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Regular Article |
From Drug Discovery, The R. W. Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania
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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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Thrombin is known to influence the behavior of all of the cells identified within the TME. For instance, thrombin activates platelets to adhere to other cells or extracellular matrix, increases vascular permeability and expression of adhesion molecules, attracts monocytes, stimulates mitogenic activity of endothelial cells and fibroblasts, and degranulates mast cells.6-9 Thrombin also influences the rate of deposition of connective tissue proteins and the development of tissue fibrosis during normal wound healing; a process similar to cellular metastasis.10,11 Many of thrombin’s effects are mediated through the seven transmembrane G-protein coupled receptors, protease-activated receptor (PAR)-1, via proteolytic cleavage of the amino-terminal extension unveiling a new amino terminus that activates the receptor through a tethered peptide ligand mechanism.12 In vitro studies have demonstrated increased tumor cell adhesion to endothelium, extracellular matrix and platelets, enhanced metastatic capacity of tumor cells, and activated cell growth and stimulation of angiogenesis in response to thrombin and PAR-1 agonist peptides.13-18 PAR-1 has been localized in smooth muscle cells,19 pancreas tumor cells,20-21 carcinoma and melanoma cell lines16 and recently in human mast cells.22 In breast carcinoma cells, the level of PAR-1 expression has been correlated to the degree of invasiveness.23 Furthermore, B16F10 melanoma cells, transfected with PAR-1, enhanced thrombin-treated tumor cell adhesion to fibronectin 2.5-fold in vitro and pulmonary metastasis as high as 39-fold in vivo compared to the control thrombin-treated tumor cells.24
Trypsin can stimulate fibroblasts to secrete procollagen, stimulate mast cells to degranulate, and is secreted by numerous tumor cell lines that are correlated with the stage and histological type of carcinoma.4,25-27 Some of the actions of trypsin are mediated by a second protease-activated receptor known as PAR-2.8,28-30 PAR-2 has been described in human tissues and tumor cell lines20,29-33 as well as in human mast cells.22 The ability of trypsin to degrade matrix proteins suggests it may participate in the processes of invasion, adhesion and metastasis; however, the presence of trypsin in tumors also suggests that PAR-2 may mediate these processes.34 Although it is clear that tumor-derived trypsin-like enzymes could directly regulate growth in an autocrine and/or paracrine manner via PAR-2 activation,30 the function of PAR-2 activation remains to be fully characterized.
The expression of PAR-1 and PAR-2 in malignant and benign human tumor tissues has not been extensively described in their histological context among the surrounding cell types forming the TME. The aim of this study was to characterize the expression of PAR-1 and PAR-2 protein and mRNA in normal, benign and malignant human tissues using immunohistochemistry and in situ hybridization, respectively. In addition, in vitro studies were used to investigate the presence of PAR-1 and PAR-2 in quiescent (hyperconfluent), proliferating (subconfluent), and wounded cultured fibroblasts. The results from these studies highlight the changes in expression of PAR-1 and PAR-2 in supporting cells in the TME as tumors gain metastatic potential.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary antibodies used in these experiments include the
following: desmin (Dako, Carpinteria, CA), endothelial cell (CD31;
Dako), fibroblast (prolyl 4-hydroxylase) (Dako), macrophage (CD68;
Dako), mast cell tryptase (Dako), non-immuno serum (Vector
Laboratories, Burlingame, CA), PAR-1 (The Robert Wood Johnson
Pharmaceutical Research Institute (RWJPRI), Spring House,
PA),35-37
PAR-2 (RWJPRI),33,35,38
smooth
muscle actin (Dako), DNA topoisomerase II
(TOPO II) (Pharmingen,
San Diego, CA)39
and vimentin (Dako).
3'-Biotinylated molecular probes used for in situ hybridization include the following: PAR-1 (5' TTC ATT TTT CTC CTC CTC CTC CTC ATC C) (Research Genetics, AL),36-37 PAR-2 (5' CAA TAA TGT AGA CGA CCG GAA GAA AGA) (Research Genetics, Huntsville, AL),38 glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) (5' GAC GCC TGC TTC TCC TCC TTC TTG) (Ransom Hill, Ramona, CA), poly d(T) (5' TTT TTT TTT TTT TTT TTT TTT TTT) (Research Genetics), lac Z (5' CAC AGC GGA TGG TTC GGA TAA TG) (Ransom Hill).
Immunohistochemistry
Commercial human checkerboard tissue slides (Dako; Biomeda, Foster City, CA) representing normal breast tissues (n = 26), benign breast fibroadenomas (n = 14), malignant breast carcinomas (n = 46), and six other non-breast human carcinomas (n = 4 to 6 of each) were deparaffinized, hydrated and processed for routine immunohistochemistry (IHC) as previously described.33 Briefly, slides were microwaved in Target buffer (Dako), cooled, placed in phosphate-buffered saline (PBS; pH 7.4) and treated with 3.0% H2O2 for 10 minutes. Slides were processed through an avidin-biotin blocking system according to the manufacturer’s instructions (Vector Laboratories) and then placed in PBS. All subsequent reagent incubations and washes were performed at room temperature.
Normal blocking serum (Vector Laboratories) was placed on all slides for 10 minutes. After briefly rinsing in PBS, primary antibodies were placed on slides for 30 minutes. The slides were washed and biotinylated secondary antibodies, goat anti-rabbit (polyclonal antibodies) or horse anti-mouse (monoclonal antibodies) were placed on the tissue sections for 30 minutes (Vector Laboratories). After rinsing in PBS, the avidin-horseradish peroxidase-biotin complex reagent (ABC; Vector Laboratories) was added for 30 minutes. Slides were washed and treated with the chromogen 3,3'-diaminobenzidine (DAB, Biomeda) twice for 5 minutes each, then rinsed in dH2O, and counterstained with hematoxylin, dehydrated in graded ethanols, cleared in xylene, coverslipped in Permount (Fisher Scientific, Pittsburgh, PA) and photographed with an Olympus BX50 light microscope. The negative controls included replacement of the primary antibody with pre-immune serum or with the same species IgG isotype non-immune serum.
Analysis of PAR-1 and PAR-2 Immunoreactivity
The tissues were scored for the intensity of PAR-1 and PAR-2
immunoreactivity to compare the relative amounts of PAR-1 and PAR-2 in
the stromal fibroblasts and epithelial cells in the normal
(n = 26), benign (n = 14)
and malignant (n = 46) breast tissues. For each
tissue, the presence of PAR-1 and PAR-2 immunoreactivity in the stromal
fibroblasts were ranked under a 20x objective according to the
following criteria: 1) no immunoreactivity (N); 2) weak, light brown
immunoreactivity (W); 3) moderate brown immunolabeling (M), and 4)
intense, dark brown immunoreactivity (S) (Table 1)
. The negative controls did not produce
observable labeling.
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To help characterize the PAR-1 and PAR-2 positive stromal cells,
we used double immunohistochemical methods (IHC:IHC) to simultaneously
detect PAR-1 or PAR-2 expression with detection of a proliferation
marker Topo II,39
a fibroblasts marker (prolyl
4-hydroxylase), an endothelial marker (CD31), and smooth muscle actin
(SMA). Protocols for IHC:IHC have been previously
described.40
Briefly, slides were first processed for
single IHC labeling protocols for detection of each marker antibody as
described above, except that the chromogen was SG (Vector
Laboratories). Without processing the slides for hematoxylin, PAR-1 or
PAR-2 antibodies were placed on the tissues for 30 minutes. After
several PBS washes, the biotinylated horse anti-mouse secondary
antibodies (Vector Laboratories) were similarly incubated. The presence
of PAR-1 or PAR-2 positive cells was visualized using an alkaline
phosphatase detection system through incubation with alkaline
phosphatase conjugated ABC followed by development using the Fast Red
chromogen (Sigma Chemical Co., St. Louis, MO). Slides were then
routinely counterstained and coverslipped with a water-based mounting
media (Dako).
In Situ Hybridization
Slides were routinely dewaxed, rehydrated, placed in 3% H2O2 for 10 minutes at room temperature, and processed for in situ hybridization (ISH) as previously described.36-38 Briefly, after a 5-minute wash in water, slides were placed in Universal buffer (Research Genetics) and the tissue sections were digested with pre- diluted pepsin (Research Genetics) for 10 minutes at 42°C. Sections were washed and then dehydrated in 100% alcohol for 1 minute. Each probe was diluted to 1.0 µg/ml in commercially formulated hybridization buffer (Biomeda) and heated for 5 minutes at 103°C in a microcentrifuge tube on a heat block. The ISH probes were maintained at 42°C in a water bath until placement onto the tissue sections. Ten microliters of probe was added to each section, and a coverslip was gently placed to cover the solution and prevent evaporation. Slides were placed in a humid chamber and incubated at 42°C for 2 hours. After hybridization, they were then immediately placed into a low stringency wash (2 x SSC) for 5 minutes at 42°C, followed by a high stringency wash (0.1 x SSC) for 5 minutes at 42°C. Sections were washed in PBS and treated with ABC for 1 hour at room temperature. After washing, sections were placed in DAB for 2 x 5 minutes, washed, briefly stained with hematoxylin, then coverslipped. Positive controls included two biotinylated mRNA oligonucleotide probes: GAPDH mRNA and a poly d(T) probe that hybridizes non-specifically to all mRNA. Negative controls included 1) the absence of probe in the probe cocktail; 2) a biotinylated probe that hybridizes to lac Z operon mRNA; and 3) pre-digestion of the tissues with RNase, DNase free (10 µg/µl, Boehringer Mannheim, Indianapolis, IN) for 2 hours at 42°C before probe hybridization.
Cell Culture
Human neonatal dermal fibroblasts and their culture media were obtained from Clonetics/BioWhittaker (Walkersville, MD). Cell suspensions (5 x 104/ml) were seeded in 4-well chamber slides (NUNC, Naperville, IL) for immunocytochemistry. Cells were incubated for either 2 days (subconfluent, proliferative conditions) or 9 days (hyperconfluent, quiescent conditions) before evaluation without serum exchange. To mimic the in vivo activation of differentiated, quiescent fibroblasts in vitro, 9-day quiescent cultured cells were subjected to scrape wounding, which was induced by the end of a pipette, and then cultured for 5 additional days without medium exchange (wound conditions). As a control, other 9-day cultures without scrapes continued to grow in parallel.
Immunocytochemistry
Four-chambered culture slides were routinely fixed with 10%
neutral buffered saline for 10 minutes at room temperature, rinsed in
PBS, and then assayed for ICC as previously
described.24,35
Hyperconfluent (quiescent), subconfluent
(proliferating), and wounded cultures were processed for ICC using
antibodies to PAR-1, PAR-2, smooth muscle actin (SMA), Topo II and
pre-immune serum. Before processing, the chambers were carefully
removed from the slides. All washing steps were performed using
Automation Buffer with Tween-20 (Research Genetics). Primary antibodies
were added to the wells for 30 minutes at room temp. After
washes, the secondary antibodies were similarly incubated on the cells.
Subsequently, the presence of the primary antibodies were detected
using the ABC followed by DAB development for 2 x 5 minutes each.
The slides were then counterstained using hematoxylin and coverslipped.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PAR-1 and PAR-2 proteins were localized in formalin-fixed,
paraffin-embedded tissues. Normal (n = 26),
benign (n = 14), and malignant
(n = 46) human breast tissues and six non-breast
carcinomas (n = 4–6 of each) were assayed
simultaneously in a multitissue format to eliminate potential staining
artifacts such as slide-to-slide and run-to-run variability. Marginal
increases of PAR-1 and PAR-2 expression were observed in the malignant
cells as compared to the normal and benign epithelial cells. Striking
changes in PAR-1 and PAR-2 expression were noted in the stromal
fibroblasts surrounding the malignant cells as compared to the
fibroblasts surrounding the normal and benign epithelial cells (Table 1)
. No PAR-1 or PAR-2 immunolabeling was observed in the stromal
fibroblasts of the benign (n = 14) or normal
(n = 26) breast tissues. In contrast, most
malignant tissues had prominent moderate to strong PAR-1
(n = 39/46) and PAR-2 (n
= 37/46) labeling in the stromal fibroblasts.
We applied additional immunohistochemical markers to further
characterize these tissues (Figure 1)
. No
immunolabeling was detected using negative control antibodies in
normal, benign and malignant breast tissue stromal fibroblasts or
epithelial cells (data not presented). SMA-positive immunolabeling was
localized in the myoepithelial cells (large arrowheads) around the
epithelial ducts and in the vascular smooth muscle cells in the normal
(Figure 1A)
and benign fibroadenoma (Figure 1B)
breast tissues. SMA
immunolabeling was absent from stromal fibroblasts in the normal
(Figure 1A)
and benign (Figure 1B)
tissues, which were immunoreactive
to the fibroblast marker (data not presented). In the malignant breast
carcinoma tissues, SMA immunolabeling (small arrowheads) was prominent
in the stromal fibroblasts surrounding the tumor cells, in addition to
the vascular smooth muscle cells (Figure 1C)
. Carcinoma cells (large
arrowheads) did not express SMA. Positive, nuclear Topo II
immunolabeling (large arrowhead), a marker for proliferating
cells,39
was sparsely observed in normal breast epithelial
cells (Figure 1D)
and absent in stromal fibroblasts (small arrowheads).
Topo II nuclear immunolabeling was observed in the benign,
fibroadenoma cells (large arrowheads), but was similarly absent in the
surrounding stromal fibroblasts (small arrowheads) in Figure 1E
. In
contrast, Topo II nuclear immunolabeling was observed in stromal
fibroblasts (small arrowheads) and tumor cells (large arrowheads) of
the malignant tissues (Figure 1F)
. Furthermore, the stromal fibroblasts
surrounding the malignant cells also expressed vimentin but did not
express desmin (data not presented).
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and
PAR-2 (Figure 1J)
immunolabeling (large arrowheads) was confined to the
normal breast ductal epithelial cells. PAR-1 (Figure 1H)
and PAR-2
(Figure 1K)
immunolabeling (large arrowheads) was also observed in the
fibroadenoma cells. In both cases, normal and benign tissues,
surrounding stromal fibroblasts (small arrowheads) did not express
detectable PAR-1 (Figure 1, G and 1H
, respectively) or PAR-2 (Figure 1, J and K
, respectively). In the breast carcinoma tissues, PAR-1 and
PAR-2 positive immunoreactivity was observed in many cell types forming
the TME such as in the malignant cells (large arrowheads) and the
stromal fibroblasts (small arrowheads) (Figure 1, I and L)
. Although
not present in these photomicrographs, PAR-1 and PAR-2 immunolabeling
was also observed in endothelial cells, vascular smooth muscle cells,
as well as in the mast cells and macrophages. The PAR-1 and PAR-2 mast
cell labeling pattern was consistent with our previous
report,22
and was similarly localized to the plasma
membrane and to the membranes of the secretory vesicles. PAR-1 and
PAR-2 positive macrophages were also observed around these cancerous
tissues. Labeling in the macrophages was observed in or on the plasma
membrane as well as intracellularly (Figure 1, I and L)
. The identity
of the mast cells and macrophages were confirmed in these tissue
sections using antibodies to mast cell tryptase and macrophages (CD68)
(data not presented). Co-Localization of PAR-1 and PAR-2 Expression in Proliferating-, Smooth Muscle Actin-, Fibroblastic-Positive Cells in Situ
The results of double immunohistochemical labeling using
antibodies to PAR-1 or PAR-2 with antibodies to Topo II demonstrated
that proliferating stromal cells expressed PAR-1 and PAR-2
immunolabeling as represented in Figures 2A and 2B
, respectively. Co-localization
of PAR-1 (Figure 2A)
or PAR-2 (Figure 2B)
immunolabeled red with black,
immunolabeled Topo II positive nuclei was observed in both malignant
cells (large arrowheads) and stromal fibroblasts (small arrowheads) in
the breast carcinoma tissues. The proliferating, PAR-1 and PAR-2
positive stromal cells surrounding the carcinoma cells also expressed
SMA (small arrowheads, Figures 2C and 2D
, respectively). This
immunophenotype was not observed in normal and benign breast tissues
(data not presented). Furthermore, these proliferating, SMA, PAR-1 and
PAR-2 stromal cells also were immunolabeled with antibodies for prolyl
4-hydroxylase, a fibroblast marker (small arrowheads, Figure 2, E and F
, respectively). These cells did not express the endothelial marker,
CD31 (Figure 2, G and H
, respectively; small arrowheads), which was
only observed in endothelium of nearby vessels (arrows).
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Other human non-breast malignant tumors demonstrated similar PAR-1
and PAR-2 expression in the tumor cells (large arrowheads), stromal
fibroblasts (small arrowheads), mast cells and macrophages (arrows), as
well as in the endothelial and vascular smooth muscle cells (Figure 3)
. Figure 3
shows PAR-1 (Figure 3, A, C and E)
and PAR-2 (Figure 3, B, D, and F)
immunolabeling in tissues
representing a gastric carcinoma (n = 4; Figure 3, A and B
), an undifferentiated carcinoma (n =
4, Figure 3, C and D
) and a lung adenocarcinoma
(n = 4, Figure 3, E and F
). PAR-1 and PAR-2
immunoreactivity was similarly present in heptacarcinomas
(n = 6), thyroid carcinomas
(n = 4) and ovarian carcinomas
(n = 6) (data not shown). Positive PAR-1 and
PAR-2 immunoreactivity was also observed on surrounding endothelial and
vascular smooth muscle cells, as well as in the stromal fibroblasts in
contrast to the absence of PAR-1 and PAR-2 immunoreactivity in the
stromal fibroblasts on the normal tissue counterparts (data not
presented).
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The PAR-1 and PAR-2 protein expression correlated well with their
respective mRNA levels in the same tissues as determined by in
situ hybridization. The localization patterns of PAR-1 (Figure 4A)
and PAR-2 (Figure 4B)
mRNA were
observed in human breast carcinoma tissues (n =
48). Figure 4A
shows the intracellular localization of PAR-1 mRNA in
the malignant tumor cells (large arrowheads) and in the surrounding
stromal fibroblasts (small arrowheads). PAR-1 mRNA was not present in
the stromal cells of the normal (n = 26) and
benign (n = 10) breast tissues (data not
presented). Similar localization patterns were observed for PAR-2 in
the same breast carcinoma tissues as shown in Figure 4B
, and PAR-2 mRNA
was also not present in the stromal cells of the normal and benign
breast tissues (data not presented). As a positive control probe, cells
also expressed GAPDH mRNA (Figure 4C)
. When the same tissues were
probed with the lac Z biotinylated mRNA probe (negative
control), no observable labeling was observed in tumor cells (large
arrowheads) or stromal fibroblasts (small arrowheads) (Figure 4D)
.
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In Vitro PAR-1 and PAR-2 Expression
Our IHC and ISH results indicated that PAR-1 and PAR-2 expression
was induced in stromal fibroblasts during the transition to a
myofibroblast phenotype. We used ICC to determine whether this
transition could be mimicked in vitro. Hyperconfluent,
fibroblast cultures (quiescent conditions) were compared to 1)
subconfluent cultures with visible mitotics (proliferative conditions),
and 2) confluent cultures subjected to a mechanical scrape and
allowed to recover for 5 days without changing the media (wound
conditions). Figure 5
(Figure 5, A
-C)
shows the lack of observable immunolabeling using negative control
antibodies in all three tissue culture conditions. SMA immunolabeling
was present in the proliferating cells (Figure 5E
, arrowheads) and in
the cells migrating over the scraped area (Figure 5F
, arrowheads), but
was absent in the confluent cultured cells (Figure 5D)
, suggesting that
the confluent conditions produced quiescent, differentiated cells that
were not myofibroblasts. Immunoreactivity to the proliferation marker,
Topo II, was present in the nuclei of the proliferating cells in the
subconfluent cultures (Figure 5H
, arrowheads) and in the cells
migrating over the scraped area in the wounded cultures (Figure 5I
,
arrowheads), but was absent in the cell nuclei of the quiescent cells
(Figure 5G)
, further confirming the quiescent, non-proliferating status
of these differentiated fibroblasts when grown to confluency.
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, respectively). However, positive PAR-1 and PAR-2
immunolabeling (arrowheads) was observed in the proliferating cells in
the subconfluent (Figures 5K and 5N
, respectively) and wounded (Figures 5, L and O
, respectively) conditions. PAR-1 and PAR-2 Expression Is Associated with Proliferating Cells in Vitro
In an effort to replicate our in situ findings in the
in vitro condition, we similarly applied double
immunohistochemical labeling using antibodies to PAR-1 or PAR-2 with
antibodies to Topo II, and demonstrated that proliferating cells
expressed PAR-1 and PAR-2 immunolabeling. Figure 5, P and Q
,
respectively, show representative examples of these results.
Co-localization of red immunolabeled PAR-1 (Figure 5P)
or red
immunolabeled PAR-2 (Figure 5Q)
with black immunolabeled Topo II
positive nuclei was observed in proliferating cultured fibroblasts
(large arrowheads). As an internal control, a nearby non-proliferating,
Topo II-negative cells (small arrowheads) did not express obvious
detectable levels of PAR-1 (Figure 5P)
or PAR-2 (Figure 5Q)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PAR-1 and PAR-2 expression has previously been shown on endothelial cells, vascular smooth muscle cells and mast cells. It is therefore not surprising to find similar results for these cell types within the TME. Activation of either PAR-1 or PAR-2 on these cells results in characteristic events associated with inflammatory responses such as generation of cytokines, expression of adhesion molecules and increased vascular permeability. However, little is known about the presence of PAR-1 and PAR-2 on macrophages. It has been reported that macrophages can secrete thrombin,41 and that thrombin has been localized in pulmonary alveolar macrophages,42 suggesting an association between macrophages and thrombin. The presence of PAR-1 and PAR-2 on human macrophages in malignant tumors in situ has not been reported previously, although PAR-2 immunoreactivity has been reported on macrophage-like cells in the adventitia of the mouse isolated ureter.43 Here, we show that macrophages express both PAR-1 and PAR-2. PAR-1 and PAR-2 activation may provide a stimulus for macrophages to proliferate, migrate and/or phagocytize degraded stromal proteins, in addition to synthesizing and secreting thrombin and growth factors into the TME.
The most striking observation from our study is the presence of PAR-1
and PAR-2 on the stromal fibroblasts surrounding the metastatic tumor
cells but not on the stromal fibroblasts surrounding the benign,
non-metastatic, or normal epithelial cells. The exact origin of the
PAR-1 and PAR-2 expressing stromal fibroblasts is unclear, ie, local
dedifferentiated stromal fibroblasts, vascular smooth muscle cells, or
migrating undifferentiated stem cells such as pericytes.44
In breast cancer, it has been shown that primary fibroblasts convert to
the myofibroblast phenotype when exposed to tumor cells; vascular
smooth muscle cells and pericytes can also differentiate to
myofibroblasts, but to a lesser extent.44
The stromal
fibroblasts associated with metastatic tumors in our study were
characterized by the positive expression of SMA, prolyl 4-hydroxylase
(a fibroblast marker), Topo II (a proliferation marker) and
vimentin, as well as the absence of the vascular markers desmin and
CD31, confirming the fibroblastic nature of these
cells.44-48
Reactive stromal myofibroblasts are frequently associated with cancers of epithelial origin, a process known as desmoplasia.49 The induction of this phenotype has not been well characterized, however in vitro studies have indicated that diffusable signals, such as transforming growth factor-ß, generated from primed or initiated carcinoma cells are involved.44,50-52 The stromal myofibroblasts, in turn, influence the invasive and metastatic potential of carcinoma cells by an unidentified mechanism1 once the carcinoma cells invade the basement membrane surrounding the epithelial cells. Elaboration of matrix degrading proteases, deposition of new extracellular matrix proteins to facilitate tumor cell adhesion, cell motility and cell proliferation,1,11 and release of cytokines and growth factors by these myofibroblasts emphasize the importance of this phenotypic change to the invasiveness of the tumor. PAR-1 and PAR-2 activation results in many of these biochemical events, indicating that they are likely participants in the balance of tumor containment and/or metastasis.53-58 Moreover, the expression of tissue factor, an essential co-factor for plasma coagulation factor VII/VIIa, was reported to be consistently observed in stromal cells of invasive breast carcinomas but not in the benign breast tumors.58 The increased presence of tissue factor/factor VIIa within the TME, which in turn can generate thrombin via the extrinsic coagulation pathway on fibroblasts, parallels our observation of increased PAR-1 expression.
Benign proliferative disorders are characterized by a continuous basement membrane separating the epithelium from the stroma, similar to the normal tissue organization.2 It is possible that the presence of a continuous basement membrane may actually quarantine any tumor-derived thrombin or trypsin from the stromal fibroblasts. Thus, the actions of thrombin and trypsin within the TME may be accentuated through up-regulation of PAR-1 and PAR-2 in the stromal fibroblasts as they de-differentiate (ie, SMA-negative to SMA-positive). The activation of PAR-1 and PAR-2 on tumor cells contributes to migration by increasing their adhesive properties and releasing urokinase, both of which are early changes during the initiation of metastasis.14,59-60
We were able to mimic our in situ observations in
vitro using cultured human dermal fibroblasts. Quiescent,
SMA-negative, non-proliferating (Topo II-negative) cell cultures did
not express detectable PAR-1 or PAR-2, similar to those of the stromal
fibroblasts in normal and benign human tissues in situ. Most
notably, we were able to mimic the transformation of PAR-1 and
PAR-2-negative to PAR-1 and PAR-2-positive fibroblasts in
vitro, after the quiescent cells were subjected to scrape wounding
indicating that cell damage relays a signal for PAR induction.
In summary, this is the first in situ histological comparative report describing the presence of PAR-1 and PAR-2 protein and mRNA in human malignant tumor cells and local mast cells, macrophages, endothelium, and vascular smooth muscle cells of the TME. More importantly, we observed PAR-1 and PAR-2 immunolabeling in the stromal fibroblasts immediately surrounding the malignant cells that was absent in the surrounding stromal fibroblasts of the normal and benign breast epithelial cells. The presence of both PARs and their activating proteases within the TME suggests an autocrine and/or paracrine cascade in the processes of cellular metastasis, perhaps as natural mechanisms of tissue injury. It will be important to investigate if there is a correlation between the relative amounts of PAR-1 or PAR-2 in the tumors cells and in the stromal fibroblasts with tumor grade, and to expand our investigations into the expression of all of the members of the PARs into other pathological tissues. Because the degree of tumor cell malignancy has been classified by the amounts of secreted thrombin or trypsin, theoretically, the amounts of PAR-1 and PAR-2 in the TME cells may also be a valid predictor of metastatic activity, thereby acquiring diagnostic and prognostic value. More importantly, these data suggest attractive targets for therapeutic approaches, whereby PAR-1 and PAR-2 antagonists and anti-thrombin and anti-tryptase agents may be directed to disrupt some of the processes of cell metastasis.
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Acknowledgements |
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Footnotes |
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Accepted for publication February 16, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IIß compared with detection of Ki-67, a marker of cellular proliferation, in human tumors. Appl Immunohist 1994, 2:177-185
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 A. Masamune, K. Kikuta, M. Satoh, N. Suzuki, and T. Shimosegawa Protease-Activated Receptor-2-Mediated Proliferation and Collagen Production of Rat Pancreatic Stellate Cells J. Pharmacol. Exp. Ther., February 1, 2005; 312(2): 651 - 658. [Abstract] [Full Text] [PDF]
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 M. R. D'Andrea, M. R. Saban, N. P. Gerard, B. K. Wershil, and R. Saban Lack of neurokinin-1 receptor expression affects tissue mast cell numbers but not their spatial relationship with nerves Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2005; 288(2): R491 - R500. [Abstract] [Full Text] [PDF]
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 L. V. M. Rao and U. R. Pendurthi Tissue Factor-Factor VIIa Signaling Arterioscler. Thromb. Vasc. Biol., January 1, 2005; 25(1): 47 - 56. [Abstract] [Full Text] [PDF]
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 L. Ge, S. K. Shenoy, R. J. Lefkowitz, and K. DeFea Constitutive Protease-activated Receptor-2-mediated Migration of MDA MB-231 Breast Cancer Cells Requires Both {beta}-Arrestin-1 and -2 J. Biol. Chem., December 31, 2004; 279(53): 55419 - 55424. [Abstract] [Full Text] [PDF]
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X. Shi, B. Gangadharan, L. F. Brass, W. Ruf, and B. M. Mueller Protease-Activated Receptors (PAR1 and PAR2) Contribute to Tumor Cell Motility and Metastasis Mol. Cancer Res., July 1, 2004; 2(7): 395 - 402. [Abstract] [Full Text] [PDF]
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D. Darmoul, V. Gratio, H. Devaud, and M. Laburthe Protease-activated Receptor 2 in Colon Cancer: TRYPSIN-INDUCED MAPK PHOSPHORYLATION AND CELL PROLIFERATION ARE MEDIATED BY EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION J. Biol. Chem., May 14, 2004; 279(20): 20927 - 20934. [Abstract] [Full Text] [PDF]
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 V. S. OSSOVSKAYA and N. W. BUNNETT Protease-Activated Receptors: Contribution to Physiology and Disease Physiol Rev, April 1, 2004; 84(2): 579 - 621. [Abstract] [Full Text] [PDF]
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T. Minami, A. Sugiyama, S.-Q. Wu, R. Abid, T. Kodama, and W. C. Aird Thrombin and Phenotypic Modulation of the Endothelium Arterioscler. Thromb. Vasc. Biol., January 1, 2004; 24(1): 41 - 53. [Abstract] [Full Text] [PDF]
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L. Ge, Y. Ly, M. Hollenberg, and K. DeFea A {beta}-Arrestin-dependent Scaffold Is Associated with Prolonged MAPK Activation in Pseudopodia during Protease-activated Receptor-2-induced Chemotaxis J. Biol. Chem., September 5, 2003; 278(36): 34418 - 34426. [Abstract] [Full Text] [PDF]
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 J. M. Carton, D. J. Uhlinger, A. D. Batheja, C. Derian, G. Ho, D. Argenteri, and M. R. D'Andrea Enhanced Serine Palmitoyltransferase Expression in Proliferating Fibroblasts, Transformed Cell Lines, and Human Tumors J. Histochem. Cytochem., June 1, 2003; 51(6): 715 - 726. [Abstract] [Full Text] [PDF]
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C. D. Major, R. J. Santulli, C. K. Derian, and P. Andrade-Gordon Extracellular Mediators in Atherosclerosis and Thrombosis: Lessons From Thrombin Receptor Knockout Mice Arterioscler. Thromb. Vasc. Biol., June 1, 2003; 23(6): 931 - 939. [Abstract] [Full Text] [PDF]
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D. Darmoul, V. Gratio, H. Devaud, T. Lehy, and M. Laburthe Aberrant Expression and Activation of the Thrombin Receptor Protease-Activated Receptor-1 Induces Cell Proliferation and Motility in Human Colon Cancer Cells Am. J. Pathol., May 1, 2003; 162(5): 1503 - 1513. [Abstract] [Full Text] [PDF]
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M. R. D'Andrea, M. R. Saban, N.-B. Nguyen, P. Andrade-Gordon, and R. Saban Expression of Protease-Activated Receptor-1, -2, -3, and -4 in Control and Experimentally Inflamed Mouse Bladder Am. J. Pathol., March 1, 2003; 162(3): 907 - 923. [Abstract] [Full Text] [PDF]
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C. K. Derian, B. P. Damiano, M. F. Addo, A. L. Darrow, M. R. D'Andrea, M. Nedelman, H.-C. Zhang, B. E. Maryanoff, and P. Andrade-Gordon Blockade of the Thrombin Receptor Protease-Activated Receptor-1 with a Small-Molecule Antagonist Prevents Thrombus Formation and Vascular Occlusion in Nonhuman Primates J. Pharmacol. Exp. Ther., February 1, 2003; 304(2): 855 - 861. [Abstract] [Full Text] [PDF]
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 Y. Li and F. H. Sarkar Gene Expression Profiles of Genistein-Treated PC3 Prostate Cancer Cells J. Nutr., December 1, 2002; 132(12): 3623 - 3631. [Abstract] [Full Text] [PDF]
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 A. F. Milia, M. B. Salis, T. Stacca, A. Pinna, P. Madeddu, M. Trevisani, P. Geppetti, and C. Emanueli Protease-Activated Receptor-2 Stimulates Angiogenesis and Accelerates Hemodynamic Recovery in a Mouse Model of Hindlimb Ischemia Circ. Res., August 23, 2002; 91(4): 346 - 352. [Abstract] [Full Text] [PDF]
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 N. Vergnolle, C. K. Derian, M. R. D'Andrea, M. Steinhoff, and P. Andrade-Gordon Characterization of Thrombin-Induced Leukocyte Rolling and Adherence: A Potential Proinflammatory Role for Proteinase-Activated Receptor-4 J. Immunol., August 1, 2002; 169(3): 1467 - 1473. [Abstract] [Full Text] [PDF]
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