Progression of Osteosarcoma from a Non-Metastatic to a Metastatic Phenotype Is Causally Associated with Activation of an Autocrine and Paracrine uPA Axis.

Liliana Endo-Munoz,Na Cai,Lilia Merida De Long,Rebecca Macklin,Pamela Mukhopadhyay,Michelle Hill,Eleni Topkas,Nicholas A Saunders,Andrew Cumming,David M Loeb

doi:10.1371/journal.pone.0133592

Liliana Endo-Munoz, Na Cai + Show 8 more

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https://doi.org/10.1371/journal.pone.0133592

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Abstract

Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS) resulting in 10–20% long-term survival. The factors and pathways regulating these processes remain unclear, yet their identification is crucial in order to find new therapeutic targets. In this study we used a multi-omics approach to identify molecules in metastatic and non-metastatic OS cells that may contribute to OS metastasis, followed by validation in vitro and in vivo. We found elevated levels of the urokinase plasminogen activator (uPA) and of the uPA receptor (uPAR) exclusively in metastatic OS cells. uPA was secreted in soluble form and as part of the protein cargo of OS-secreted extracellular vesicles, including exosomes. In addition, in the tumour microenvironment, uPA was expressed and secreted by bone marrow cells (BMC), and OS- and BMC-derived uPA significantly and specifically stimulated migration of metastatic OS cells via uPA-dependent signaling pathways. Silencing of uPAR in metastatic OS cells abrogated the migratory response to uPA in vitro and decreased metastasis in vivo. Finally, a novel small-molecule inhibitor of uPA significantly (P = 0.0004) inhibited metastasis in an orthotopic mouse model of OS. Thus, we show for the first time that malignant conversion of OS cells to a metastatic phenotype is defined by activation of the uPA/uPAR axis in both an autocrine and paracrine fashion. Furthermore, metastasis is driven by changes in OS cells as well as in the microenvironment. Finally, our data show that pharmacological inhibition of the uPA/uPAR axis with a novel small-molecule inhibitor can prevent the emergence of metastatic foci.

Highlights

Osteosarcoma (OS) is the most commonly diagnosed paediatric primary bone malignancy [1]
The results of our multi-omics analysis are supported by early studies reporting that urokinase plasminogen activator (uPA) expression correlated with an invasive or metastatic phenotype in human OS [14,15,16,17,18,19]. These studies provided initial evidence for the potential importance of the uPA/ uPA receptor (uPAR) axis in OS growth and metastasis, they were limited to the use of a rat OS cell line and did not address the mechanism by which uPA/uPAR contributed to metastasis
Most importantly, we show that a novel pharmacological inhibitor of uPA/uPAR selectively prevents pulmonary metastasis in an orthotopic model of OS metastasis

Summary

Introduction

Osteosarcoma (OS) is the most commonly diagnosed paediatric primary bone malignancy [1]. The most frequent complication is the development of metastatic disease [2], with up to 80% of patients having clinically undetectable metastasis at the time of diagnosis [3]. Nhmrc.gov.au), and by the Cancer Council Queensland The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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