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[Cancer Research 66, 5224-5233, May 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Mammary Gland Tissue Targeted Overexpression of Human Protease-Activated Receptor 1 Reveals a Novel Link to ß-Catenin Stabilization

Yong-Jun Yin1, Vered Katz1, Zaidoun Salah1, Myriam Maoz1, Irit Cohen1, Beatrice Uziely1, Hagit Turm1, Sorina Grisaru-Granovsky1, Hiromu Suzuki2 and Rachel Bar-Shavit1

1 Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem, Israel and 2 Department of Public Health, Sapporo Medical University S1, Chuo-ku, Sapporo, Japan

Requests for reprints: Rachel Bar-Shavit, Department of Oncology, Hadassah-Hebrew University Hospital, P.O. Box 12000, Jerusalem 91120, Israel. Phone: 972-2-677-7563; Fax: 972-2-642-2794; E-mail: barshav{at}md.huji.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protease-activated receptor 1 (PAR1) is emerging with distinct assignments in tumor biology. We show that tissue targeted overexpression of hPar1 in mice mammary glands results in precocious hyperplasia, characterized by a dense network of ductal side branching and accelerated proliferation. These glands exhibit increased levels of wnt-4 and wnt-7b and a striking ß-catenin stabilization. Nuclear localization of ß-catenin is observed in hPar1 transgenic mouse tissue sections but not in the wild-type, age-matched counterparts. PAR1 induces ß-catenin nuclear localization also in established epithelial tumor cell lines of intact ß-catenin system (transformed on the background of mismatch repair system; RKO cells). We propose hereby that PAR1-mediated ß-catenin stabilization is taking place primarily via the increase of Wnt expression. Enforced expression of a specific Wnt antagonist family member, secreted frizzled receptor protein 5 (SFRP5), efficiently inhibited PAR1-induced ß-catenin stabilization. Likewise, application of either SFRP2 or SFRP5 on epithelial tumor cells completely abrogated PAR1-induced ß-catenin nuclear accumulation. This takes place most likely via inhibition of Wnt signaling at the level of cell surface (forming a neutralizing complex of "Receptors-SFRP-Wnt"). Furthermore, depletion of hPar1 by small interfering RNA (siRNA) vectors markedly inhibited PAR1-induced Wnt-4. The striking stabilization of ß-catenin, inhibited by SFRPs on one hand and Wnt-4 silencing by hPar1 siRNA on the other hand, points to a novel role of hPar1 in Wnt-mediated ß-catenin stabilization. This link between PAR1 and ß-catenin may bear substantial implications both in developmental and tumor progression processes. (Cancer Res 2006; 66(10): 5224-32)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Protease-activated receptors (PAR) form a family of G protein–coupled receptors encoding their own ligands and uniquely activated via proteolytic cleavage (1). Each of the four PAR family members is activated via proteolytic cleavage, exposing an internal ligand distinct for every PAR protein (2). PARs act as sensitive sensors to the constantly changing extracellular proteases regardless of whether they are present in a soluble or microenvironment-immobilized forms. In addition to the classic role of thrombin receptor PAR1 in hemostasis, thrombosis, and vascular biology, it emerges with distinct assignments in tumor biology and angiogenesis (3–7). We have previously shown that PAR1 is involved both in malignant breast carcinoma tumor progression (3) and the physiologic invasion of placenta trophoblasts into the uterine decidua (4). A direct correlation exists between levels of hPar1 expression and tumor advancement in both clinically obtained biopsy specimens and differentially metastatic cell lines (3, 8). In-fact, hPar1 plays an active role in breast carcinoma invasion because antisense silencing of the gene abrogates efficiently metastatic breast carcinoma cells from invading Matrigel-coated filters in vitro (3).

In parallel, a cDNA expression library screen based on the loss of anchorage-dependent growth and focus-forming activity in NIH 3T3 cells has identified PAR1 as a novel oncogene (9). With these observations, PAR1 joins other G protein–coupled receptors, including mas and g2a, which behave as oncogenes (10, 11). The oncogenic properties of PAR1 accompany a collection of data showing that hPar1 is overexpressed in a wide range of epithelial tumors, pointing altogether to the central role of PAR1 in carcinoma invasion.

The canonical wnt/Wingless signaling pathway directs cell fate in many cell types and plays a central role in development and in tumor progression. Wnt proteins are soluble glycoproteins initiating cell signaling through binding to receptor complexes composed of Frizzled proteins and LDL receptor–related protein (LRP) 5/6 (12, 13). The core of the Wnt pathway is the stability of ß-catenin. Accumulation of ß-catenin in the cytoplasm leads ultimately to its transport to the nuclei where it forms functional complexes with lymphoid enhancer factor (LEF)/T-cell factor (TCF) transcription factors (14, 15). Because the LEF/TCF DNA-binding proteins are incapable of activating gene transcription alone, ß-catenin acts as a bridging cofactor, enabling the performance of LEF/TCF (for review, see ref. 16). A growing list of genes has presently been identified as downstream targets of ß-catenin nuclear activity. Among these are c-Myc (17) and cyclin D1 (18–20).

To gain further insight into the causal relationship between hPar1 expression, breast tumor formation, and mammary gland development, we have established a line of mice carrying MMTV-long terminal repeat (LTR)-SV40-driven hPar1 designed to overexpress in the mammary glands. Whereas mammary tissues can be used to study discrete developmental remodeling aspects of the breast, they also provide an opportunity to dissect the contribution of individual genes in normal and malignant mammopoiesis. We examined the phenotype of hPar1 transgenic (tg) mice with respect to breast morphogenesis and evaluated levels of distinct Wnt gene expression and ß-catenin stabilization. We hereby propose a novel link between hPar1, ß-catenin, and Wnt generation. This is based on the combined analyses of mammary gland tissue samples and epithelial cancer cell lines. By the use of either secreted frizzled receptor proteins (SFRP), members of the Wnt antagonist family, or hPar1 small interfering RNAs (siRNA), we provide evidence on the involvement of Wnts in PAR1-induced ß-catenin stabilization. Enforced expression of SFRP5, as also application of either SFRP2, SFRP5, or both, effectively abrogated PAR1-induced ß-catenin stabilization. This mode of inhibition points to the presence of an autocrine Wnt signaling loop initiated by PAR1. Ectopic overexpression of members of SFRP antagonist sequesters the PAR1-induced Wnts and binds Frizzled receptors through the homology site of cysteine-rich domain. Thus, SFRPs antagonize Wnt signaling on the level of cell surface. Our data are in line with the elegant studies by Bafico et al. (21) on the Wnt autocrine loop in human tumorigenicity. In parallel, hPar1 siRNA constructs depleting hPar1 levels efficiently inhibited Wnt-4 expression. Altogether, we conclude that PAR1-induced ß-catenin stabilization is mediated primarily via the induced Wnt generation. The novel link between PAR1, ß-catenin stabilization, and Wnt may impinge significantly both on developmental and tumor progression processes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of MMTV-hPar1-tg
The coding sequence of the full-length human Par1 gene from pcDNA3-hPar1 was subcloned into MMTV-SV40-BSSK (kindly provided by Dr. R.G. Pestell, Department of Developmental and Molecular Biology and Medicine, Albert Einstein College of Medicine, New York, NY). Briefly, pcDNA3-hPar1 was digested with HindIII and EcoRI to isolate the 1.4-kb full-length hPar1. In parallel, MMTV-SV40-BSSK was digested with HindIII and EcoRI, and full-length hPar1 was ligated into digested MMTV-SV40-BSSK after the MMTV-LTR followed by the SV40 poly(A) site (see Fig. 1 ). The product was named the MMTV-hPar1 construct. The purified MMTV-hPar1 plasmid was prepared for microinjection by digestion with SpeI. It was then injected into the pronucleus of fertilized C57BL/6 mouse oocytes and transferred to a pseudopregnant CB6/F1 mice uterus (initial animal manipulations were carried out by the Transgenic Facility Unit of Hadassah-Hebrew University Medical School, Jerusalem, Israel). Mice were maintained on a CB6/F1 background. For timed pregnancies, male and female mice were mated overnight and female mice were scored for vaginal plugs the next morning, representing pregnancy day 1.


Figure 1
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  		Figure 1. Generation of MMTV-hPar1-tg mice (hPar1-tg) targeted to the mammary glands. A, schematic representation of MMTV-SV40-BSSK-hPar1 construct for mammary-specific expression (MMTV-LTR-hPar1). The long form of the MMTV-LTR is used to drive selective targeted expression and the SV40 splicing and polyadenylation fragment enhances export and translation. Full-length human Par1 DNA was inserted between the sites of HindIII and EcoRI. B, genotyping of the founder mice was carried out by Southern blotting. The tail DNA was digested with BamHI and EcoRI, and a transgene fragment was detected via Southern blot. Estimated copies of hPar1 gene 300 pg (30 copies), 100 pg (10 copies), lines 1-4 (L1-L4), and wt. C, whole-mount hematoxylin staining of wt and hPar1-tg mammary glands at different developmental stages. The epithelial tissue derived from the hPar1-tg mammary glands displays increased lateral branching and pervasive intraductal hyperplasia in virgin (V-13w) and pregnant mammary glands (days 8 and 12 of pregnancy, respectively) compared with age-matched wt mice. D, histologic analyses of H&E staining showing the fine histology of the same stages depicted in the whole-mount staining of wt and hPar1-tg mammary glands. V, virgin; P, pregnancy.

 
Southern blot Analysis of Genomic DNA
Genomic DNA was prepared using proteinase K (10 µg) and cut (overnight at 37°C) with BamHI and EcoRI (see Fig. 1B), separated by electrophoresis on a 1% agarose-TAE gel, and transferred to Hybond-N+ membranes (Amersham Pharmacia Biotech UK Limited, London, United Kingdom). The blots were then hybridized (overnight at 65°C) with [{alpha}-32P]dCTP probe (Rediprimer II, Amersham Biosciences UK) for human Par1 (1.9 kb BamHI- and EcoRI-digested fragment of MMTV-hPar1). Blots were washed and exposed to X-ray films. The approximate transgene copy number was determined by adding 100 pg (10 copies) or 300 pg (30 copies) of MMTV-hPar1 to genomic DNA from a wt mouse.

Plasmid Purification
The plasmids pCMV-HA, pCMV-HA-SFRP5, and pcDNA3-Myc-wt-ß-catenin (22) were purified using the Qiagen plasmid purification kit (Qiagen GmbH, Hilden, Germany):

Mammary Gland Whole-Mount Staining
Whole-mount staining of mammary glands was done as described (23). Briefly, the entire left no. 3 mammary gland was removed and flattened on a tissue capsule, fixed in Telly's fixative, defatted in three changes of acetone, hydrated in 95% ethanol, and stained with hematoxylin (0.65 g FeCl3, 67.5 mL H2O, and 8.7 mL 10% hematoxylin in 95% ethanol, pH 1.25). Glands were rinsed in water, destained in acid ethanol, dehydrated in increasing ethanol concentrations, stored indefinitely in methyl salicylate, and photographed using a Zeiss microscope.

Histology and Immunohistochemistry Assay
The entire left no. 4 mammary gland was removed and fixed with 4% formaldehyde. Sections of 5 µm were prepared and stained with H&E. For immunoperoxidase staining, paraffin-embedded sections were gradually dehydrated. Antigen retrieval was achieved by microwaving the sections (5 minutes), then incubating overnight (4°C) with the following antibodies: proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), ß-catenin (610153, BD Transduction Laboratories, Franklin Lakes, NJ), and anti c-Myc (9E10, Santa Cruz Biotechnology) and visualized using Broad Spectrom (AEC) Kit (Zymed Laboratories, Inc., San Francisco, CA).

In situ Hybridizations
RNA probes were transcribed and labeled by T7 RNA polymerase (for antisense orientation) or T3 RNA polymerase (for sense orientation as control) using DIG-UTP labeling mix (Boehringer Mannheim, Mannheim, Germany) as previously described (3).

Western blot Analysis
Entire right no. 4 mammary gland tissues were removed, homogenized in radioimmunoprecipitation assay buffer, incubated (20 minutes, 4°C) for adequate lysis, and centrifuged at 10,000 x g (20 minutes, 4°C). Lysates (50 µg) were resolved by 10% SDS-PAGE and transferred to Immobilon-P membrane (Millipore, Bedford, MA). Membranes were then blocked and probed with polyclonal anti–cyclin D1 (Santa Cruz Biotechnology), monoclonal anti-ß-catenin (Transduction Laboratories, Lexington, KY), or monoclonal anti-ß-actin (Sigma-Aldrich Israel Ltd., Rehovot, Israel) primary antibodies in 1% bovine serum albumin in TTBS. After extensive washes, blots were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase (Pierce, Rockford, IL). Immunoreactive bands were detected by the enhanced chemiluminescence reagent using Supersignal (Pierce).

PAR1 Activating Peptide
SFLLRNP. H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-NH2.

Nuclear Fraction
Cells were washed with PBS, scraped, and collected. The cell pellet was resuspended in buffer A [10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L DTT, and protease inhibitors 5 mg/mL aprotinin, cocktail, and 10 mg/mL leupeptin]. The cells were incubated on ice (15 minutes); after which, a 10% solution of NP40 was added. The homogenate was centrifuged at 10,000 x g (30 seconds at 4°C). The pellet was rotated (15 minutes at 4°C) with buffer C [20 mmol/L HEPES (pH 7.9), 420 mmol/L KCl, 1 mmol/L EDTA, and 1 mmol/L DTT, with protease inhibitors 5 mg/mL aprotinin, cocktail, and 10 mg/mL leupeptin]. The tubes were centrifuged at 15,000 x g (15 minutes at 4°C) and the protein contents of the supernatants were evaluated.

Imunoprecipitation
Mammary gland lysates (600 µg) of total protein were used for immunoprecipitation using goat anti-mouse Wnt-4 antibody (10 µg/mL; R&D Systems, Inc., Minneapolis, MN). After overnight incubation, protein G-sepharose beads (50 µL; Amersham Pharmacia Biotech, London, United Kingdom) were added to the suspension, which was subsequently rotated at 4°C for 1 hour. Elution of the reactive proteins was done by resuspending the beads in 2x protein sample buffer followed by boiling for 5 minutes. The supernatant was then resolved on a 10% SDS-polyacrylamide gel and treated as indicated above for Western blotting.

Reverse Transcriptase-PCR
Entire right no. 4 mammary tissues were removed and total RNA was isolated using the TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the instructions of the manufacturer. First-strand cDNA was synthesized from 1 µg of total RNA (1 hour, 42°C) using MMLV reverse transcriptase and oligo-dT (both from Promega, Heidelberg, Germany). cDNA was subjected to PCR amplification with Taq polymerase (Bioline, London, United Kingdom) using a specific set of primers in a total volume of 20 µL. PCR reaction was done by denaturation at 94°C for 3 minutes and 24 to 30 cycles of amplification (94°C for 30 seconds, 55-65°C for 1 minute, and 72°C for 1 minute). All the PCR reactions were followed by a 10-minute extension at 72°C at the final step. Glyceraldehyde-phosphate dehydrogenase (GAPDH) and L19 primers were used as housekeeping genes for of the human and mouse control. Primers used were L19: 5'-CTGAAGGTGAAGGGGAATGTG-3' (sense), 5'-GGATAAAGTCTTGATGATCTC-3' (antisense; 24 cycles); human Par1: 5'-GCCAGAATCAAAAGCAACAA-3' (sense), 5'-GAGATGAATGCAGGAAGTTGTTT-3' (antisense; 30 cycles); mouse wnt-4: 5'-AGGAGTGCCAATACCAGTTCC-3' (sense), 5'-TGTGAGAAGGCTACTCCATA-3' (antisense; 30 cycles); and mouse wnt-7b: 5'-CAAGGCTACTACAACCAGGC-3' (sense), 5'-CACCTCCACCTGCACCGCTG-3' (antisense; 30 cycles). The PCR products were run electrophoretically on a 1% agarose-TAE gel and visualized by ethidium bromide under UV light.

siRNA Constructs
We used U6 promoter-driven and lentivirus (pLentilox 3.7)-mediated delivery cassette of siRNA, specific for hPar1. For this, a sequence of 19 nucleotides of the hPar1 coding region was selected for stem-and-loop oligonucleotide siRNA. The selected sequences were submitted to a BLAST search against the human genome to ensure that it was not targeted. To construct the hairpin, stem-and-loop siRNA expression cassette, appropriate DNA oligonucleotides were synthesized. The oligos were composed of the following: 19 bases of hPar1 coding sequence, the loop sequence linker (9 bases), reverse complement of the 19 bases of hPar1 coding region, and a terminator sequence poly(T). The sticky end of the XhoI site was added to the antisense strand oligos. Both sense and antisense sequences were phosphorylated at the 5' ends. The sense sequence oligos were annealed to their respective antisense oligos. siRNA cassette sequences were then ligated into pLentilox 3.7 vector (Van Parij's laboratory). We created four such siRNA cassettes from the hPar1 gene.

Preparation of Lentivirus
The lentivirus particles were generated by a three-plasmid expression system in which 293T cells were cotransfected with the following three vectors: packaging (CMV{Delta} R8.91), envelope (CMV-VSV-G), and the transfer vector P lentilox 3.7. One day before transfection, 293T cells were plated to ~60% confluency. On the next day, the medium was changed to fresh medium and cells were transfected with the three plasmids using FuGENE 6 transfection reagent. Medium was changed to fresh medium 24 hours posttransfection. On days 2 and 3 posttransfection, medium was collected to recover viral particles. The collected medium was centrifuged for 1 hour at 40,000 rpm to concentrate the viral particles and concentrated to 100x.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mammary gland tissue targeted overexpression of hPar1-tg mice show precocious hyperplasia. To assess the effects of elevated hPar1 expression in mammary epithelial cells in vivo, we generated transgenic mice which overexpress hPar1 targeted to the mammary glands. An expression cassette construct was engineered in which the full-length cDNA for hPar1 was placed under transcriptional control of the MMTV-LTR, followed by an SV40 polyadenylation (SV40 poly A) site (Fig. 1A). The construct was injected into the pronucleus of fertilized CB57BL/6 mouse oocytes and transferred into pseudopregnant CB6/F1 foster mothers. Of the 60 resulting pups, 4 transgenic founders passed the transgene to progeny, generating four independent transgenic lines (MMTV-LTR-hPar1 L1-L4, Fig. 1B). We chose lines L2 and L4, which exhibited high hPar1 levels by Southern blot analysis, for further studies.

To determine the effects of hPar1 expression on mammary gland morphogenesis, histologic preparations of mammary tissues from various developmental stages were analyzed. Whole mounts of mammary glands from wild-type (wt) CB6/F1 mice were compared with age-matched transgenic littermates (Fig. 1C and D). The mammary glands of hPar1-tg mice showed increased branching with moderate to marked lobuloalveolar development. During normal mammary development, rudiments of the ducts formed at birth and grow slowly until onset of puberty, when terminal end buds form and ductal elongation and bifurcation begin. Normally, in virgin wt animals, the degree of ductal side branching and the number of terminal end buds increase with age (until ~6 weeks of age, then they begin to decline); however, the overall ductal complexity observed in hPar1-overexpressing glands was consistently greater than that of their age-matched wt counterparts (Fig. 1C, V-13w; 3W-10W of virgin mice not shown). During early pregnancy, precocious development of hPar1-tg glands was even more striking. Lobuloalveolar structures become evident in wt females during early pregnancy (Fig. 1C, wt P8d and P12d). This phenotype commenced earlier in the hPar1-overexpressing glands, exhibiting a high level of ductal network complexity and increased alveolar buds (see Fig. 1C, hPar1-tg; P8d and P12d).

H&E staining of tissue sections revealed accelerated lobuloalveolar development of the hPar1-overexpressing mammary glands as compared with wt mice (Fig. 1D). In situ hybridization analysis with hPar1 Dig-labeled riboprobes revealed abundant expression of hPar1 in the transgenic animals at all ages examined. This expression is confined to the luminal epithelium and is absent from the myoepithelial layer (see arrow in Fig. 2A , P4d, high magnification). It is of interest that the luminal site at which hPar1 is expressed is also the site of wnt-4, which was elegantly shown to play a major role in mammary gland ductal side branching (24, 25). Reverse transcriptase-PCR (RT-PCR) analysis of RNA extracted from the mammary glands of individual mice showed a similar pattern: high hPar1 expression levels in the hPar1-tg mammary glands (with a slightly elevated expression during pregnancy) as compared with no expression in age-matched wt animals (Fig. 2B). RT-PCR analysis revealed that expression of hPar1 in both lines (e.g., L2 and L4) was confined to the mammary glands where it was present at high levels (see Fig. 2C).


Figure 2
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  		Figure 2. Tissue localization and pattern of hPar1 expression in hPar1-tg mammary glands. A, in situ hybridization analysis of hPar1 expression in sections of mammary gland in the hPar1-tg and wt mice. hPar1-specific expression is seen in 13w virgin mice and in pregnant mice at 4, 8, and 12 days. No hPar1 was observed in either wt virgin or through the pregnancy period (data not shown) and 12-day pregnant wt (bottom). High magnification shows hPar1 expression confined specifically to the luminal epithelial layer of the mammary gland (arrow in the square). B, RT-PCR analysis of hPar1 expression in different development stages of the transgenic mice (5w, 8w 10w, 13w, P4d, and P8d) compared with age-matched wt mice. C, RT-PCR analysis of hPar1 expression in different organs of MMTV-LTR-hPar1 transgenic mice. Organs from hPar1-tg mice were removed, RNA was isolated, and RT-PCR analysis was done as described in Materials and Methods. PCR for hPar1 and L19 (control gene) was done. D, i, mammary glands overexpressing hPar1 display increased epithelial proliferation. Mammary glands from wt and hPar1-tg mice were examined for PCNA expression by immunohistochemistry. The staining was done at 5w, 10w, and 13w of the virgin stage, and 4 and 8 days of pregnancy, respectively. ii, histogram indicates the proliferation index, defined as the number of PCNA-positive nuclei per total number of nuclei. The values represent the average proliferation index for three different mice mammary glands.

 
Whether increased ductal network complexity in hPar1-overexpressing mammary glands is attributed solely to ductal side branching or due to on-going proliferation was next addressed. For this, we immunostained sections of normal and hPar1-overexpressing mammary glands with an antibody to PCNA, a marker for S-phase cells (Fig. 2D, i and ii). The proliferation index is defined as the number of PCNA-positive nuclei of alveolar epithelial cells divided by the total number of nuclei. Whereas increased proliferation is seen in normal wt mammary already at 5 weeks of age, the proliferation index in the transgenic mice shows a 3-fold increase. The difference in proliferation index between wt and hPar1-overexpressing animals is particularly pronounced between postnatal weeks 10 and 13 (e.g., virgin 10W and 13W) when proliferation of alveolar epithelia is normally arrested in wt animals (Fig. 2D, i and ii). During pregnancy, proliferation of alveolar epithelia resumes also in the wt mice; however, we observe an accelerated proliferation rate in the hPar1-overexpressing mice.

Wnt-4 and wnt-7b are overexpressed in hPar1-tg mammary glands. Because one of the mechanisms of mammary gland hyperplasia suggested an essential role for Wnt-4 in ductal side branching (24), we thought to screen for levels of mouse wnts in normal wt versus transgenic mice mammary glands. Wnt encodes a large family of secreted glycoproteins and binds to a complex of Frizzled family of seven-transmembrane receptors (15, 16, 18) and LRP5/6 coreceptors (12, 13, 21). Whereas wnt exerts its effects on embryonic cells and in pathologic carcinoma progression, the proximal steps of the signaling cascade beyond Frizzled are less well understood.

The expression of wnt in mouse mammary glands was initially analyzed by RT-PCR. We have examined an entire spectrum of mouse wnts (i.e., wnt-7a, -5b, -6, and -10b, known to be present in the epithelia, as well as wnt-2 and -5a, present in mouse mammary stroma). Our data revealed specific enhancement of wnt-4 and wnt-7b in hPar1-tg mammary glands. No change was detected in wnt-2, -5a, -6, as well as in wnt-7a, -5b, and -10b. Normally, low levels of wnt-4 are observed in the virgin wt mice, which increased slightly during pregnancy. In comparison, hPar1-overexpressing mice exhibit greater levels of wnt-4 in the virgin mammary glands, most visibly at 10 weeks of age. An additional increase in wnt-4 levels is observed throughout pregnancy in the transgenic animals (Fig. 3A ). To substantiate the effects of hPar1 overexpression on wnt-4 levels, we did immunoprecipitation analysis on mammary gland protein lysates (Fig. 3B). The results confirm a profound increase in the levels of Wnt-4 protein when compared with age- and gestation-matched controls, especially in pregnant hPar1-overexpressing mammary glands at 4 and 8 days of pregnancy. We cannot currently explain why a regulated increase in wnt-4 levels is observed in hPar1-tg, low in virgin mice, and significantly increased during pregnancy because hPar1 levels are similarly expressed throughout these stages. It may be that hormones present during pregnancy may be involved in the process. In addition to the increase in wnt-4, we found enhancement in the levels of wnt-7b but no effect on levels of other wnts normally expressed in the mouse mammary glands (24). This suggests that hPar1 specifically regulates the expression of at least the two mouse wnt genes either directly or indirectly.


Figure 3
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  		Figure 3. Increasing mouse Wnt and ß-catenin expression in hPar1-tg mammary glands. A, RT-PCR analysis of mouse wnt-4 and wnt-7b expression in mammary glands of wt and hPar1-tg mice. B, immunoprecipitation analysis of mouse wnt-4 expression in hPar1-tg and wt mice. The transgenic mice show higher expression levels in 4- and 8-day pregnant mice compared with the wt mice. The same amount of protein was loaded and blotted with ß-actin as control. The immunoprecipitation data seem to be specific because normal immunoglobulin G (IgG) did not show any protein precipitation. C, increased cyclin D1 induction in hPar1-tg mice mammary glands. Western blot analysis of cyclin D1 expression in wt and hPar1-tg mammary glands. ß-Actin was used as a control for protein loading. The average relative expression levels are shown by a histogram where wt mice were given an arbitrary value of 1.00.

 
One target of transcriptional activation by the wnt pathway is cyclin D1 (19, 26). Cyclin D1, the major G1 cyclin expressed in mammary epithelial cells, is deficient in mice resistant to breast cancers induced by the neu and ras oncogenes, implying its tight connection to the Neu-Ras pathway transformation (20). We found increased cyclin D1 expression in the hPar1-overexpressing mammary glands as compared with wt counterparts (Fig. 3C). At 4 days of pregnancy, cyclin D1 levels are 4.5-fold higher in the transgenic mice than in wt animals, and at 8 days of pregnancy, they remain elevated 2.5 times higher in the transgenic animals.

Accumulation of ß-catenin in hPar1-overexpressing mice. To explore the possible signaling pathway downstream of wnt, we evaluated the expression levels of ß-catenin in the mammary glands overexpressing hPar1 and age-matched controls. Western blot analysis showed a striking accumulation of ß-catenin in the mammary gland tissues of both virgin and pregnant hPar1 transgenes as compared with minimal levels in the mammary tissue of wt controls (Fig. 4B ). Immunohistochemistry staining revealed abundant localization of ß-catenin in the nuclei of alveolar epithelial cells, taking place only in hPar1-overexpressing glands, as compared with none in age-matched wt animals (Fig. 4A). This shows that hPar1 overexpression leads to stabilization of ß-catenin that is ultimately imported to the nuclei, where it may further regulate gene transcription.


Figure 4
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  		Figure 4. hPar1-tg mammary glands show increased ß-catenin expression. A, immunohistochemical staining shows ß-catenin localization in the mammary glands of hPar1-tg and wt mice. At 10 days of pregnancy, nuclear ß-catenin staining is observed in hPar1-tg mice but not in wt mice (x40). Bottom, higher magnification (x100). B, Western blot analysis of ß-catenin expression in hPar1-tg mammary glands at different developmental stages compared with age-matched wt mammary glands.

 
To address the molecular interrelation between hPar1 and ß-catenin stabilization, we examined transformed epithelial cells in parallel to the transgenic-mice tissues. It is important to note that most breast cancer cell lines have impaired ß-catenin signaling machinery system as shown in a wide screening done by van de Wetering et al. (27). It has been reported that only 1 of the 15 breast cancer cell lines analyzed showed TCF-mediated ß-catenin activity although most of them expressed ß-catenin. We therefore chose to do analyses in a panel of cell lines, including colorectal cancer cell lines, widely used for studies in wnt/ß-catenin signaling machinery. These cell lines express high hPar1 levels. When HT-29 colon cancer cells were processed to obtain discrete cell fractions of either membrane or cytoplasm after PAR1 activation, the following pattern was seen. Whereas the majority of ß-catenin was initially localized at the membrane pool, a significant increase in ß-catenin cytoplasmic fraction was seen as soon as 1 hour after PAR1 activation and remained elevated at 2 hours activation (Fig. 5A ). A similar pattern of kinetics is obtained when HCT116 cells were analyzed (data not shown). When the nuclear fraction of HCT116 cells was isolated and analyzed for levels of ß-catenin, an increase is seen as soon as 2 hours after PAR1 activation, a significant elevation after 4 hours, and remaining enhanced up to 24 hours of examination (Fig. 5B). Similarly, when these cells were pretreated with LiCl (a known glycogen synthase kinase 3ß inhibitor) followed by SFLLRNP (a synthetic peptide of PAR1 internal ligand) for PAR1 activation, an induced enhancement of ß-catenin stabilization was observed. The induction was seen as early as 30 minutes after activation (3-fold), remaining up to 5 hours (3.2-fold; Fig. 5C). Thus, we notice a further induction in ß-catenin stabilization, following PAR1 activation, regardless of whether pretreated with LiCl or without. These colon cancer cell lines, however, harbor mutations in either antigen-presenting cells (APC; e.g., HT-29 cells, truncated APC) or ß-catenin (e.g., HCT116, mutated amino acid 45), resulting in a constitutively activated ß-catenin (28). We did, therefore, a control experiment using a transformed cancer cell line of intact ß-catenin machinery system, the RKO cells. RKO is a colorectal cancer cell line transformed on the basis of gene instability (29). This cell line displays microsatellite instability for hypermethylation of the hMLH1 promoter but expresses wt APC, ß-catenin, and p53 (28, 30). Thus, RKO is a colon cancer cell line on the background of a defective mismatch repair system. We analyzed the effect of PAR1 in RKO cells to eliminate the possibility that PAR1-induced ß-catenin takes place on the background of a constitutively activated ß-catenin/TCF-regulated transcription system. Whereas ß-catenin was not detected initially in these cells, pretreatment with either LiCl or MG132 (an inhibitor of the proteasomal system) followed by PAR1 activation showed a further enhancement in nuclear ß-catenin (Fig. 5Di). Semiquantitative RT-PCR analysis shows the levels of hPar1 in various types of epithelial tumor cell lines. The data show high hPar1 levels in HCT116 and HT-29, very low levels in RKO and SW480, and none in CaCO2 cells (Fig. 5Dii).


Figure 5
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  		Figure 5. Activation of PAR1 induces ß-catenin stabilization. A, cytoplasmic accumulation of ß-catenin. Separate fractions of membrane and cytoplasm in HT-29 colorectal cells show marked accumulation of ß-catenin in the cytoplasmic fraction after thrombin PAR1 activation. Significant enhancement is observed 1 hour after activation and remains elevated at 2 hours. B, nuclear accumulation of ß-catenin. The nuclear fraction of HCT116 cells shows consistent accumulation of ß-catenin after PAR1 activation (as indicated by addition of SFLLRNP), initiated 2 hours after activation and remaining elevated up to 24 hours of examination. C, LiCl pretreatment (2 hours) of HT-29 cells followed by PAR1 activation (for the indicated periods of time) results in enhanced levels of ß-catenin in the cell nuclei. Histogram shows the estimated levels of ß-catenin before and after PAR1 activation, obtained either with or without LiCl pretreatment (for 2 hours). D, i, PAR1 activation induces ß-catenin levels in RKO cells. RKO cells pretreated with either LiCl or MG132 before (–) and after (+) PAR1 activation. Whereas no ß-catenin is observed in nontreated RKO cells, it is seen following both treatments. After PAR1 activation, a further increase in ß-catenin levels is noticed. ii, semiquantitative RT-PCR analysis of colon cancer epithelial cell lines. E, nuclear ß-catenin levels in MDA 231 breast cancer cells. PAR1 activation induces ß-catenin levels in breast cancer cells, similar to the effects seen in colorectal cell lines, before and after LiCl pretreatment.

 
Essentially, induced ß-catenin levels are observed also in MDA231 breast cancer cell lines. Activation of PAR1 either before or after LiCl pretreatment showed elevation in ß-catenin levels (Fig. 5E). Taken together, we conclude that PAR1 activation leads to stabilization of ß-catenin analyzed either in a breast cancer cell line or in colon cancer cells transformed regardless of whether on the background of intact ß-catenin system or constitutively activated ß-catenin.

Enforced overexpression of SFRP5 and SFRP2 abrogate ß-catenin stabilization following PAR1 activation. To examine whether PAR1 activation leads primarily to increased Wnt expression that ultimately induces ß-catenin stabilization, we have analyzed members of the Wnt antagonist family, SFRPs, on PAR1-induced ß-catenin stabilization. For this purpose, we cotransfected the HCT116 cell line with Myc-wt-ß-catenin and SFRP5 plasmids. As seen in Fig. 6A , PAR1 activation led to a marked increase in the ectopically transfected ß-catenin detected by the Myc tagging of nuclear ß-catenin after PAR1 activation. This increase is abrogated in the presence of enforced overexpression of SFRP5. The neutralizing effect of SFRP indicates that PAR1 activation induced ß-catenin stabilization primarily via the induction of wnt. In the presence of SFRP5, it sequesters Wnt, which is generated by the activation of PAR1, binds to the Frizzled cell-surface receptors through the sequence homology domain cysteine-rich domain, and forms a complex also with LRP5/6 coreceptor (21, 31–33). It is postulated that by forming a neutralizing complex with the extracellular domains of both receptors, the SFRPs are effective in antagonizing Wnt signaling machinery (21).


Figure 6
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  		Figure 6. SFRPs suppress PAR1-induced ß-catenin stabilization. A, HCT116 cells were cotransfected with Myc-wt-ß-catenin and SFRP5. Nuclear fractions were isolated and levels of ß-catenin were analyzed by Western blot analysis before and after PAR1 activation. Detection of the ectopically inserted wt-ß-catenin was followed by its Myc tagging (i.e., anti-Myc antibodies; 9E10, Santa Cruz Biotechnology). Induced nuclear accumulation in Myc-wt-ß-catenin is seen after PAR1 activation (b) as compared with (a). Whereas no effect on ß-catenin levels is seen following mock plasmid cotransfection (data not shown), the cotransfection of Myc-wt-ß-catenin with SFRP5 to the cells following PAR1 activation resulted in distinct inhibition of ß-catenin levels. B, i, application of conditioned medium (CM) containing SFRP5 or SFRP2 or both efficiently abrogates PAR1-induced ß-catenin stabilization. Nuclear fractions of HT-29 cells were isolated either before (a and d) or after (b, c, e, f, g, and h) PAR1 activation, following incubation of some of the cells with either SFRP2-CM (e, g, and h) or SFRP5-CM (d, e and g, h). Detection of endogenous ß-catenin levels is evaluated by the use of anti-ß-catenin antibodies. The amount of nuclear proteins applied is monitored by a nuclear housekeeping control gene, lamin. ii, RT-PCR analysis showing levels of SFRP2 and SFRP5 following transfection (+) and levels before transfection (–). iii, histogram analyses of SDS-PAGE band intensity (nearly 2-fold induction is observed by PAR1 activation, which is reduced to a background level in the presence of SFRPs). C and D, hPar1 siRNA inhibits PAR1-induced Wnt-4. RT-PCR analysis of Wnt-4 in either RKO (C) or HT-29 cells (D) following PAR1 activation. Levels of wnt-4 were compared with a housekeeping control gene, GAPDH. Infection of siRNA constructs in RKO (Cii) or HT-29 cells (Dii) potently inhibited PAR1-induced wnt-4 levels. Inhibition of PAR1-induced wnt-4 is obtained under conditions where hPar1 is depleted [using the siRNA constructs i and iv as shown for HT-29 cells (Dii); similarly seen in RKO-depleted hPar1 cells (data not shown)].

 
In parallel, 293T cells were transfected with either SFRP5, SFRP2, or both and the conditioned medium containing secreted SFRP5, SFRP2, or both was collected, providing a source for SFRPs. Equal transfection yields were observed following SFRP2 and SFRP5 plasmid transfections (Fig. 6Bb). When these conditioned media were administered to HT-29 cells before and after PAR activation, the following outcome was observed: whereas enhanced stabilization of ß-catenin was seen after PAR1 activation, it was efficiently abolished in the presence of conditioned medium containing either SFRP5, SFRP2, or both (Fig. 6B). This indicates that SFRPs (at least SFRP2 and SFRP5) can efficiently inhibit PAR1-induced ß-catenin stabilization.

siRNA-mediated hPar1 silencing inhibits PAR1-induced Wnt-4 generation. We next evaluated whether a direct link between PAR1 and wnt-4 exists. For this purpose, we have examined levels of wnt-4 expression in both RKO and HT-29 cells. Enhanced levels of wnt-4 are seen as soon as 4 hours after PAR1 activation, which were significantly elevated by 12 hours (Fig. 6Ci). The same pattern is seen in HT-29 cells, but with a different kinetics. Increased levels of wnt-4 take place earlier, showing increased levels as soon as 1 hour after PAR1 activation with a maximal induction observed by 4 hours (Fig. 6Di).

When constructs of anti-hPar1 RNA interfering sequences (siRNA) ligated into pLentilox 3.7 viral vector (Van Parij's laboratory) were infected into either RKO or HT-29 cells (Fig. 6Cii and Dii, respectively), effective depletion of hPar1 is noted in two of the four constructs examined (Fig. 6Dii). Under these conditions of hPar1-depleted levels, a significant abrogation of PAR1-induced wnt-4 enhancement is observed.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To evaluate the causal relationship between hPar1 gene expression, breast tissue morphogenesis, and hyperplasia, we generated mice overexpressing hPar1 in their mammary glands. These glands exhibit precocious hyperplasia and recapitulate the hallmarks of Wnt signaling pathway as shown by ß-catenin stabilization and nuclear localization. Altogether, the properties portrayed by these glands point to a novel molecular trail elicited by hPar1. The fact that SFRPs, members of Wnt antagonist family, effectively abrogate PAR1-induced ß-catenin stabilization shows that hPar1 expression and activation lead primarily to Wnt generation, which further induces ß-catenin stabilization via an autocrine loop (21). Once generated, it is postulated that secreted Wnts reactivate their cognate cell-surface receptors affecting downstream cell signaling. In addition, we show that viral constructs of hPar1 siRNA, depleting hPar1 levels, inhibit effectively PAR1-induced Wnt-4 generation. This is the first demonstration of a direct link between hPar1 and Wnt-4. Neutralization of hPar1 and, hence, the abrogation of Wnt-4 take place regardless of whether on the background of intact ß-catenin system but defective in mismatch repair system or in cells with mutated APC on the background of constitutively activated ß-catenin. Whether Wnt-4 promotes a canonical or a noncanonical signaling machinery is controversial. Recently, Wnt-4 was shown to promote, at least in a specific tissue context of Madin-Darby canine kidney epithelial cells, the canonical ß-catenin pathway (34). The association between hPar1 and wnt-7b remains to be fully elucidated. The molecular pathway by which PAR1 expression and activation induce Wnt3 remains as well to be shown.

It is intriguing to note that a recent study (35) also showed a mechanism of ß-catenin stabilization for another class of G protein–coupled receptors belonging to the lysophosphatidic acid receptors. Lysophosphatidic acid receptors are shown to play a central part in several types of cancer (36, 37). Thus, PAR1 G protein–coupled receptor joins at least lysophosphatidic acid 2 and 3 receptors, acting via ß-catenin stabilization and shown to induce increased expression of c-Myc and cyclin D1, two of the downstream target genes of ß-catenin transcriptional activity. A common theme is thus emerging, pointing to the significance of G-proteins as the proximal missing piece in the Frizzled seven-transmembrane receptor/s signaling puzzle (under our current investigation).

The Wnt family of secreted glycoproteins act through two cell-surface receptors, the Frizzled and the single-transmembrane protein LRP5/6 (or Arrow). These components are essential for efficiently transducing signals from Wnt, an extracellular ligand, to the intracellular pathway that stabilizes ß-catenin. The current model suggests that Wnt protein binds to the extracellular domains of both LRP and Frizzled receptors, forming membrane-associated hetero-oligomers that interact with both Dishevelled (via the intracellular portion of Frizzled) and Axin (via the intracellular domain of LRP; refs. 12, 13, 38). The molecular mechanism, however, by which these two types of membrane receptor synergies transmit Wnt signals is yet unknown.

The fact that exogenous administration of specific Wnt antagonists (i.e., SFRPs) inhibits PAR1-induced ß-catenin stabilization, further afflicting downstream components, implies a Wnt autocrine loop. We show now that overexpression of SFRP5 confers inhibition of PAR1-induced ß-catenin stabilization by either enforced SFRP5 expression or the ectopic application of SFRP5 and/or SFRP2 or both. This result is in line with the above studies (21, 22) showing an autocrine loop of Wnt signaling. It has been shown, for example, that colorectal cancer develops on the background of SFRP gene neutralization (following the hypermethylated SFRP promoter silencing; refs. 22, 39). Whereas in the presence of functionally active SFRP, Wnt signaling is attenuated, the normal equilibrium is disrupted when SFRPs are hypermethylated and silenced. Furthermore, functionally active SFRPs suppress Wnt signaling also in cells possessing constitutively activated mutations of ß-catenin (22). The discrete pattern of individual SFRP gene expression in hPar1-tg mice is under current investigation.

The hPar1-overexpressing mammary glands show a phenotype of precocious hyperplasia. We cannot currently explain the lack of tumor formation in the hPar1-tg mammary gland mice model. Our preliminary data show that orthotopic injection of MCF-7-hPar1 stable clones to the mammary fat pads induces tumors [data generated in our laboratory as also by Boire et al. (40)]. The orthotopic injections of MCF-7 clones overexpressing various hPar1 forms (e.g., full-length, Y397Z superactive or truncated form devoid the entire cytoplasmic tail) show advanced tumor formation in the hPar1 full-length, compared with very little or none in truncated hPar1 or mock-transfected cells.4 It is possible that cross-mating of the hPar1-tg mice with known models of mammary gland tumor progression (e.g., transgenes overexpressing the erBb 1-4 gene family) may result in accelerated rates of tumor formation (currently being explored in our laboratory).

The extracellular matrix barrier surrounding the myoepithelial mammary gland ducts plays an active role as a depot site for bioactive molecules, among which are proangiogenic and growth factors as well as adhesion molecules (41). Indeed, loss of the myoepithelium and extracellular matrix is associated with invasive characteristics in breast cancer (42, 43). hPar1 overexpression in the mammary glands elicits increased lateral budding and precocious mammary hyperplasia, suggesting the induction of stochastic events by hPar1, a gene that plays a central part in the invasive phenotype.

We hypothesize that several PAR1 ligand activators are present either in the stroma microenvironment or in neighbor host cells surrounding the invading cancer cells. For example, circulating prothrombin is abundantly present in wt mouse mammary glands (data not shown). It is possible that other candidates belonging to the coagulation cascade (44–46) may functionally activate PAR1. Recently, an intriguing observation suggests that matrix metalloprotease 1 (MMP-1), derived from the stromal microenvironment, activates PAR1 at the same thrombin cleavage/activation site (40). Immobilization of proteases to the stroma microenvironment allows the sustained and localized functions of proteolytic enzymes such as cathepsin G, elastase, and plasmin, as well as MMP-1, all of which may eventually have an effect on hPar1 signaling (47–49).

We propose that hPar1 expression and activation elicit primarily wnt expression and the striking stabilization of ß-catenin. Our studies highlight hPar1 gene as a potent target for cancer therapy because neutralization of the gene may effectively inhibit initiation of oncogenicity via wnt generation, ß-catenin stabilization, and LEF/TCF transcriptional response (i.e., capable of inducing an array of responsive genes-downstream). Elucidation of this fundamental core molecular pathway may lead nonetheless to a better understanding of cancer progression.


   Acknowledgments
 
Grant support: U.S. Army grant DAMD17-00-1-0277, Israel Science Foundation, founded by the Israel Academy of Sciences and Humanities, and the Israel Cancer Association and Israel Cancer Research Fund (R. Bar-Shavit).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Uri Gat for the reagents and for stimulating discussions and Dr. Cliff Tabin (Department of Genetics, Harvard Medical School, Boston, MA) for the generous gift of the mouse wnt-4 plasmid.


   Footnotes
 
3 Manuscript in preparation. Back

4 Cohen et al., manuscript in preparation. Back

Received 11/29/05. Revised 2/ 9/06. Accepted 3/ 6/06.


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 Materials and Methods
 Results
 Discussion
 References
 

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