PAR Genes: Molecular Probes to Pathological Assessment in Breast Cancer Progression
Taking the issue of tumor categorization a step forward and establish molecular imprints to accompany histopathological assessment is a challenging task. This is important since often patients with similar clinical and pathological tumors may respond differently to a given treatment. Protease-activated receptor-(1) (PAR(1)), a G protein-coupled receptor (GPCR), is the first member of the mammalian PAR family consisting of four genes. PAR(1) and PAR(2) play a central role in breast cancer. The release of N-terminal peptides during activation and the exposure of a cryptic internal ligand in PARs, endow these receptors with the opportunity to serve as a "mirror-image" index reflecting the level of cell surface PAR(1&2)-in body fluids. It is possible to use the levels of PAR-released peptide in patients and accordingly determine the choice of treatment. We have both identified PAR(1) C-tail as a scaffold site for the immobilization of signaling partners, and the critical minimal binding site. This binding region may be used for future therapeutic modalities in breast cancer, since abrogation of the binding inhibits PAR(1) induced breast cancer. Altogether, both PAR(1) and PAR(2) may serve as molecular probes for breast cancer diagnosis and valuable targets for therapy.
SAGE-Hindawi Access to Research
Pathology Research International
Volume 2011, Article ID 178265, 6pages
PAR Genes: Molecular Probes to Pathological Assessment in
Breast Cancer Progression
Beatrice Uziely,1Hagit Turm,1Myriam Maoz,1Irit Cohen,1Bella Maly,2
and Rachel Bar-Shavit1
1Departments of Oncology, Hadassah-University Hospital P.O. Box 12000, Jerusalem 91120, Israel
2Departments of Pathology, Hadassah-University Hospital P.O. Box 12000, Jerusalem 91120, Israel
Correspondence should be addressed to Rachel Bar-Shavit, firstname.lastname@example.org
Received 15 September 2010; Accepted 4 January 2011
Academic Editor: Beiyun Chen
Copyright © 2011 Beatrice Uziely et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Taking the issue of tumor categorization a step forward and establish molecular imprints to accompany histopathological
assessment is a challenging task. This is important since often patients with similar clinical and pathological tumors may respond
diﬀerently to a given treatment. Protease-activated receptor-1(PAR1), a G protein-coupled receptor (GPCR), is the ﬁrst member of
the mammalian PAR family consisting of four genes. PAR1and PAR2play a central role in breast cancer. The release of N-terminal
peptides during activation and the exposure of a cryptic internal ligand in PARs, endow these receptors with the opportunity to
serve as a “mirror-image” index reﬂecting the level of cell surface PAR1&2-in body ﬂuids. It is possible to use the levels of PAR-
released peptide in patients and accordingly determine the choice of treatment. We have both identiﬁed PAR1C-tail as a scaﬀold
site for the immobilization of signaling partners, and the critical minimal binding site. This binding region may be used for future
therapeutic modalities in breast cancer, since abrogation of the binding inhibits PAR1induced breast cancer. Altogether, both PAR1
and PAR2may serve as molecular probes for breast cancer diagnosis and valuable targets for therapy.
The classiﬁcation of a tumor diﬀerentiation level is rou-
tinely based on histopathological criteria whereby poorly
diﬀerentiated tumors generally exhibit the worst prognoses.
However, the underlying molecular pathways that regulate
the level of breast tumor development are as yet poorly
described. Until now the pathological tissue criteria that
entail tissue traits have not been deﬁned by an appropriate
set of genes. A challenging task is to take the issue of breast
tumor categorization a step forward and establish molecular
imprints to accompany histopathological assessment. This
is important since often patients with similar clinical and
pathological tumors may have a markedly diﬀerent outcome
in response to a given treatment. These diﬀerences are
encoded by and stem from the tumor genetic proﬁle .
Individual gene signature may complement or replace the
traditional pathological assessment in evaluating tumor
behavior and risk. This is the basis for optimizing our
approach to personalized care whereby genomic ﬁnger prints
may reﬁne the prediction of the course of disease and the
response to treatment . Oncotype Dx is a clinically vali-
dated and widely used multigene assay (there are also other
commercially available gene panels such as Mammaprint;
Agendia Amsterdam, Netherland, and THEROS H/I; Bio-
theranostics, San Diego, CA), that quantiﬁes the likelihood
of breast cancer recurrence. This gene proﬁle has been
developed speciﬁcally for women with hormone receptor-
positive (estrogen and progesterone receptor; ER, PR) and
lymph node-negative disease. The gene proﬁle consists of 21
genes that are associated with disease recurrence. Sixteen are
cancer-related genes and 5 serve as reference genes. This gene
panel is used to calculate the recurrence score (RS), a number
that correlates with the speciﬁc likelihood of breast cancer
recurrence within 10 years from the original diagnosis.
Therefore, an ongoing goal is to identify important genes that
play a central part in breast cancer biology and determine
their relative function during the course of breast cancer pro-
gression . Identiﬁcation of these genes will signiﬁcantly
contribute to the prospect of treatment making choices.
Protease-activated receptor-1(PAR1), a G protein-coup-
led receptor (GPCR), is the ﬁrst and prototype member of
2Pathology Research International
the mammalian PAR family consisting of four genes. The
activation of PAR1involves the release of an N-terminal
peptide and the exposure of an otherwise hindered ligand,
resulting in an exclusive mode of activation. This mode
of activation serves as a general paradigm for the entire
PAR family [4–6]. While a well-known classical observation
points to a close link between hyperactivation of the coagu-
lation system and cancer malignancies, the molecular mech-
anism that governs procoagulant tumor progression remains
poorly deﬁned [7–10]. Thrombin is a main eﬀector of the
coagulation cascade. In addition to cleaving ﬁbrinogen, it
also activates cells through at least three PARs: PAR1,PAR
and PAR4. In contrast, PAR2is activated by multiple trypsin-
like serine proteases including the upstream coagulant pro-
teases VIIa—tissue factor (TF) and Xa, but not by thrombin.
It is now becoming well established that human Par1,hPar
1, plays a central role in epithelial malignancies [13,14,16].
PAR2, the second member of the family, is also emerging with
central assignments in breast cancer [11,12]. High levels of
hPar1expression are directly correlated with epithelia tumor
progression in both clinically obtained biopsy specimens
and a wide spectrum of diﬀerentially metastatic cell lines
[13,14]. PAR1also plays a role in the physiological invasion
process of placental cytotrophoblasts during implantation
into the uterus deciduas . Trophoblast invasion shares
many features with the tumor cell invasion process. It diﬀers,
however, by the time-limited hPar1expression, which is
conﬁned to the trophoblast-invasive period and is shut oﬀ
immediately thereafter, when there is no need to invade
. This strongly supports the notion that the hPar1gene
is part of an invasive gene program. Surprisingly, the zinc-
dependent matrix-metalloprotease 1 (MMP-1), a collagenase
that eﬃciently cleaves extra cellular matrix (ECM) and base-
ment membrane components, has been shown to speciﬁcally
activate PAR1. PAR1-MMP1 axis may thus provide a
direct mechanistic link between PAR1and tumor metastasis.
The mechanism that leads to hPar1gene overexpression in
tumor is yet unclear and under current extensive investi-
gation. Although the impaired internalization of PAR1that
results with persistent signaling and invasion was previously
suggested for several breast cancer lines , an imbalanced
expression between hPar1repressors and activators was
proposed, suggesting transcriptional regulation . We
found that the mechanism of hPar1overexpression involves
enhanced transcriptional activity, whereby enhanced RNA
chain elongation takes place in the aggressive cancer cells as
compared with the nonaggressive, low metastatic potential
cells . Indeed, we have identiﬁed the Egr-1 transcription
factor as a critical DNA-binding protein eliciting hPar1
expression in prostate cancer cells and the wt p53 tumor
suppressor as an hPar1transcription repressor [19,20]. The
wt form of p53 thus acts as a ﬁne-tuning regulator of hPar1
in cancer progression.
2. Prognostic Parameters of PARs
The PARs act as delicate sensors of extra cellular protease
gradient to allow the cells to respond to a proteolytically
modiﬁed environment. The fact that PAR1gene and protein
overexpression are associated with the aggressiveness of a
tumor, in vivo, reﬂect its potential role in cancer dissemi-
nation. Furthermore, it assigns PAR1as an attractive target
for anticancer therapy. On the other hand, the release of an
N-terminal peptide during activation and the exposure of
an otherwise cryptic internal ligand in PARs endow these
receptors with the opportunity to serve as a “mirror-image”
index reﬂecting in body ﬂuids the level of PARs on the
surface of cancer cells. Hence, PAR1and PAR2peptides in
the blood directly imitate PAR expression serving as a faithful
indicator for the extent of cancer progression. While the
overexpression of both PAR1and PAR2takes place on the
surface of cancer cells that are being constantly turned over
in the body, yet there is no current information as to the half
-life of the released peptides. It is envisioned that measuring
the level of released peptides may underline the severity of
cancer. Another aspect is that the followup levels of PAR1-
released peptides may be instrumental in demonstrating the
eﬀectiveness of a given treatment. For example, determining
the level of the released PAR1and PAR2, through repeated
measurements in the blood stream, may serve as a base
line for a patient, and a sensitive indicator for response
to a treatment. If the released PAR peptides are becoming
gradually low and ﬁnally disappear, it may reassure that the
tumor is indeed regressing until ﬁnally the cancer disappears.
In contrast, if the level remains unchanged, it may indicate
that the tumor is progressing despite of a given treatment.
A critical aspect, however, that needs to be addressed is the
prospect of high released PAR1&2 peptides present during
inﬂammation [21,22]. Therefore, the repeated followup
of PAR released peptides is necessary for the purpose of
demonstrating that during inﬂammation the high PAR-
released peptide level is transient and disappears when the
inﬂammatory response is over. In contrast, in the case of a
tumor, the level of PAR-released peptides remains constantly
high. The relative contribution of PAR1versus PAR2during
the process of tumor progression is as yet unknown and
is under current investigation. One approach to decisively
address this issue is by immunohistological staining (of anti-
PAR1and anti-PAR2antibodies, separately) utilizing tissue
microarray biopsy specimens on a large pool of primary
breast cancer biopsy specimens representing invasive carci-
noma. Such analysis will determine the relative percentage
of PAR-positive individuals in a given cancer patient pool.
Whether PARs join the triple negative population (ER-, PR-,
and Her-2/Neu, an indicator of disease aggressiveness)—
or perhaps stands independently as a prognostic marker—
needs to be evaluated.
3. PARs as Target for Therapy
Importantly, PAR1cellular traﬃcking and signal termination
appear to occur in a diﬀerent mode than other GPCRs.
Instead of recycling back to the cell surface after ligand
stimulation, activated PAR1is sorted to the lysosomes where
it is degraded [23,24]. While cellular traﬃcking of PAR1
impinges on the extent and mode of signaling, the identiﬁca-
tion of individual PAR1signaling partners and their contri-
bution to breast cancer progression remain to be elucidated.
Pathology Research International 3
Breast cancer progression
Steps in epithelia tumor progression
Normal epithelium Dysplasia Carcinoma In-Situ (high grade) Invasive carcinoma
PAR 1&2(+) PAR 1&2(−)PAR1&2(+) PAR1&2(−)PAR1&2(+) PA R1&2(−)PA R1&2(+) PAR1&2(−)
Figure 1: Steps in breast cancer progression. Subtypes deﬁnition of breast cancer according to ER, PR, and Her2/neu status. Additional
categorization is suggested including PARs status.
We have adopted the approach of utilizing a truncated
form of hPar1gene devoid of the entire cytoplasmic tail to
demonstrate the signiﬁcant role of PAR1signaling in breast
tumor progression. This was demonstrated in a xenograft
mice model of mammary gland tumor development, in vivo
. Along this line of evidence, we have identiﬁed PAR1
C-tail as a scaﬀold site for the immobilization of signaling
partners. In addition to identifying key partners, we have
determined the hierarchy of binding and established a region
in PAR1C-tail critical for breast cancer signaling. This
minimal binding domain may provide a potent platform
for future therapeutic vehicles in treating breast cancer. The
above-described outcome is a brief summary of the detailed
experimental approach illustrated bellow.
The functional outcome of MCF7 cells overexpressing
various hPar1constructs in vivo was assessed by orthotopic
mammary fat pad tumor development. MCF7 cells over-
expressing either persistent hPar1Y397Z or wt hPar1con-
structs (e.g., MCF7/Y397Z hPar1;MCF7/wt hPar1)markedly
enhanced tumor growth in vivo following implantation into
the mammary glands, whereas MCF7 cells overexpressing
truncated hPar1, devoid of the entire cytoplasmic tail,
behaved similarly to control MCF7 cells in vector-injected
mice, which developed only very small tumors. The tumors
obtained with MCF7/wt hPar1and MCF7/Y397Z hPar1were
5 and 5.8 times larger, respectively, than tumors produced by
the MCF7/empty vector-transfected cells. Histological exam-
ination (H&E staining) showed that while both MCF7/wt
hPar1and MCF7/Y397Z hPar1tumors inﬁltrated into the
fat pad tissues of the breast, the MCF7/Y397Z hPar1tumors
further inﬁltrated the abdominal muscle. In contrast, tumors
produced by empty vector or truncated hPar1-transfected
cells were capsulated, with no obvious cell invasion. Tumor
growth can also be attributed to blood vessel formation [26,
27]. The hPar1-induced breast tumor vascularization was
assessed by immunostaining with antilectin and anti-CD31
antibodies, showing that both MCF7/Y397Z hPar hPar1and
MCF7/wt hPar1tumors were intensely stained. In contrast,
only few blood vessels were found in the small tumors
of empty vector or truncated hPar1. Thus, both MCF7/wt
4Pathology Research International
COOH Etk binding
SH2 SH3 PK NH2
Schematic presentation of PAR1
C-tail scaﬀold interactions with the signalingpartners
Etk/Bmxand Shc: identiﬁcation of the minimal binding
motif in PAR1C-tail
SH2 SH3 PK NH2
Figure 2: Activation of PAR1leads to the association of Etk/Bmx with PAR1C-tail. This association is mediated through Etk/Bmx PH-
domain enabling next the binding of Shc. The site of the “signal binding” domain (e.g., Etk/Bmx, as a prime signaling partner) in PAR1has
been identiﬁed. Insertion of successive replacement of A residues forming a PAR1mutant incapable of binding Etk/Bmx showed impaired
capabilities of PAR1induced invasion and migration. This site provides therefore a platform for the development of future therapeutic
medicaments in breast cancer.
hPar1and MCF7/Y397Z hPar1cells were shown to eﬀectively
induce breast tumor growth, proliferation, and angiogenesis,
while the MCF7/truncated hPar1and MCF7/empty vector-
expressing cells had no signiﬁcant eﬀect. This experimental
results highlight the signiﬁcance of PAR1signaling in PAR1-
induced breast cancer progression.
4. Antibody Array for Protein-Protein
Interactions Reveals Signaling Candidates
Next, in order to identify speciﬁc PAR1signaling com-
ponents, the following approach was utilized. To detect
the putative mediator(s) linking PAR1to potential signal-
ing pathway, we examined a custom-made antibody-array
membranes. When aggressive breast carcinoma MDA-MB-
435 cells (with high hPar1levels) were incubated with the
antibody-array membranes before and after PAR1activation
(15 minutes), the following results were obtained. Several
activation-dependent proteins which interact with PAR1,
including ICAM, c-Yes, Shc, and Etk/Bmx, were identiﬁed.
Of these proteins, we chose to focus here on Etk/Bmx and
The epithelial tyrosine kinase (Etk), also known as Bmx,
is a nonreceptor tyrosine kinase that is unique by virtue of
being able to interact with both tyrosine kinase receptors and
GPCRs . This type of interaction is mainly attributed
to the pleckstrin homology (PH) which is followed by the
Src homology SH3 and SH2 domains and a tyrosine kinase
site . Etk/Bmx-PAR1interactions were characterized
by binding of lysates exhibiting various hPar1forms to
GST-PH-Etk/Bmx. While Y397Z hPar1and wt hPar1showed
speciﬁc association with Etk/Bmx, lysates of truncated hPar1
or JAR cells (lacking PAR1) exhibited no binding. A tight
association between the PAR1C-tail and Etk/Bmx was
obtained, independent of whether wt or kinase-inactive
Etk/Bmx (KQ) was used [29,30].
5. Hierarchy of Binding
Next, we wished to determine the chain of events mediating
the signaling of PAR1and the binding of Shc and Etk/Bmx to
PAR1C-tail. Shc is a well-recognized cell signaling adaptor
known to associate with tyrosine-phosphorylated residues.
To this end, analysis of MCF7 cells that express little to
no hPar1were ectopically forced to overexpress hPar1gene.
When coimmunoprecipitation with anti-PAR1antibodies
following PAR1activation was performed, surprisingly, no
Pathology Research International 5
Shc was detected in the PAR1immunocomplex. Shc asso-
ciation with PAR1was fully rescued only when MCF7 cells
were initially cotransfected with Etk/Bmx, resulting with
abundant assembly of Shc in the immunocomplex. Thus,
Etk/Bmx is a critical component that binds ﬁrst to activated
PAR1C-tail enabling the binding of Shc. Shc may bind either
to phosphorylated Etk/Bmx, via its SH2 domain, or in an
unknown manner to the PAR1C-tail, provided that Etk/Bmx
is present and is PAR1-bound complex. One cannot, how-
ever, exclude the possibility that Bmx binds ﬁrst to Shc, and
only then the complex of Etk/Bmx-Shc binds to PAR1.
The functional consequences of the Etk/Bmx binding
was further evaluated by inserting mutations to the “signal-
binding” site. We prepared hPar1constructs with successive
replacement of the designated seven residues (378-384;
CQRYVYS) with A, termed as hPar1-7A. This HA tagged
mutant, HA-hPar1-7A, completely failed to immunoprecip-
itate Etk/Bmx. In contrast, in the presence of HA-wt hPar1,
potent immunoprecipitation was obtained. We thus con-
clude that the critical region for Etk/Bmx binding to PAR1
C-tail resides in the vicinity of CQRYVYS. The physiological
signiﬁcance of PAR1-Etk/Bmx binding is emphasized by the
following outcome. Activated MCF7 cells that express hPar1-
7A mutant failed to invade Matrigel-coated membranes. In-
contrast, a potent invasion was obtained by activated wt
hPar1. This outcome highlights the fact that by preventing
the binding of a key signaling partner to PAR1C-tail, eﬃcient
inhibition of PAR1pro-oncogenic functions, including the
loss of epithelial cell polarity, migration, and invasion, is
obtained (see Figures 1and 2for wt and mutated PAR1C-
tail and the ability to form a scaﬀold complexes with the
signaling partners). Elucidation of the PAR1C-tail binding
domain may therefore provide a potent platform for future
therapeutic vehicles in treating breast cancer.
The same approach may be utilized to identify a prime
signaling partner for PAR2. This will eventually lead to
characterization of a minimal PAR2C-tail binding region.
Generation of peptides that can enter the cells via adding
Tat or penetratin, or alternatively, addition of either myris-
toylation, or another lipid moiety, will assist the peptides
to cross the cell membrane. These peptides may prove
as eﬀective therapeutic inhibitors of PARs-induced breast
cancer growth and development. Along this line of evidence,
successful PAR1-derived peptides termed “pepducin” were
developed by the group of Kuliopulos A . This group has
demonstrated that PAR1-induced breast tumor in a mouse
model, in vivo, is blocked by the cell-penetrating lipopeptide
“pepducin,” P1pal-7, which is a potent inhibitor of cell via-
bility in breast carcinoma cells expressing PAR1.Ithasbeen
shown that P1pal-7 is capable of promoting apoptosis in
breast tumor xenografts and signiﬁcantly inhibits metastasis
to the lung.
In summary, PARs may provide a timely eﬀective chal-
lenge for developing valuable prognostic vehicles and also
critical targets for therapy in breast cancer. While the PAR
prognostic vehicles stem from the extracelluar portion of the
receptors, we oﬀer the intracellular C-tail site as potential
targets for therapy in breast cancer. What is the relative
contribution of PAR1versus PAR2in breast cancer tumor
growth and development is yet an open question and a
subject of current evaluation.
Conﬂict of Interests
The authors have declared that no conﬂict of interests exists.
This work was supported by grants from the Israel Science
Foundation (Grant no. 1313/07), Fritz-Thyssen Foundation,
and Israel Cancer Research Fund (granted to R. B.). The
funds had no role in the study design, data collection,
analysis, decision to publish, or preparation of the paper.
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