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Mammary Gland Tissue Targeted Overexpression of Human Protease-Activated Receptor 1 Reveals a Novel Linkto B-Catenin Stabilization

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Mammary Gland Tissue Targeted Overexpression of Human Protease-Activated Receptor 1 Reveals a Novel Linkto B-Catenin Stabilization Research Article Mammary Gland Tissue Targeted Overexpression of Human Protease-Activated Receptor 1 Reveals a Novel Linkto B-Catenin Stabilization Yong-Jun Yin,1 Vered Katz,1 Zaidoun Salah,1 Myriam Maoz,1 Irit Cohen,1 Beatrice Uziely,1 Hagit Turm,1 Sorina Grisaru-Granovsky,1 Hiromu Suzuki,2 and Rachel Bar-Shavit1 1Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem, Israel and 2Department of Public Health, Sapporo Medical University S1, Chuo-ku, Sapporo, Japan Abstract receptor PAR1 in hemostasis, thrombosis, and vascular biology, it Protease-activated receptor 1 (PAR1) is emerging with distinct emerges with distinct assignments in tumor biology and angio- assignments in tumor biology. We show that tissue targeted genesis (3–7). We have previously shown that PAR1 is involved both overexpression of hPar1 in mice mammary glands results in in malignant breast carcinoma tumor progression (3) and the precocious hyperplasia,characterized by a dense network of physiologic invasion of placenta trophoblasts into the uterine ductal side branching and accelerated proliferation. These decidua (4). A direct correlation exists between levels of hPar1 glands exhibit increased levels of wnt-4 and wnt-7b and expression and tumor advancement in both clinically obtained a striking B-catenin stabilization. Nuclear localization of biopsy specimens and differentially metastatic cell lines (3, 8). In- B-catenin is observed in hPar1 transgenic mouse tissue fact, hPar1 plays an active role in breast carcinoma invasion sections but not in the wild-type,age-matched counterparts. because antisense silencing of the gene abrogates efficiently metastatic breast carcinoma cells from invading Matrigel-coated PAR1 induces B-catenin nuclear localization also in estab- lished epithelial tumor cell lines of intact B-catenin system filters in vitro (3). (transformed on the background of mismatch repair system; In parallel, a cDNA expression library screen based on the loss of RKO cells). We propose hereby that PAR1-mediated B-catenin anchorage-dependent growth and focus-forming activity in NIH stabilization is taking place primarily via the increase of Wnt 3T3 cells has identified PAR1 as a novel oncogene (9). With these expression. Enforced expression of a specific Wnt antagonist observations, PAR1 joins other G protein–coupled receptors, family member,secreted frizzled receptor protein 5 (SFRP5 ), including mas and g2a, which behave as oncogenes (10, 11). The efficiently inhibited PAR1-induced B-catenin stabilization. oncogenic properties of PAR1 accompany a collection of data Likewise,application of either SFRP2 or SFRP5 on epithelial showing that hPar1 is overexpressed in a wide range of epithelial tumor cells completely abrogated PAR1-induced B-catenin tumors, pointing altogether to the central role of PAR1 in nuclear accumulation. This takes place most likely via carcinoma invasion. inhibition of Wnt signaling at the level of cell surface (forming The canonical wnt/Wingless signaling pathway directs cell fate in a neutralizing complex of ‘‘Receptors-SFRP-Wnt’’). Further- many cell types and plays a central role in development and in more,depletion of hPar1 by small interfering RNA (siRNA) tumor progression. Wnt proteins are soluble glycoproteins vectors markedly inhibited PAR1-induced Wnt-4. The striking initiating cell signaling through binding to receptor complexes stabilization of B-catenin,inhibited by SFRPs on one hand and composed of Frizzled proteins and LDL receptor–related protein Wnt-4 silencing by hPar1 siRNA on the other hand,points to a (LRP) 5/6 (12, 13). The core of the Wnt pathway is the stability of h h novel role of hPar1 in Wnt-mediated B-catenin stabilization. -catenin. Accumulation of -catenin in the cytoplasm leads This link between PAR1 and B-catenin may bear substantial ultimately to its transport to the nuclei where it forms functional implications both in developmental and tumor progression complexes with lymphoid enhancer factor (LEF)/T-cell factor processes. (Cancer Res 2006; 66(10): 5224-33) (TCF) transcription factors (14, 15). Because the LEF/TCF DNA- binding proteins are incapable of activating gene transcription h Introduction alone, -catenin acts as a bridging cofactor, enabling the performance of LEF/TCF (for review, see ref. 16). A growing list Protease-activated receptors (PAR) form a family of G protein– of genes has presently been identified as downstream targets of coupled receptors encoding their own ligands and uniquely h-catenin nuclear activity. Among these are c-Myc (17) and cyclin activated via proteolytic cleavage (1). Each of the four PAR family D1 (18–20). members is activated via proteolytic cleavage, exposing an internal To gain further insight into the causal relationship between ligand distinct for every PAR protein (2). PARs act as sensitive hPar1 expression, breast tumor formation, and mammary gland sensors to the constantly changing extracellular proteases regard- development, we have established a line of mice carrying MMTV- less of whether they are present in a soluble or microenvironment- long terminal repeat (LTR)-SV40-driven hPar1 designed to over- immobilized forms. In addition to the classic role of thrombin express 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 Requests for reprints: Rachel Bar-Shavit, Department of Oncology, Hadassah- of individual genes in normal and malignant mammopoiesis. We Hebrew University Hospital, P.O. Box 12000, Jerusalem 91120, Israel. Phone: 972-2-677- examined the phenotype of hPar1 transgenic (tg) mice with respect 7563; Fax: 972-2-642-2794; E-mail: [email protected]. I2006 American Association for Cancer Research. to breast morphogenesis and evaluated levels of distinct Wnt gene doi:10.1158/0008-5472.CAN-05-4234 expression and h-catenin stabilization. We hereby propose a novel Cancer Res 2006; 66: (10). May 15, 2006 5224 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. hPar1 Induced b-Catenin Stabilization link between hPar1, h-catenin, and Wnt generation. This is based Materials and Methods on the combined analyses of mammary gland tissue samples and Generation of MMTV-hPar1-tg epithelial cancer cell lines. By the use of either secreted frizzled The coding sequence of the full-length human Par1 gene from pcDNA3- receptor proteins (SFRP), members of the Wnt antagonist family, or hPar1 was subcloned into MMTV-SV40-BSSK (kindly provided by Dr. R.G. hPar1 small interfering RNAs (siRNA), we provide evidence on the Pestell, Department of Developmental and Molecular Biology and Medicine, involvement of Wnts in PAR1-induced h-catenin stabilization. Albert Einstein College of Medicine, New York, NY). Briefly, pcDNA3-hPar1 Enforced expression of SFRP5, as also application of either SFRP2, was digested with HindIII and EcoRI to isolate the 1.4-kb full-length hPar1. SFRP5, or both, effectively abrogated PAR1-induced h-catenin In parallel, MMTV-SV40-BSSK was digested with HindIII and EcoRI, and stabilization. This mode of inhibition points to the presence of an full-length hPar1 was ligated into digested MMTV-SV40-BSSK after the autocrine Wnt signaling loop initiated by PAR1. Ectopic over- MMTV-LTR followed by the SV40 poly(A) site (see Fig. 1). The product was expression of members of SFRP antagonist sequesters the PAR1- named the MMTV-hPar1 construct. The purified MMTV-hPar1 plasmid was prepared for microinjection by digestion with SpeI. It was then injected into induced Wnts and binds Frizzled receptors through the homology the pronucleus of fertilized C57BL/6 mouse oocytes and transferred to a site of cysteine-rich domain. Thus, SFRPs antagonize Wnt signaling pseudopregnant CB6/F1 mice uterus (initial animal manipulations were on the level of cell surface. Our data are in line with the elegant carried out by the Transgenic Facility Unit of Hadassah-Hebrew University studies by Bafico et al. (21) on the Wnt autocrine loop in human Medical School, Jerusalem, Israel). Mice were maintained on a CB6/F1 tumorigenicity. In parallel, hPar1 siRNA constructs depleting hPar1 background. For timed pregnancies, male and female mice were mated levels efficiently inhibited Wnt-4 expression. Altogether, we conc- overnight and female mice were scored for vaginal plugs the next morning, lude that PAR1-induced h-catenin stabilization is mediated representing pregnancy day 1. primarily via the induced Wnt generation. The novel link between Southern blot Analysis of Genomic DNA PAR1, h-catenin stabilization, and Wnt may impinge significantly Genomic DNA was prepared using proteinase K (10 Ag) and cut both on developmental and tumor progression processes. (overnight at 37jC) with BamHI and EcoRI (see Fig. 1B), separated by Figure 1. Generation of MMTV-hPar1-tg mice (hPar1-tg) targeted to the mammary glands. A, schematic representation of MMTV-SV40-BSSK-hPar1construct 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 Par1DNA 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. www.aacrjournals.org 5225 Cancer Res 2006; 66: (10).
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