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Department of Clinical Research (V.C.-L.L., S.E.A., M.G.-K.T.)
Department of General Surgery (E.H.N., E.H.-L.N) Singapore General
Hospital Republic of Singapore 169608
Department of
Anatomy (B.H.B.) National University of Singapore Republic of
Singapore 169608
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(ER-) negative breast cancer cell model by transfecting
PR cDNA into ER-- and PR-negative MDA-MB-231 cells in order that the
functions of progesterone can be studied independently of estrogens. We
have demonstrated using this model that progesterone markedly inhibited
cell growth. We have also discovered that progesterone induced
remarkable changes in cell morphology and specific adhesion structures.
Progesterone-treated cells became considerably more flattened and well
spread than vehicle-treated control cells. This was associated with a
striking increase of stress fibers, both in number and diameter, and
increased focal contacts as shown by the staining of focal adhesion
proteins paxillin and talin. There were also distinct increases in
tyrosine phosphorylation of focal adhesion protein paxillin and focal
adhesion kinase in association with increased focal adhesion.
The staining of tyrosine-phosphorylated proteins was concentrated at
focal adhesions in progesterone-treated cells. More
interestingly, monoclonal antibody (Ab) to ß1 integrin was able to
inhibit progesterone-induced cell spreading and formation of actin
cytoskeleton. To our knowledge, this is the first report describing a
direct effect of progesterone in inducing spreading and adhesion of
breast cancer cells, and ß1-integrin appeared to play an essential
role in the effect. It is known that the initial step of tumor
metastasis is the breakaway of tumor cells from primary tumor mass when
they lose the ability to attach. Hence, progesterone-induced cell
spreading and adhesion may have significant implications in tumor
metastasis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Since T47D breast cancer cells were found to constituitively express
high levels of PR independent of estrogens (11), the cell line and its
variants have been the major models by which to study the functions of
progesterone in the regulation of cell growth and other cellular
processes (12, 13). We have established ER-independent expression of PR
by stably transfecting PR cDNA into ER-- and PR-negative breast
cancer cell line MDA-MB-231, which has recently been reported to
express ER-ß mRNA (14). The resulting PR-positive but ER--negative
cell model allows us to assess PR-mediated progesterone-regulated
cellular processes independent of estrogen and ER. We have reported
that progestins markedly inhibit cell proliferation of these PR
transfectants (15). The findings are similar to what was reported for
the growth-inhibitory effects of progestins in T47D cells (1) and in a
PR-negative subline T47D-Y transfected with either the B or A isoforms
of PR (T47D-YB and T47D-YA) (13). In this report, the novel finding
that progesterone induced remarkable cell spreading and focal adhesion
in PR-transfected MDA-MB-231 cells is described. Progesterone-induced
cell adhesion was associated with increased tyrosine phosphorylation of
focal adhesion protein paxillin and focal adhesion kinase (FAK) and
inhibited by Ab to ß1-integrin.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A comprehensive report on the effects of progesterone on the growth of PR-transfected MDA-MB-231 cells (ABC28) has been previously published (15). We have described in that report that progesterone markedly inhibited the growth of ABC28 cells in a concentration-dependent fashion. Maximal inhibition of cell growth was observed with 10-9 M of progesterone, and the cell number was reduced by a maximum of 70% as compared with the vehicle-treated controls.
Although progesterone markedly inhibited the growth of PR-transfected
MDA-MB-231 cells, progesterone-treated cells did not exhibit any sign
of apoptotic or necrotic death as shown in Fig. 1
. Instead, progesterone induced
remarkable changes in cell morphology and specific adhesion structures
in these cells. The majority of PR-transfectant cells in control medium
appeared rounded in shape and attached to the substratum poorly (Fig. 1, a and c). In contrast, progesterone-treated cells became
considerably flattened and more spread with much larger cell surface
than the vehicle-treated control (Fig. 1, b and d). The effect on cell
spreading began to be visible after 8 h of progesterone treatment
as more cells were flattened and adhered to the substratum (Fig. 1b).
The cell spreading and flattening were very prominent after 48 h
treatment (Fig. 1d). The flattened and well-spread morphology of
progesterone-treated cells is best illustrated in the micrograph (Fig. 2b), as compared with the much more
rounded control cells (Fig. 2a). Progesterone had no detectable effect
on vector-transfected control cells CTC15 or the parental cell line
MDA-MB-231-CL2 cells (not shown).
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). In contrast, progesterone-treated
cells were characterized by a very well developed stress fiber system,
and the new stress fibers appeared to be increased both in number and
diameter (Fig. 3b). To confirm that progesterone induced specific focal
adhesion, cells were stained with Ab to focal adhesion proteins, talin
and paxillin, after 48 h of treatment. Progesterone-treated cells
(Fig. 4b for talin and Fig. 4d for
paxillin) showed increased focal contacts as evidenced by the much more
prominent staining of focal adhesion proteins than that in
vehicle-treated controls (Fig. 4a for talin and Fig. 4c for paxillin).
The nuclei regions of the cells showed strong staining by the Abs, and
we are not sure of the significance of this staining. Figure 4, e and
f, showed cells stained with Ab to talin (red) and paxillin
(red), respectively, in conjunction with
FITC-phalloidin, the probe to filamentous actin. It can be seen that
focal adhesion proteins, talin and paxillin, colocalized with the ends
of stress fibers, thus playing the role of anchoring the cytoskeleton
to the extracellular matrix and hence enabling the cells to adhere and
spread.
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). Lanes in
panel A were probed with antityrosine Ab, and lanes in panel B were
probed with Ab to FAK and paxillin, respectively, to reveal the amount
of immunoprecipitated protein analyzed. There were increased tyrosine
phosphorylations of FAK and paxillin in progesterone-treated
cells (+ lanes, Fig. 5, panel A) as compared with
the vehicle-treated controls (-lanes, Fig. 5a, panel A) after
normalization with the amount of immunoprecipitated protein analyzed
(panel B). Densitometry data of two experiments revealed that there was
an average of 62% increase in tyrosine phosphorylation of FAK after
8 h and 24 h of 1 nM progesterone treatment, and
100% and 240% increases, respectively, in tyrosine phosphorylation of
paxillin after 8 h and 24 h of 1 nM progesterone
treatment. It is to be noted that cell spreading began to be visible
after 8 h of progesterone treatment and by 24 h the
phenomenon was well established. Hence, increased tyrosine
phosphorylation by progesterone accompanies the function of induction
of focal adhesion by progesterone. This observation is consistent with
the idea that tyrosine phosphorylation of focal adhesion proteins is
involved in the development of new focal adhesions. Indeed, the
staining of Ab to phosphotyrosine in progesterone-treated cells was
concentrated at focal contacts as shown by colocalization of the
staining of Ab to phosphotyrosine (Fig. 4g
) with that of the focal
adhesion protein paxillin (Fig. 4h).
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2, 3, 4,
5, and 6. It was revealed that monoclonal Ab to ß1-integrin
dramatically reversed progesterone-induced cell spreading and adhesion
when it was incubated for 24 h with the cells that had been
treated with progesterone for 24 h when the focal adhesions had
already been established. Similar inhibition of progesterone-induced
spreading and adhesion by ß1-integrin Ab was demonstrated when the Ab
was added at the same time as progesterone. Cells treated with Ab to
ß1-integrin became smaller and less protracted (Fig. 1e) as compared
with progesterone-treated cells without ß1-integrin Ab (Fig. 1d).
There was also considerable reduction in the amount of stress fibers in
ß1-integrin Ab-treated cells (Fig. 3c) as compared with the cells
treated with progesterone alone (Fig. 3b).
Quantitative assessment of reversing effect of ß1-integrin Ab on
progesterone-induced cell spreading is shown in Table 1. Images of light micrographs of 76–115
cells were acquired via a charge-coupled device (CCD) camera,
and the surface area of individual cells was analyzed by KS 400
software (Kontron Instruments Ltd.). Progesterone-treated
cells were, on average, 3.4 times larger in area than the
ethanol-treated controls (P < 0.00001). ß1-Integrin
Ab at 1:100 was able to reduce progesterone-induced spreading by nearly
40% after 24 h treatment (P < 0.0005). These
observations suggest that the ß1-integrin subunit is critically
involved in progesterone-induced cell spreading and adhesion. This is
in line with the belief that integrins function to establish bridges
between extracellular matrix proteins and cytoskeleton at focal
adhesion sites.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-negative model in
revealing the function of progesterone.
There has been recent evidence suggesting the involvement of
progesterone in cell-cell adhesion. Estrogen was shown to suppress the
expression of E-cadherin and - and ß-actenin in Ishikawa cells of
well differentiated endometrial cancer cells, which could lead to
decreased cell-to-cell adhesion. The use of progestins reversed the
suppressions induced by estrogen (19). In T47D cells, progestins
induced the expression of desmoplakins (3), which are essential
intracellular attachment proteins that connect intermediate filaments
with the desmosomes, which in turn interact with transmembrane linker
proteins to hold the adjacent membranes together. These findings
suggest promoting mechanisms of progesterone for cell-cell interaction.
However, these T-47D cells displayed much more rounded morphology after
progestin treatment, which is opposite to what we have found in
PR-transfected MDA-MB-231 cells. Another recent finding was that in
transgenic mice carrying an imbalance in the native ratio of A to B
forms of PR, the mammary glands exhibited decreased cell-cell adhesion
(20). This is an in vivo demonstration of the role of
progesterone in cell-cell adhesion that is mediated by a balanced
effort of A to B forms of PR. It remains to be determined whether the
function of progesterone in inducing focal adhesion reported here
reflects a normal physiological function and whether it can also be
reproduced in other PR-positive but ER-negative cell models.
Progesterone-induced cell adhesion was associated with increased tyrosine phosphorylation of FAK and focal adhesion protein paxillin (16, 17). FAK was first identified to be concentrated at focal adhesions in 1992 (21). Substantial evidence suggests that FAK is capable of autophosphorylation in response to integrin clustering (21, 22, 23, 24). Upon activation, FAK can itself phosphorylate paxillin, which will then serve to recruit additional signaling molecules to focal contacts and hence to catalyze the formation of focal adhesion assemblies and to initiate signals that may direct the activation of other cellular signaling pathways. In PR-transfected MDA-MB-231 cells, ß1-integrin was shown to play an essential role in progesterone-induced cell spreading and formation of actin cytoskeleton as Ab to ß1-integrin inhibited progesterone-induced cell adhesion. Our preliminary results also revealed that the ß1-integrin Ab was able to reverse progesterone-induced tyrosine phosphorylation of FAK and paxillin. Hence, ß1-integrin may be responsible for the activation of FAK, which will then trigger the generation of other signaling molecules such as the phosphorylation of paxillin in the focal contacts as is generally proposed for the model of signal transduction in focal adhesion (22, 23).
It is to be noted that the magnitude of ß1-integrin Ab-mediated reduction of progesterone-induced cell spreading was about 40%. The inability of ß1-integrin Ab to completely reverse progesterone-induced focal adhesion may be because the functional efficiency of ß1-integrin Ab is not 100% or there may be other mechanisms working in parallel with ß1-integrin to promote cell adhesion.
At present, we can only speculate on the mechanisms of integrin activation by progesterone. Progestins have been shown to increase the expression of laminin receptor mRNA in T47-D cells (24), and ß1 integrin-associated heterodimers are well known laminin receptors (25, 26). However, our study revealed no progesterone-induced changes in the expression of ß1-integrin or in tyrosine phosphorylation of ß1-integrin in these PR-transfected MDA-MB-231 cells that are known to express high level of ß1-integrin (26, 27). We hypothesize that progesterone induces focal adhesion by activating ß1-integrin via intermediate signaling molecules, notably the growth factor-mediated signaling pathways. This hypothesis is supported by several lines of evidence. First, progesterone is known to mediate the expression of a number of growth factors and their receptors. These include fibroblast growth factor (28), epidermal growth factor (29), transforming growth factor (30), and insulin-like growth factors (IGF) and IGF binding proteins (IGFBPs) (31, 32, 33, 34). Second, several reports suggest that growth factors are involved in cell adhesion. IGF-I has been reported to stimulate the formation of adhesion structures. In SH-SY5Y neuroblastoma cells, IGF-I induces lamellapodia extension and formation of stress fibers, and this is associated with increased tyrosine phosphorylation of FAK and paxillin (35). IGF-I also stimulates chemotaxis of breast cancer cells lines MCF-7 and MDA-MB-231 in which specific types of integrins are required for the IGF-I-mediated response (36). Furthermore, direct interactions between IGFBP-I and integrins have been reported both in vitro (37) and in vivo (38). Accordingly, recent evidence also highlighted the importance of interactions between integrins and classical growth factor signaling pathways, with several reports showing integration of integrin and growth factor signal transduction pathways (39, 40, 41).
Direct evidence of progesterone acting as a mediating factor of growth factor signaling in breast tissue has emerged recently. Progesterone was shown to up-regulate type I growth factor receptors, and selectively amplify downstream MAPK cascade (42). Progesterone also primes breast cells for growth factors’ action. For example, T47D cells primed by progestins for approximately 48 h become highly sensitive to the proliferative effect of epidermal growth factor (EGF) that is not mitogenic in these cells in the absence of progesterone (42, 43). In bovine mammary tissue transplanted to nude mice, progesterone significantly augmented the mitogenic effect of EGF (44). In agreement with published results, preliminary results from our laboratory also suggested progesterone-dependent effect of EGF on cell spreading in the PR-transfected MDA-MB-231 cells. EGF-mediated activation of ß1-integrin by progesterone is currently under investigation in our laboratory.
Cell adhesion is a key process in the establishment of tissue structure and differentiation. Complex and coordinated reductions and increases in adhesion have been proposed to be necessary for tumor invasion and metastasis. The findings that progesterone induced cell-extracellular matrix adhesion suggest that this hormone may play a significant role in the process of tumor invasion and metastasis and the effect may be mediated by integrins. Integrins are generally believed to promote the cell-substrate adhesion. Ab to ß1-integrin significantly inhibited the adhesion of MDA-MB-231 cells to extracellular matrix, bone matrix, and to human umbilical vein endothelial cells (27, 28, 45). ß1-Integrin Ab also inhibited the attachment of rat mammary tumor cells on the lymph node stroma (46). The integrin-promoted attachment can be positively or negatively related to metastasis, depending on whether the integrin functions to adhere the tumor cells to basement membrane surrounding the primary tumor or whether it functions to aid in adhesion at a secondary site. Since the initial step of metastasis is believed to be the detachment of tumor cells from the primary tumor mass when the cells lose the ability to attach (47), increased adherence of tumor cells to basement membrane may prevent the tumor to metastasize to a secondary site. Experiments are underway to study the effect of progesterone on cell invasion in ABC28 cells both in vitro and in vivo.
It is also interesting to note that morphogenesis of human mammary cells in collagen gel was prevented by ß1-integrin Ab (48, 49, 50) and progesterone is well known to be involved in mammary morphogenesis (2). It would be interesting to determine whether in vivo development of lobule-alveolar structure of the mammary gland requires the activation of ß1-integrin by progesterone.
In conclusion, we have demonstrated that progesterone induces remarkable focal adhesion in PR-transfected MDA-MB-231 cells. In association with progesterone-induced focal adhesion was the increased tyrosine phosphorylation of focal adhesion protein paxillin and FAK. Ab to ß1-integrin distinctively inhibited progesterone-induced focal adhesion and tyrosine phosphorylation of FAK and paxillin, suggesting that ß1-integrin plays an important role in progesterone-induced focal adhesion. This is the first report describing a direct effect of progesterone on focal adhesion. The results provide new directions to which the therapeutic potential of progesterone in breast cancer can be explored. It remains to be studied whether progesterone also induces focal adhesion in physiological situation in which both PR and ER are naturally present.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2-6 were from Becton Dickinson and Co. (San Jose, CA) Mouse monoclonal
antiphosphotyrosine (PY20) Ab was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All fluorescence-conjugated
secondary Abs were purchased from Roche Molecular Biochemicals
(Indianapolis, IN). All tissue culture reagents were obtained
from Life Technologies (Gaithersburg, MD). Tissue culture
plastic wares were from Corning, Inc. (Corning, NY).
Cell Culture
All cells were routinely maintained in phenol red containing
DMEM supplemented with 5% FCS, 2 mM glutamine, and 40
mg/liter gentamicin. For all experiments, cells were grown in phenol
red-free DMEM supplemented with 5% dextran charcoal-treated FCS
to remove the endogenous steroid hormones that might interfere with the
analysis. Cells were treated with progesterone from 1000-fold stock in
ethanol. This gave a final concentration of ethanol of 0.1%. Treatment
controls received 0.1% ethanol only.
Transfection
PR expression vectors hPR1 and hPR2 were generous gifts of
Professor P. Chambon (Institute of Genetics and Molecular and Cellular
Biology, Strasbourg, France). Vectors hPR1 and hPR2 contained
human PR cDNA coding for PR isoform B and isoform A, respectively, in
pSG5 plasmid (51). Vector pBK-CMV (Stratagene, La Jolla,
CA) containing the neomycin-resistant gene was cotransfected with hPR1
and hPR2 into MDA-MB-231-CL2 cells using Lipofectin reagent (Life Technologies, Inc.). Neomycin-resistant clones selected in
medium containing G418 were screened for vector pSG5 sequence by PCR
and for PR using the PR enzyme immunoassay kit from Abbott Laboratories (North Chicago, IL). Eight PR-positive clones
expressing both PR isoforms A and B were isolated and characterized.
They showed similar responses to progesterone treatment. For simplicity
of interpretation, the effects of progesterone on clone ABC28 that
expressed approximately 660 fmol PR per mg protein were described in
this report. The cells at passage numbers 5–30 were used. Cells stably
transfected with both vectors pBK-CMV and pSG5 were used as
transfection controls.
Light Microscopy
Cells were grown in six-well plates and treated with either
progesterone in 0.1% ethanol or 0.1% ethanol for the required period
of time before they were viewed and photographed under an AXIOVERT 35
phase contrast microscope (Carl Zeiss, Thornwood, NY).
Quantitative measurement of cell sizes was according to that described
by Bay and Tay (52). Light micrographs containing 76–115 cells taken
at magnifications of 100 times were analyzed. Image acquisition was
performed via a Variocam CCD camera mounted on a copy stand and
analyzed with KS 400 software (Kontron Instruments Ltd.,
Eching, Germany).
Immunofluorescence Microscopy
Cells were grown on glass coverslips in six-well plates and
treated with progesterone in 0.1% ethanol or 0.1% ethanol for 48
h. After rinsing with PBS, the cells were fixed in 4% paraformaldehyde
for 10 min and permeabilized with 0.2%Triton X-100 for 10 min. This
was followed by incubation with 2% normal horse serum in PBS for
1 h to block nonspecific binding. All the subsequent incubations
with Ab were carried out in PBS containing 2% normal horse serum. Ab
to paxillin, talin, and phosphotyrosine alone or in combinations (for
colocalization) were incubated with the cells overnight at 4 C,
followed by incubation with FITC- or rhodamine-conjugated sheep
antimouse, antirat, or antirabbit IgG at room temperature for 1 h.
For F-actin staining, the fixed and permeabilized cells were incubated
with 10 µg/ml FITC-phalloidin in PBS for 1 h at room
temperature. After washing in PBS, the coverslips were mounted on
slides with fluorescence mounting media from DAKO Corp.
(Carpinteria, CA). Stained cells were viewed and photographed using the
model LSM 510 Carl Zeiss confocal laser scanning
microscope.
Immunoprecipitation
Cells (1–5 x 106) grown on 100-mm
petri dishes were lysed with 200 µl cold lysis buffer (50
mM Tris, 150 mM NaCl, 2 mM EDTA,
0.5 mM EGTA, 1 mM sodium vanadate, 0.1% sodium
deoxycholate, 0.5% Triton X-100, and a cocktail of protease inhibitors
for serine, cysteine, and metalloproteases, pH 7.5) at 4 C for 30 min
before they were scraped and harvested. The protein supernatants were
collected by centrifugation at 30,000 x g for 30 min
and the protein concentrations in the lysates were determined using a
protein assay kit (Bio-Rad Laboratories, Inc.). Protein
(400 µg) was incubated with Ab against paxillin, FAK, or integrin
ß1 in lysis buffer overnight at 4 C. The Ab-bound proteins were
precipitated with protein A/G Sepharose. The protein A/G Sepharose
beads were then boiled for 5 min in sample buffer, and the supernatants
containing the protein of interest were analyzed by Western blotting
using ECL kit (Amersham Pharmacia Biotech, Arlington
Heights, IL). After probing with antiphosphotyrosine Ab, the membrane
was stripped in buffer containing 62.5 mM
Tris-HCl, pH 6.7, 2% SDS, and 100 mM
ß-mercaptoethanol for 30 min at 55 C. The membrane was reprobed with
the respective Ab of interest to determine the relative amounts of each
protein expressed.
Antibody Inhibition
Cells grown on six-well plates were treated with 1
nM progesterone or 0.1% ethanol for 48 h. Ab to
integrins 2, 3, 4, 5, 6, ß1, or ß4 were added to
cells at 24 h after progesterone treatment and were incubated for
a further 24 h. Effects of these integrin Ab to
progesterone-induced cell spreading and focal adhesion were viewed and
photo- graphed.
Scanning Electron Microscopy
Cells on glass coverslips were fixed in 100 mM
cacodylate buffer containing 5% glutaraldehyde, pH 7.2, for 20 min.
After extensive washing with cacodylate buffer, coverslips were
incubated for 15 min in 1% osmium tetroxide in cacodylate buffer and
then dehydrated by successive 5-min incubations in 50%, 75%, and 95%
ethanol. The coverslips were incubated 3 times for 5 min each in 100%
ethanol, and then were dried in a Balzer’s CPD 030 critical point
dryer using liquefied carbon dioxide. Coverslips were sputter coated
with 20 nM gold before viewing in a scanning electron
microscope.
Statistical Analysis
Differences between treatments were tested by ANOVA. When
significant differences were detected by ANOVA, multiple comparisons
among means were performed by the least significant difference test.
Correlation analysis was performed using the program in
Excel.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was funded by the National Medical Research Council, Republic of Singapore.
Received for publication March 3, 1999. Revision received October 25, 1999. Accepted for publication November 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2ß1 integrin. J Cell Sci 102:437–446This article has been cited by other articles:
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, Ethanol-treated controls; 
, 1
nM progesterone-treated group.