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 (PAR1), a G protein-coupled receptor (GPCR), is the first member of the mammalian PAR family consisting of four genes. PAR1 and PAR2 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 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 PAR1 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 PAR1 induced breast cancer. Altogether, both PAR1 and PAR2 may serve as molecular probes for breast cancer diagnosis and valuable targets for therapy.


Introduction
The classification of a tumor differentiation level is routinely 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 [1]. 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 [2]. Oncotype Dx is a clinically validated and widely used multigene assay (there are also other commercially available gene panels such as Mammaprint; Agendia Amsterdam, Netherland, and THEROS H/I; Biotheranostics, San Diego, CA), that quantifies the likelihood of breast cancer recurrence. This gene profile has been developed specifically for women with hormone receptorpositive (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 progression [3]. Identification of these genes will significantly contribute to the prospect of treatment making choices.
Protease-activated receptor-1 (PAR 1 ), a G protein-coupled receptor (GPCR), is the first and prototype member of the mammalian PAR family consisting of four genes. The activation of PAR 1 involves 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][5][6]. While a well-known classical observation points to a close link between hyperactivation of the coagulation system and cancer malignancies, the molecular mechanism that governs procoagulant tumor progression remains poorly defined [7][8][9][10]. Thrombin is a main effector of the coagulation cascade. In addition to cleaving fibrinogen, it also activates cells through at least three PARs: PAR 1 , PAR 3 , and PAR 4 . In contrast, PAR 2 is activated by multiple trypsinlike serine proteases including the upstream coagulant proteases VIIa-tissue factor (TF) and Xa, but not by thrombin. It is now becoming well established that human Par 1 , hPar 1 , plays a central role in epithelial malignancies [13,14,16]. PAR 2 , the second member of the family, is also emerging with central assignments in breast cancer [11,12]. High levels of hPar 1 expression are directly correlated with epithelia tumor progression in both clinically obtained biopsy specimens and a wide spectrum of differentially metastatic cell lines [13,14]. PAR 1 also plays a role in the physiological invasion process of placental cytotrophoblasts during implantation into the uterus deciduas [15]. Trophoblast invasion shares many features with the tumor cell invasion process. It differs, however, by the time-limited hPar 1 expression, which is confined to the trophoblast-invasive period and is shut off immediately thereafter, when there is no need to invade [13]. This strongly supports the notion that the hPar 1 gene is part of an invasive gene program. Surprisingly, the zincdependent matrix-metalloprotease 1 (MMP-1), a collagenase that efficiently cleaves extra cellular matrix (ECM) and basement membrane components, has been shown to specifically activate PAR 1 [16]. PAR 1 -MMP1 axis may thus provide a direct mechanistic link between PAR 1 and tumor metastasis. The mechanism that leads to hPar 1 gene overexpression in tumor is yet unclear and under current extensive investigation. Although the impaired internalization of PAR 1 that results with persistent signaling and invasion was previously suggested for several breast cancer lines [17], an imbalanced expression between hPar 1 repressors and activators was proposed, suggesting transcriptional regulation [18]. We found that the mechanism of hPar 1 overexpression 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 [19]. Indeed, we have identified the Egr-1 transcription factor as a critical DNA-binding protein eliciting hPar 1 expression in prostate cancer cells and the wt p53 tumor suppressor as an hPar 1 transcription repressor [19,20]. The wt form of p53 thus acts as a fine-tuning regulator of hPar 1 in cancer progression.

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 PAR 1 gene and protein overexpression are associated with the aggressiveness of a tumor, in vivo, reflect its potential role in cancer dissemination. Furthermore, it assigns PAR 1 as 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, PAR 1 and PAR 2 peptides in the blood directly imitate PAR expression serving as a faithful indicator for the extent of cancer progression. While the overexpression of both PAR 1 and PAR 2 takes 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 PAR 1released peptides may be instrumental in demonstrating the effectiveness of a given treatment. For example, determining the level of the released PAR 1 and PAR 2 , 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 PAR 1&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 PARreleased 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 PAR 1 versus PAR 2 during 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-PAR 1 and anti-PAR 2 antibodies, separately) utilizing tissue microarray biopsy specimens on a large pool of primary breast cancer biopsy specimens representing invasive carcinoma. 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 markerneeds to be evaluated.

PARs as Target for Therapy
Importantly, PAR 1 cellular 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 PAR 1 is sorted to the lysosomes where it is degraded [23,24]. While cellular trafficking of PAR 1 impinges on the extent and mode of signaling, the identification of individual PAR 1 signaling partners and their contribution to breast cancer progression remain to be elucidated. We have adopted the approach of utilizing a truncated form of hPar 1 gene devoid of the entire cytoplasmic tail to demonstrate the significant role of PAR 1 signaling in breast tumor progression. This was demonstrated in a xenograft mice model of mammary gland tumor development, in vivo [25]. Along this line of evidence, we have identified PAR 1 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 PAR 1 C-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 hPar 1 constructs in vivo was assessed by orthotopic mammary fat pad tumor development. MCF7 cells overexpressing either persistent hPar 1 Y397Z or wt hPar 1 constructs (e.g., MCF7/Y397Z hPar 1 ; MCF7/wt hPar 1 ) markedly enhanced tumor growth in vivo following implantation into the mammary glands, whereas MCF7 cells overexpressing truncated hPar 1 , 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 hPar 1 and MCF7/Y397Z hPar 1 were 5 and 5.8 times larger, respectively, than tumors produced by the MCF7/empty vector-transfected cells. Histological examination (H&E staining) showed that while both MCF7/wt hPar 1 and MCF7/Y397Z hPar 1 tumors infiltrated into the fat pad tissues of the breast, the MCF7/Y397Z hPar 1 tumors further infiltrated the abdominal muscle. In contrast, tumors produced by empty vector or truncated hPar 1 -transfected cells were capsulated, with no obvious cell invasion. Tumor growth can also be attributed to blood vessel formation [26,27]. The hPar 1 -induced breast tumor vascularization was assessed by immunostaining with antilectin and anti-CD31 antibodies, showing that both MCF7/Y397Z hPar hPar 1 and MCF7/wt hPar 1 tumors were intensely stained. In contrast, only few blood vessels were found in the small tumors of empty vector or truncated hPar 1 Figure 2: Activation of PAR 1 leads to the association of Etk/Bmx with PAR 1 C-tail. This association is mediated through Etk/Bmx PHdomain enabling next the binding of Shc. The site of the "signal binding" domain (e.g., Etk/Bmx, as a prime signaling partner) in PAR 1 has been identified. Insertion of successive replacement of A residues forming a PAR 1 mutant incapable of binding Etk/Bmx showed impaired capabilities of PAR 1 induced invasion and migration. This site provides therefore a platform for the development of future therapeutic medicaments in breast cancer. hPar 1 and MCF7/Y397Z hPar 1 cells were shown to effectively induce breast tumor growth, proliferation, and angiogenesis, while the MCF7/truncated hPar 1 and MCF7/empty vectorexpressing cells had no significant effect. This experimental results highlight the significance of PAR 1 signaling in PAR 1induced breast cancer progression.

Antibody Array for Protein-Protein Interactions Reveals Signaling Candidates
Next, in order to identify specific PAR 1 signaling components, the following approach was utilized. To detect the putative mediator(s) linking PAR 1 to potential signaling pathway, we examined a custom-made antibody-array membranes. When aggressive breast carcinoma MDA-MB-435 cells (with high hPar 1 levels) were incubated with the antibody-array membranes before and after PAR 1 activation (15 minutes), the following results were obtained. Several activation-dependent proteins which interact with PAR 1 , including ICAM, c-Yes, Shc, and Etk/Bmx, were identified. Of these proteins, we chose to focus here on Etk/Bmx and Shc. 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 [28]. 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 [29]. Etk/Bmx-PAR 1 interactions were characterized by binding of lysates exhibiting various hPar 1 forms to GST-PH-Etk/Bmx. While Y397Z hPar 1 and wt hPar 1 showed specific association with Etk/Bmx, lysates of truncated hPar 1 or JAR cells (lacking PAR 1 ) exhibited no binding. A tight association between the PAR 1 C-tail and Etk/Bmx was obtained, independent of whether wt or kinase-inactive Etk/Bmx (KQ) was used [29,30].

Hierarchy of Binding
Next, we wished to determine the chain of events mediating the signaling of PAR 1 and the binding of Shc and Etk/Bmx to PAR 1 C-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 hPar 1 were ectopically forced to overexpress hPar 1 gene. When coimmunoprecipitation with anti-PAR 1 antibodies following PAR 1 activation was performed, surprisingly, no Shc was detected in the PAR 1 immunocomplex. Shc association with PAR 1 was 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 PAR 1 C-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 PAR 1 C-tail, provided that Etk/Bmx is present and is PAR 1 -bound complex. One cannot, however, exclude the possibility that Bmx binds first to Shc, and only then the complex of Etk/Bmx-Shc binds to PAR 1 .
The functional consequences of the Etk/Bmx binding was further evaluated by inserting mutations to the "signalbinding" site. We prepared hPar 1 constructs with successive replacement of the designated seven residues (378-384; CQRYVYS) with A, termed as hPar 1 -7A. This HA tagged mutant, HA-hPar 1 -7A, completely failed to immunoprecipitate Etk/Bmx. In contrast, in the presence of HA-wt hPar 1 , potent immunoprecipitation was obtained. We thus conclude that the critical region for Etk/Bmx binding to PAR 1 C-tail resides in the vicinity of CQRYVYS. The physiological significance of PAR 1 -Etk/Bmx binding is emphasized by the following outcome. Activated MCF7 cells that express hPar 1 -7A mutant failed to invade Matrigel-coated membranes. Incontrast, a potent invasion was obtained by activated wt hPar 1 . This outcome highlights the fact that by preventing the binding of a key signaling partner to PAR 1 C-tail, efficient inhibition of PAR 1 pro-oncogenic functions, including the loss of epithelial cell polarity, migration, and invasion, is obtained (see Figures 1 and 2 for wt and mutated PAR 1 Ctail and the ability to form a scaffold complexes with the signaling partners). Elucidation of the PAR 1 C-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 PAR 2 . This will eventually lead to characterization of a minimal PAR 2 C-tail binding region. Generation of peptides that can enter the cells via adding Tat or penetratin, or alternatively, addition of either myristoylation, 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 PAR 1 -derived peptides termed "pepducin" were developed by the group of Kuliopulos A [31]. This group has demonstrated that PAR 1 -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 viability in breast carcinoma cells expressing PAR 1 . It has been 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 challenge 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 PAR 1 versus PAR 2 in breast cancer tumor growth and development is yet an open question and a subject of current evaluation.