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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:940.)
© 2003 American Heart Association, Inc.

ATVB in Focus

Human Protease-Activated Receptor 1 Expression in Malignant Epithelia

A Role in Invasiveness

Yong-Jun Yin; Zaidoun Salah; Sorina Grisaru-Granovsky; Irit Cohen; Sharona Cohen Even-Ram; Myriam Maoz; Beatrice Uziely; Tamar Peretz; Rachel Bar-Shavit

From the Department of Oncology Hadassah-University Hospital, Jerusalem, Israel.

Correspondence to Rachel Bar-Shavit, PhD, Department of Oncology, Hadassah-University Hospital, POB 12000, Jerusalem 91120, Israel. E-mail barshav{at}md.huji.ac.il

Series Editor: Marschall S. Runge
ATVB In Focus Extracellular Mediators in Atherosclerosis and Thrombosis

Previous Brief Reviews in this Series:

•Brasier AR, Recinos A III, Eledrisi MS. Vascular inflammation and the renin-angiotensin system. 2002;22:1257–1266.
•Moser M, Patterson C. Thrombin and vascular development: a sticky subject. 2003;23:922–930.
•Major CD, Santulli RJ, Derian CK, Andrade-Gordon P. Extracellular mediators in atherosclerosis and thrombosis: lessons from thrombin receptor knockout mice. 2003;23:931–939.

	Abstract

Top Abstract Introduction PAR1 Expression Is Associated... Molecular Mechanisms of PAR1... PAR1 Expression Correlates With... PAR1 Induces VEGF and... hPar1 and Mammary Gland... Future Perspective References

 
While protease-activated receptors (PARs) play a traditional role in vascular biology, they emerge with surprisingly new assignments in tumor biology. PAR1 expression correlates with the invasion properties of breast carcinoma, whereas human PAR1 antisense reduces their ability to migrate through Matrigel. Part of the molecular mechanism of PAR1 invasion involves the formation of focal contact complexes on PAR1 activation. PAR1 induces angiogenesis in animal models in vivo and exhibits an oncogenic phenotype of enhanced ductal complexity when overexpressed in mouse mammary glands.

Key Words: PAR1 • epithelia • invasion • metastasis • angiogenesis

	Introduction

Top Abstract Introduction PAR1 Expression Is Associated... Molecular Mechanisms of PAR1... PAR1 Expression Correlates With... PAR1 Induces VEGF and... hPar1 and Mammary Gland... Future Perspective References

 
In addition to its central role in hemostasis and thrombosis, the serine protease thrombin is a potent mitogen of vascular endothelial and smooth muscle cells.^1,2 Along with endothelial dysfunction, massive proliferation of vascular smooth muscle cells (VSMCs) is one of the prominent characteristics of atherosclerosis, suggesting a role for thrombin in the etiology of the disease. While vascular proliferation, inflammation, apoptosis, and extracellular matrix alterations all contribute to the pathobiology of atherosclerosis, the precise role of each of these processes is poorly understood. In this review, we will consider the role of the thrombin receptor in cell proliferation and invasiveness, which is likely to be relevant to atherosclerotic VSMC proliferation.

Most thrombin-regulated cellular events are mediated via the protease activated receptors PAR1, PAR3, and PAR 4.³ The PARs are a family of seven transmembrane G-protein coupled receptors activated via proteolytic cleavage. This unique mode of activation appears to involve exposure of autoligated sites present on the receptor themselves.^4–7 While the full repertoire of protease signaling through PARs remains to be determined, this family plays a distinct role in epithelial cell biology. Analysis of PAR1 in the context of normal epithelial function as well as under pathological conditions highlights a new function for this gene (and possibly other members of the PAR family) in normal development and in tumor progression (Figure).

View larger version (60K): [in this window] [in a new window]   Steps in tumor carcinoma progression and metastasis. Normal epithelia consist of polarized sheets of cells with well organized junctions underlined by a basement membrane. In carcinoma in situ, epithelial cells proliferate locally, but are still confined by the basement membrane. The transition from epithelial to mesenchyme stage (EMT) involves further alterations that can induce local dissemination, emergence through the basement membrane, intravasation and extravasation of lymph or blood vessels, and the establishment of metastatic colonies at distant sites. PAR1 is present at high levels in DCIS but not in normal epithelia or dysplastic carcinoma. Active signaling initiated by PAR1 association with vß5 induces reorganization of the cytoskeleton and focal adhesion complex formation, which fosters invasion and metastasis. Part of the scheme is modified from Thiery, JP (Nature Reviews June 2002 volume 2 (6)p. 442 to 454).

Epithelial cells associate into intact polarized sheets and communicate through an intricate network of cell-cell junctions and cell-extracellular matrix (ECM) interactions. This architectural restraint of cell-cell junctions underlined by a basement membrane serves as extra-strict boundaries to maintain normal cellular behavior. A critical event in malignant tumor development is the acquisition of the ability to invade through basement membranes, enter the circulatory system, and re-emerge from blood vessels to establish metastatic colonies at distant sites. This task is accomplished via a well orchestrated set of events including the recruitment of enzymes to remodel targeted locations of the basement membrane microenvironment. We have shown that PAR1 has central roles in breast carcinoma invasion and metastasis⁸ as well as in other types of carcinomas (eg, bladder, ovary, prostate and colon) (S. Grisaru-Granovsky and Z. Salah, unpublished observations). In addition, recent studies by Martin et al⁹ identified PAR1 as a potent oncogene in a cDNA expression library screen for genes that induce focus forming activity and transformation in NIH3T3 cells. Thus PAR1 joins the list of G-protein coupled receptors that harbor oncogenic potential such as, mas and g2a. This property of human PAR1 (hPAR1) was attributed to ectopic overexpression of the receptor, as also demonstrated for Wnt-1 and HER2/Neu (erbB2),^10,11 rather than to a specifically inserted mutation as in the case of adenomatous polyposis coli (APC).¹⁰ A putative oncogenic role for PAR1 is further supported by our observations that hPAR1 is overexpressed in a series of carcinoma biopsy specimens and in a collection of differentially metastatic cell lines.^8,12

	PAR1 Expression Is Associated With Metastatic Potential

Top Abstract Introduction PAR1 Expression Is Associated... Molecular Mechanisms of PAR1... PAR1 Expression Correlates With... PAR1 Induces VEGF and... hPar1 and Mammary Gland... Future Perspective References

 
A direct correlation has been established between the levels of PAR1 expression and the invasion properties of breast carcinomas.⁸ In situ hybridization performed on paraffin-embedded sections from a broad range of archival biopsy specimens showed a differential pattern of distribution. Invasive carcinoma specimens, selected from typical infiltrating ductal carcinoma (IDC) with vascular invasion and lymph node metastases, revealed high PAR1 expression, confined to the carcinoma of the primary tumors. Lower levels of expression were observed in high-grade ductal carcinoma in situ (DCIS) of comedo type. In contrast, very little or no PAR1 was observed in low-grade DCIS of solid type and in the premalignant atypical intraductal hyperplasia (AIDH). No PAR1 expression was observed in normal mammary sections obtained from reduction mammoplasty. Likewise, a panel of mammary carcinoma cell lines was surveyed for a correlation between PAR1 expression and established metastatic potential.⁸ The cell lines included a near-normal diploid immortalized breast epithelial cell line originating from fibrocystic disease (MCF10A), and six tumor cell lines with different doubling times, tumorigenicity, and metastatic potential in nude mice. In these cell lines, high levels of PAR1 expression were found in the highly metastatic cell lines, moderate PAR1 levels in moderately metastatic cell lines, and no expression was present in nonmetastatic cells.

PAR1 expression does not merely correlate with tumor progression but appears likely to play an active role during metastatic breast carcinoma cell invasion. This is suggested by the fact that introduction of PAR1 antisense into the aggressively metastatic MDA-435 cells markedly reduces their ability to migrate through Matrigel (a reconstituted basement membrane) in vitro.¹³ In the complementary experiment, cell lines overexpressing PAR1 exhibit increased invasion through Matrigel.¹² These results identify PAR1 as a potential cellular target for cancer therapies.

	Molecular Mechanisms of PAR1 in Tumor Invasion: PAR1 Cooperates With vß5 Integrin to Promote Cytoskeletal Reorganization

Top Abstract Introduction PAR1 Expression Is Associated... Molecular Mechanisms of PAR1... PAR1 Expression Correlates With... PAR1 Induces VEGF and... hPar1 and Mammary Gland... Future Perspective References

 
To begin to evaluate the molecular mechanisms by which PAR1 promotes tumor invasion and metastasis, stable clones of nonmetastatic melanoma cells (SB-2) overexpressing hPAR1 were established. These clones exhibited increased adhesion to extracellular matrix components, accompanied by reorganization of cytoskeletal F-actin toward a morphology favoring migration.¹² Activation of PAR1 by TRAP (100 µmol/L) increased the phosphorylation of focal adhesion kinase and paxillin and induced the formation of focal contact complexes. In addition, PAR1 activation affected the distribution of cell-surface integrins without altering their level of expression: vß5, but not vß3 or 5ß1, was specifically recruited to focal contact sites. PAR1-overexpressing cells showed selective reciprocal coprecipitation of paxillin with vß5 but not with vß3. This co-immunoprecipitation failed to occur in cells containing the truncated form of PAR1 lacking the entire cytoplasmic portion of the receptor.¹² Thus, the PAR1 cytoplasmic tail is essential for recruitment of vß5 integrin. PAR1 overexpressing cells were invasive in vitro, as reflected by migration through a Matrigel barrier, and invasion was further enhanced by ligand activation of PAR1. Moreover, the application of anti-vß5 antibodies specifically attenuated this PAR1-induced invasion.¹² We conclude that activation of PAR1 may foster tumor cell invasion via a novel mechanism involving cooperation with vß5 integrin. While we observed induced invasion following PAR1 activation, Kamath et al¹⁴ have reported that activation of PAR1 inhibited the invasion and migration of MDA231 breast cancer cells. By the use of a truncated version of PAR1, devoid of the entire cytoplasmic tail, one may resolve this controversy.

	PAR1 Expression Correlates With Normal Physiological Invasion During Placental Implantation

Top Abstract Introduction PAR1 Expression Is Associated... Molecular Mechanisms of PAR1... PAR1 Expression Correlates With... PAR1 Induces VEGF and... hPar1 and Mammary Gland... Future Perspective References

 
Placental implantation into the uterus wall in early pregnancy is an example of a normal physiological invasion process. We hypothesized that genes such as PAR1 are part of an invasive program that should be turned off when the period of invasion is over. We therefore analyzed PAR1 expression during and after placenta implantation. Indeed, PAR1 is exclusively expressed during the time-limited invasion period and is completely shut off thereafter.¹⁵ In the first trimester of pregnancy, specialized placental epithelial cells, the cytotrophoblasts (CTBs), differentiate, proliferate, and invade to initiate placental implantation. To address the involvement of PAR1 in a regulated invasion process, we examined PAR1 expression in CTBs in human placental tissue samples (obtained from elective terminations of pregnancies). In contrast to the PAR1 overexpression observed in the tumor biopsy specimens, the temporal and spatial pattern of PAR1 expression during placental implantation is tightly controlled. PAR1 is detected predominantly in the cytotrophoblast layer between 7 and 10 weeks of gestation and is undetectable by 12 weeks.¹⁵ During placental implantation, CTBs differentiate, proliferate, and invade the uterine wall and the spiral arteries deep into the stroma to establish proper fetal-maternal interactions.¹⁶ Although poorly understood, the molecular basis of CTB invasion shares many features with tumor cell invasion.^16–18 Furthermore, the pathobiology of several disorders of pregnancy involves dysregulated trophoblastic invasion. Shallow invasion of the uterine wall leads to preeclampsia and restriction of fetal growth¹⁹ while excessive proliferation and invasion may result in gestational trophoblastic disease, ranging from partial hydatidiform moles to choriocarcinoma.²⁰ Trophoblast cells from complete hydatidiform moles (CHMs) proliferate rapidly, often show cytologic atypia, and bear an 20% risk of becoming malignant.²⁰ This condition provides an opportunity to study the expression of PAR1 in pathologically over-proliferative placental villi. PAR1 mRNA and protein expression in biopsies of CHMs (12 to 14 weeks of gestation) were compared with age-matched normal placentas. In CHMs, the trophoblast cells exhibit high levels of hPAR1 mRNA and protein, while very little or no expression is detected in the age-matched controls.¹⁵ These results suggest that hPAR1 expression is part of an invasive program for both normal placental development and pathological trophoblastic disorders such as CHM.

	PAR1 Induces VEGF and Tumor Angiogenesis

Top Abstract Introduction PAR1 Expression Is Associated... Molecular Mechanisms of PAR1... PAR1 Expression Correlates With... PAR1 Induces VEGF and... hPar1 and Mammary Gland... Future Perspective References

 
The formation of new blood vessels is a critical determinant of tumor progression. Although the association between the protease thrombin and angiogenesis has been previously documented, the role of PAR1 in tumor angiogenesis and its mechanism of activation are largely unknown. Using in vivo injection of either Matrigel plugs containing PAR1-expressing cells or of rat prostatic carcinoma cells transfected with a tetracycline-inducible PAR1 expression vector, we showed that PAR1 significantly enhances both angiogenesis and tumor growth.²¹ Several VEGF splice forms are induced in cells expressing PAR1 and activation of PAR1 markedly augments the expression of VEGF mRNAs and of functional VEGF proteins. Because neutralizing anti-VEGF antibodies potently inhibited PAR1-induced endothelial cell proliferation,²¹ we conclude that PAR1-induced angiogenesis requires VEGF. Specific inhibitors of PKC, Src, and PI3K inhibit PAR1-induced VEGF expression, suggesting the participation of these kinases in the process. In addition, oncogenic transformation by genes known to be part of PAR1 signaling machinery is sufficient to increase VEGF expression in NIH3T3 cells. These data identify players in the signaling process evoked by PAR1 activation and support the novel notion that initiation of the PAR1 signaling pathway, either by ligation of PAR1 or by the direct activation of downstream signaling components, is sufficient to induce VEGF and hence angiogenesis.

	hPar1 and Mammary Gland Morphogenesis

Top Abstract Introduction PAR1 Expression Is Associated... Molecular Mechanisms of PAR1... PAR1 Expression Correlates With... PAR1 Induces VEGF and... hPar1 and Mammary Gland... Future Perspective References

 
To further investigate the role of hPAR1 in breast cancer and normal development of the breast epithelia, we have established a line of mice carrying hPAR1 transgenes specifically overexpressed in the mammary glands (by using MMTV-LTR–derived construct) (Y.-J. Yin, unpublished observations). Mammary glands from transgenic animals exhibited grossly hyperplastic features compared with nontransgenic littermates. The growing branch ends from virgin transgenic animals showed enhanced complexity of alveolar side branching compared with normal virgin glands, a difference that is accentuated in pregnant hPar1^+/- mice. This phenotype is reminiscent of the effect of overexpression of several different oncogenes in the mouse breast.²² In situ hybridization analysis in sections of virgin mammary glands and during early pregnancy showed abundant hPar1 expression at the luminal phase of the mammary epithelium. Likewise, quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed high levels of hPAR1 expression in 5-, 8- and 13-week-old virgin hPAR1^+/- animals and further elevated hPAR1 levels in pregnant mice (pregnant for 4, 8, and 12 days).

In normal wild-type mice, Wnt-4 expression is low in the virgin gland and increases during pregnancy,^23,24 suggesting Wnt-4 may play a role in the induction of ductal side branching. Wnt-4 expression is markedly induced in the hPar1^+/- overexpressing glands compared with age-matched wild-type mice. This was demonstrated by both RT-PCR (at 5 and 13 weeks in virgin mice and during pregnancy at 4, 8, and 12 days of gestation) and immunoblotting assays. Immunohistochemistry detected Wnt-4 confined to the epithelial compartment of mammary tissue from 13-week virgin and 4- and 8-day pregnant hPar1^+/- animals, compatible with the localization and expression of PAR1 in these mice. Wnts encode secreted glycoproteins that carry short-range signals between cells and bind to members of the Frizzled family of 7-transmembrane receptors. Wnt 7b levels were also increased compared with normal controls in both virgin and pregnant hPar1^+/- mammary tissue at the same time points as Wnt-4. The expression of other Wnt family members (Wnt 5a, 5b, 6, and 7a) in the mammary tissue of hPar1^+/- animals was analyzed by RT-PCR and was not different from controls. The presence of elevated Wnt-4 and Wnt-7 in mammary glands from hPar1^+/- mice suggests that PAR1 directly or indirectly controls their expression.

The possibility that PAR1 overexpression in the mammary gland induces alveologenesis (ie, proliferation in the lobulo-alveolar structures) in addition to ductal side-branching was also investigated. Higher ductal network complexity results from enhanced alveoli proliferation, thus the epithelial cell proliferation rate was assessed in situ by immunostaining using antibodies to the proliferating cell nuclear antigen (PCNA). The proliferation index is defined as the number of PCNA-positive nuclei of alveolar epithelial cells divided by the total number of nuclei. These studies revealed that the proliferation of alveolar bud epithelium is significantly enhanced in hPar1^+/- mice as compared with wild-type counterparts. In virgin hPAR1^+/- mice, enhanced PCNA staining was observed at 5 weeks, declined at weeks 8 and 10, and was elevated again at 13 weeks. During pregnancy, active alveoli proliferation takes place in wild-type mice, and PCNA staining is significantly increased in the alveolar bud epithelium. In the pregnant hPAR1 mice (at 4, 8, and 12 days of gestation), PCNA staining is further elevated. In contrast to the alveolar epithelium, there were no obvious differences between wild-type and hPAR1^+/- mice in the proliferation of the ductal epithelium in virgin or pregnant mice. Overall, the mammary glands of hPar1^+/- transgenic mice show enhancement of alveoli proliferation and ductal side branching, both of which contribute to increased network complexity in the mammary ducts.

	Future Perspective

Top Abstract Introduction PAR1 Expression Is Associated... Molecular Mechanisms of PAR1... PAR1 Expression Correlates With... PAR1 Induces VEGF and... hPar1 and Mammary Gland... Future Perspective References

 
Epithelial-mesenchymal transitions (EMTs) occur during critical phases of embryonic development, and a similar transition occurs during the progression of carcinoma toward malignancy. Alterations of the normally strict epithelial architecture may reflect critical early events which ultimately lead to invasion and metastasis. The interrelation between PAR1 and E-cadherin and its signaling machinery (-, ß- and -catenins) is of primary interest. How does PAR1 affect epithelial cell morphology? Our preliminary data suggest that PAR1 suppresses E-cadherin expression. Does the mechanism involve regulators of E-cadherin expression, such as Snail and SiP1? Both of these proteins bind E-cadherin promoter regions and act as transcriptional repressors.^25–28 Alternatively, promoting aberrant methylation of the normally unmethylated 5'-CpG-rich areas in E-cadherin is another way hPAR1 might regulate E-cadherin. This epigenic mechanism is associated with the transcriptional silencing of genes, including E-cadherin, in various forms of cancer.²⁹

Another important factor to consider when studying the role of PAR1 in tumor progression is the interactions between cancer cells and their microenvironment.³⁰ The stroma microenvironment of the tumor can have profound influence on tumor progression. Whereas normal stroma can postpone or prevent tumorigenesis, abnormal stromal components can promote tumor development. This stromal effect is significant enough that in some cases, reintroduction of normal stroma can suppress transformed phenotype.³¹ Even after prolonged passaging, teratocarcinoma cells are still capable of differentiating and generating normal mice on contact with a normal microenvironment.³¹ Therefore, tumor progression may be clinically reversible if the appropriate context and signaling are supplied. It is feasible that serine-proteases capable of activating PAR1 are present in the vicinity of the tumor. Prothrombin/thrombin, factor Xa as also the tissue factor (TF) TF-VIIa complex are possible candidates to serve as signaling cues for PAR1.³² In the context of endothelial cells, for example, activated protein C (APC), a trypsin like enzyme, was shown (along with other receptors) to activate PAR1.³³ It remains to determine whether these proteins are expressed in the stroma of the tumor. Future studies will explore the involvement of PAR1 in epithelial morphology and the influence of the stromal microenvironment on PAR1 expression, activation, and function.

	Acknowledgments

 
We are indebted to Dr Susan Lewis for editing the overview. These studies were supported by grants from CapCURE, the US Army, the Israel Science Foundation (ISF), and Israel Cancer Association (to R.B.-S.).

Received December 26, 2002; accepted February 21, 2003.

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Top Abstract Introduction PAR1 Expression Is Associated... Molecular Mechanisms of PAR1... PAR1 Expression Correlates With... PAR1 Induces VEGF and... hPar1 and Mammary Gland... Future Perspective References

 
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