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

Abstract

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 B-catenin stabilization. Nuclear localization of B-catenin is observed in hPar1 transgenic mouse tissue sections but not in the wild-type, age-matched counterparts. PAR1 induces B-catenin nuclear localization also in established epithelial tumor cell lines of intact B-catenin system (transformed on the background of mismatch repair system; RKO cells). We propose hereby that PAR1-mediated B-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 B-catenin stabilization. Likewise, application of either SFRP2 or SFRP5 on epithelial tumor cells completely abrogated PAR1-induced B-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 B-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 B-catenin stabilization. This link between PAR1 and B-catenin may bear substantial implications both in developmental and tumor progression processes. (Cancer Res 2006; 66(10): 5224-33)

Protease-activated Receptor-1 (PAR1) Acts via a Novel Gα13-Dishevelled Axis to Stabilize β-Catenin Levels

“…G␣ 13 , but Not G␣ 12 , Is Involved in PAR1-mediated ␤-Catenin Stabilization-We have recently established a novel link between PAR1 and ␤-catenin stabilization, shown both in TG mouse mammary gland tissues and transformed epithelial cells (4). Because only 1 of 15 breast cancer cell lines analyzed showed Tcf-mediated ␤-catenin transcription activity (44), we chose colorectal cancer lines, widely used for studies of the Wnt/␤-catenin pathway.…”

“…MMTV-hPar1-TG Mice-Transgenic (TG) mammary-directed hPar1 mice were generated as previously described (4). Briefly, the MMTV-hPar1 construct (full-length hPar1 gene from pcDNA3, subcloned into MMTV-SV40-BSSK, subsequent to MMTV-LTR) was used to generate CB6/F1 mice targeted to overexpress hPar1 in the mammary glands.…”

“…In addition to the traditional role of PAR1 in thrombosis, hemostasis, and vascular biology, its role in tumor biology is currently emerging (1)(2)(3). We have established a novel link between hPar1 and ␤-catenin stabilization, both in transgenic mouse mammary glands and in a wide spectrum of tumor cell lines (4). Protease-activated receptors are part of a large seven-transmembrane-spanning G protein-coupled receptor (GPCR) 2 family (5,6), shown to couple to G␣ i/o , G␣ q , or G␣ 12/13 within the same cell type (7).…”

PAR1 plays a role in epithelial malignancies: Transcriptional regulation and novel signaling pathway

“…A truncated form of hPar 1 , lacking its cytoplasmic tail, failed to activate the Lef/Tcf transcriptional system. These data propose a central role for the hPar 1 gene in mammary gland hyperplasia and reveal a novel pathway triggered by PAR 1 signaling for the functional stabilization of b-catenin, a core protein in the canonical Wnt signaling pathway, eliciting transcriptional activity (26). While it is well established that Wnt-induced signaling leads to b-catenin accumulation, the role of PAR 1 in b-catenin stabilization is unknown.…”

“…The ability to delete or overexpress genes in the mouse genome allowed identification of function-associated mediators involved in mammary gland growth and development. To examine the function of the hPar 1 gene in breast cancer progression, we established a line of transgenic mice carrying the hPar 1 gene designed to overexpress in the mammary glands (26). These glands grossly exhibited a phenotype of hyperplastic features, reminiscent of the overexpression of several known oncogenes.…”