF-7 cells. HRG rapidly increased the steady-state levels
of uPAR mRNA several-fold over the undetectable uPAR mRNA level in
control cells. Cotreatment of cultures with cycloheximide superinduced
the uPAR mRNA, and cycloheximide stabilized the uPAR mRNA (Fig. 4B)
. HRG induction of uPAR mRNA was sensitive to Act-D, a
transcription inhibitor. Pretreatment of cells with Act-D abolished the
HRG-mediated induction of uPAR mRNA, suggesting that HRG may regulate
uPAR expression at a transcriptional level (Fig. 4B)
.
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. These results indicated that HRG-mediated
up-regulation of uPAR, was accompanied by an increase in the level of
uPAR expression, and may play a role in HRG regulation of the invasion
of breast cancer cells.
HRG Regulation of uPA and uPAR Promoter Activities.
To further confirm the role of HRG in the transcriptional regulation of
the uPA and uPAR, we transiently transfected MCF-7 cells with a
chimeric CAT gene fused with the 5' region of the uPA- or
uPAR-promoter (32, 33, 34)
. In the transfected MCF-7 cells
treated with HRG, there was a significant stimulation of both the uPA-
and uPAR-promoter activities (Fig. 5A)
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B (37)
activation in HRG-treated
MCF-7 cells. Inclusion of dominant-negative mutants of MEK and
p38MAPK suppressed both the constitutive and
HRG-inducible stimulation of uPAR promoter activity (Fig. 5B)
. Inclusion of dominant-negative NF-B also inhibited
the constitutive uPAR promoter activity; that inhibition, however, was
partially overcome by HRG treatment. Next, we evaluated whether
HRG-inducible signaling pathways were necessary in the activation of
uPA promoter. As shown in Fig. 5C
, uPA promoter activity was
below the detection limits in cells cotransfected with
dominant-negative mutants of MEK and NF- 66. Inclusion of the
dominant-negative p38MAPK mutant inhibited the
constitutive uPA promoter activity; this inhibition, in contrast to
that of uPAR promoter, was modestly overcome by HRG treatment (Fig. 5C)
. Blocking the PI-3 kinase pathway by dominant-negative
p85 [which effectively blocks the activation of PI-3 kinase pathway
(36)
] did not significantly affect the HRG-induced
stimulation of uPAR or uPA promoters. In brief, these results suggested
the possible involvement of MEK, p38MAPK, and
NF- 66, but not PI-3 kinase, in the baseline regulation of uPAR- and
uPA-promoter activities.
Regulation of HRG-mediated Cell Invasion by the uPA-uPAR System.
In addition to proteolysis, the uPA/uPAR system plays an important role
in cell motility (reviewed in Ref. 8
). Because HRG is a
potent mitogen (5, 6, 7)
and induces the expression of uPA
and uPAR expression (this study), and because uPA is known to stimulate
cell motility in MCF-7 cells (40
, 41)
, we explored whether
uPA/uPAR system acts as a mediator during HRG-induced cell motility and
invasion for cells grown in Matrigel. The modulation in cell shape and
membrane-bound uPA was subsequently measured. The formation of motile
structures and an increase in the level of membrane-bound uPA
immunostaining accompanied HRG stimulation of MCF-7 cells (Fig. 6A)
. Parallel quantitation of the ability of cells to
invade was confirmed by the Boyden chamber invasion assay (Fig. 6B)
. Together, these results suggested a significant
increase in the level of membrane-bound uPA upon HRG treatment.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our finding that incubation of breast cancer cells with a uPAR-inhibitor significantly suppresses HRG-induced invasion is important because it suggests that autocrine/paracrine stimulation of the uPA-uPAR pathway by cellularly derived factors such as HRG is involved in the regulation of uPAR expression in breast cancer cells. In breast cancer cells such as MCF-7, HRG may also up-regulate the expression and secretion of uPA. This raises the possibility that functional autocrine interaction exists between uPA and uPAR.
We also demonstrated that HRG stimulation of breast cancer cells was accompanied by the simultaneous stimulation of expression of uPA mRNA and uPA protein and of uPA secretion. Regulation of uPA by HRG appears to be pretranslational; it was completely inhibited by Act-D. This view is further supported by the observation that HRG can stimulate uPA-promoter activity in breast cancer cells. This was in contrast to other growth factors where the effect could be at the level of uPAR mRNA stability. Lund et al. (42) reported that phorbol ester 12-myristate 13-acetate and TGF-ß1 increased the stability of the uPAR mRNA in the human lung cancer cell line A549. Further, Shetty et al. (43) demonstrated that the increase in uPA mRNA in mesothelioma cells by the phorbol ester was attributable to a 4-fold increase in mRNA half-life. In brief, our observations raise the possibility that HRG uses the uPA/uPAR autocrine pathway to transduce its signal(s), leading to invasion.
Additional support for the idea that HRG regulates the uPAR pathway is provided by earlier published studies showing that highly invasive breast cancer MDA-MB-231 cells, which secrete HRG (44) , express very high levels of the uPA/uPAR system. These observations also raise the possibility that reported variations in the levels of basal uPA/uPAR expression among different breast cancer cells are caused in part by variable degrees of interaction between endogenous HRG and its receptors, HER3 and HER4. It is possible that the endogenous HRG, the overexpressed HER2 family members, or both may act as one potential cellular factor that controls the action of the uPA/uPAR system in breast cancer, and may have functional implications in the deregulation of the uPA pathway in breast cancer. In addition, it is also possible that the reported variability in the localization of uPA or uPAR on tumor or stroma cells may be influenced by the presence or absence of soluble factors such as heregulin.
The observed induction of the uPA/uPAR pathway in breast cancer epithelial cells by HRG, a paracrine growth factor that is secreted by the mesenchymal cells, may have functional implications in the invasion of breast cancer cells. Any potential up-regulation of uPAR by HRG in breast cancer epithelial cells is likely to promote further the ability of tumor cells to invade their surrounding environment because uPA ligation to uPAR may trigger proteolytic pathways and, thus, the ability of tumor cells to degrade the extracellular milieu. Our findings that HRG regulates the levels of expression of uPA and its receptor and the invasion of breast cancer epithelial cells open a new avenue for investigation to closely link HRG-signaling, the urokinase pathway, and breast cancer invasion.
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ACKNOWLEDGMENTS |
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B mutants, and Dan Medina for providing the HC11 cells. |
FOOTNOTES |
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1 This study was supported in part by NIH Grants
CA80066 and CA65746, the Breast Cancer Research Program of the
University of Texas M. D. Anderson Cancer (to R. K.), and NIH Grants
CA58311 and CA10845 (to D. B.). 
2 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. E-mail: rkumar{at}notes.mdacc.tmc.edu
3 The abbreviations used are: EGF, epidermal
growth factor; HER, human EGF receptor; HRG, heregulin-ß1; uPA,
urokinase plasminogen activator; uPAR, urokinase
plasminogen activator receptor; CAT, chloramphenicol acetyltransferase;
MEK, MAP/ERK kinase; p38MAPK, p38 mitogen-activated protein
kinase; PI-3 kinase, phosphatidylinositol-3 kinase; Act-D,
actinomycin D; NF, nuclear factor; ECM, extracellular matrix.
Received 2/ 7/00. Accepted 10/30/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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