Understanding how the tumor microenvironment participates in inhibiting or supporting tumor growth is critical for the development of novel therapies. Osteosarcoma (OS) metastasizes almost exclusively to the lung, an organ where Fas ligand (FasL) is constitutively expressed. This chapter focuses on our studies dedicated to the interaction of OS cells with the lung microenvironment. We will summarize our studies conducted over the past 20 years showing the importance of the Fas/FasL signaling pathway to the establishment and progression of OS metastases in the lung. We demonstrated that the FasL+ lung microenvironment eliminates Fas-positive (Fas+) OS cells that metastasize to the lungs, through apoptosis induced by Fas signaling following interaction of Fas on the tumor cell surface with FasL on the lung epithelial cells. Expression of the Fas receptor on OS cells inversely correlated with the ability of OS cells to form lung metastases. Blocking this pathway interferes with this process, allowing Fas+ cells to grow in the lung. By contrast, upregulation of Fas on Fas- OS cells inhibited their ability to metastasize to the lung. We demonstrated how the FasL+ lung microenvironment can be leveraged for therapeutic intent through the upregulation of Fas expression. To this end, we demonstrated that the histone deacetylase inhibitor entinostat upregulated Fas expression on OS cells, reduced their ability to form lung metastases, and induced regression of established micrometastases. Fas expression in OS cells is regulated epigenetically by the microRNA miR-20a. We showed that expressions of Fas and miR-20a are inversely correlated, and that delivery of anti-miR-20a in vivo to mice with established osteosarcoma lung metastases resulted in upregulation of Fas and tumor regression. Therefore, targeting the Fas signaling pathway may present therapeutic opportunities, which target the lung microenvironment for elimination of OS lung metastases. We have also shown that in addition to being critically involved in the metastatic potential, the Fas signaling pathway may also contribute to the efficacy of chemotherapy. We demonstrated that the chemotherapeutic agent gemcitabine (GCB) increased Fas expression in both human and mouse OS cells in vitro. In vivo, aerosol GCB therapy induced upregulation of Fas expression and the regression of established osteosarcoma lung metastases. The therapeutic efficacy of GCB was contingent upon a FasL+ lung microenvironment as aerosol GCB had no effect in FasL-deficient mice. Manipulation of Fas expression and the Fas pathway should be considered, as this concept may provide additional novel therapeutic approaches for treating patients with OS lung metastases.
Keywords: C-FLIP; Entinostat; FADD (Fas-associated death domain); FDN (Fas-associated death domain e negative); Fas; FasL; Gemcitabine (GCB); Histone deacetylase inhibitors; Methylation; Osteosarcoma (OS); Pulmonary metastasis; miR-20a.