Abstract 3948: An inhibitor of uPA reduces osteosarcoma metastasis by blocking signaling in tumour cells and the bone marrow microenvironment .
Authors: Endo-Munoz Liliana B. , Cai Na , Cumming Andrew , Loo Dorothy , Hill Michelle , Mukhopadhyay Pamela , Saunders Nicholas A. 
Published: August 2014

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Pulmonary metastasis is the major complication of OS and results in just 10-20% long-term survival. We have shown that metastatic potential is an inherent characteristic of OS, and that the bone microenvironment contributes to OS metastasis (Endo-Munoz et al. Cancer Res 70:7063-72, 2010). Specifically, loss of osteoclasts increases the metastatic potential of OS, and bone marrow cells (BMC) increase the migration of OS cells. However, the factors regulating inherent and BMC-mediated metastasis have not been elucidated. We have used a multi-omics approach to identify genes and proteins that may mediate OS metastasis. We found very high levels of mRNA and protein expression and secretion of the urokinase plasminogen activator (uPA) and its receptor (uPAR) exclusively in metastatic OS. Addition of uPA-rich OS conditioned medium to metastatic OS cells significantly (P &lt; 0.007) increased their basal in vitro migration to levels similar to those observed with recombinant human uPA. On the other hand, overall migration was significantly (P &lt; 0.005) inhibited by addition of a neutralizing mAb against uPAR or an uPA inhibitor. Silencing of uPAR also abrogated the response to uPA and decreased metastasis in an orthotopic model, indicating that uPA/uPAR signaling acts in a positive feedback loop in the regulation of inherent OS metastasis. To investigate whether BMC could also be signaling through uPA/uPAR, we performed a comparative transcriptomic analysis of metastatic and non-metastatic OS cell lines before and after treatment with BMC conditioned medium, and found that uPA was the most significantly (FC = 7.97, B = 18.82) upregulated gene after BMC treatment. Moreover, immunohistochemistry on femurs of mice bearing OS tumours showed high expression of uPA in the bone marrow as well as in the leading edges of the tumour. In addition, BMC conditioned medium containing high levels of secreted uPA also significantly increased metastatic OS cell migration. Our data indicate that increased uPA gene expression induced by BMC could contribute to enhanced secretion of uPA by OS cells, which in turn could augment signaling through uPAR to increase migration. In addition, BMC secrete uPA into the microenvironment to further increase migration of OS tumour cells in close proximity. Finally, we used a therapeutic inhibitor of uPA in an orthotopic mouse model of OS. The uPA inhibitor reduced metastasis significantly (P = 0.0004) in the treated group. This opens the possibility that uPA inhibitors, which are already in clinical trials for a number of cancers, may also be a useful therapeutic in the treatment for OS metastasis.

Citation Format: Liliana B. Endo-Munoz, Na Cai, Andrew Cumming, Dorothy Loo, Michelle Hill, Pamela Mukhopadhyay, Nicholas A. Saunders. An inhibitor of uPA reduces osteosarcoma metastasis by blocking signaling in tumour cells and the bone marrow microenvironment . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3948. doi:10.1158/1538-7445.AM2013-3948