Blocking Wnt/LRP5 Signaling by a Soluble Receptor Modulates the Epithelial to Mesenchymal Transition and Suppresses Met and Metalloproteinases in Osteosarcoma Saos-2 Cells Yi Guo, 1 Xiaolin Zi, 2 Zach Koontz, 1 Alison Kim, 1 Jun Xie, 1 Richard Gorlick, 3 Randall F. Holcombe, 4 Bang H. Hoang 1 1 Department of Orthopaedic Surgery, University of California, Irvine, 101 The City Drive South, Orange, California 92868 2 Department of Urology, University of California, Irvine, 101 The City Drive South, Orange, California 92868 3 Department of Pediatrics, Albert Einstein College of Medicine, Montefiore Medical Center, 111 East 210th Street, Bronx, New York 10467 4 Department of Medicine, University of California, Irvine, 101 The City Drive South, Orange, California 92868 Received 27 April 2006; accepted 10 November 2006 Published online 22 February 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20356 ABSTRACT: We previously reported the Wnt receptor low-density lipoprotein receptor-related protein 5 (LRP5) was frequently expressed in osteosarcoma (OS) tissue and correlated with metastasis and a lower disease-free survival. Subsequent in vitro analysis revealed that dominant- negative, soluble LRP5 (sLRP5) can reduce in vitro cellular invasion. In the current study, we examined the molecular mechanisms of blocking canonical Wnt signaling by sLRP5 in Saos-2 osteosarcoma cells. Transfection of sLRP5 caused a marked up-regulation of E-cadherin in this cell line. This increase in E-cadherin, seen primarily at the cell–cell contact borders, was associated with down-regulation of Slug and Twist, transcriptional repressors which mediate cancer invasion and metastasis. In contrast, N-cadherin, a mesenchymal marker, was reduced by sLRP5. In addition, blocking Wnt signaling by sLRP5 modulated other epithelial and mesenchymal markers (keratin 8 and 18, fibronectin), suggesting a reversal of epithelial– mesenchymal transition (EMT) seen during cancer progression. SLRP5 also reduced the expression of matrix metalloproteinase (MMP) 2 and 14, consistent with a decrease in invasive capacity. SLRP5 transfection decreased both Met expression and hepatocyte growth factor (HGF)-induced cell motility. Taken together, these results support a role for Wnt/LRP5 signaling in invasiveness of a subset of OS cells. ß 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:964– 971, 2007 Keywords: low-density lipoprotein receptor-related protein 5; cadherin; Wnt signal- ing; osteosarcoma; metastasis INTRODUCTION Osteosarcoma (OS), the most common primary bone cancer, has a high tendency to metastasize. Despite multi-modality therapy, this malignancy still carries a recurrence rate of 30%–40%, most commonly manifested as metastatic disease to the lung. The mechanisms of progression and metas- tasis are only beginning to be elucidated in this cancer. Emerging evidence indicates that multiple signaling pathways and their interplay are involved in the metastatic process. In the canonical Wnt pathway, Wnt ligands bind Frizzled and LRP5/6 receptors and activate an intracellular cascade, leading to translocation of b-catenin into the nucleus. Nuclear b-catenin forms complexes with the T-cell factor/lymphoid enhan- cer factor (TCF/LEF) family of transcription factors to promote expression of target genes. Upon Wnt/ Frizzled/LRP interaction at the cell surface, the intracellular enzyme glycogen synthase kinase-3b (GSK-3b) is inactivated by phosphorylation. Together, GSK-3b, Axin and the adenomatous polyposis coli (APC) tumor-suppressor promote the degradation of cytoplasmic b-catenin. Down- stream targets of the Wnt pathway include cell cycle regulators, oncogenes, and matrix degrading enzymes. 1–3 Osteosarcoma metastasis has been linked to expression of the Wnt co-receptor LRP5. 4 LRP5 mRNA in OS tissue samples correlated with the occurrence of metastasis and an inferior 964 JOURNAL OF ORTHOPAEDIC RESEARCH JULY 2007 Correspondence to: Bang H. Hoang (Telephone: 714-456-7801; Fax: 714-456-7547; E-mail: bhhoang@uci.edu) ß 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.