The Fas/FasL Signaling Pathway: Its Role in the Metastatic Process and as a Target for Treating Osteosarcoma Lung Metastases.

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.

Inhaled granulocyte-macrophage colony stimulating factor for first pulmonary recurrence of osteosarcoma: effects on disease-free survival and immunomodulation. a report from the Children's Oncology Group.

PURPOSE: Osteosarcoma most commonly recurs in the lung. Based on preliminary data on the antitumor effects of granulocyte-macrophage colony stimulating factor (GM-CSF) in animal models, and promising phase I trials, we embarked on a feasibility study of inhaled GM-CSF in patients with first isolated pulmonary recurrence of osteosarcoma. EXPERIMENTAL DESIGN: Forty-three eligible patients received inhaled GM-CSF at doses from 250 to 1,750 microg twice daily on alternate weeks. Following two cycles, patients underwent thoracotomy to resect tumor and analyze pulmonary nodules for expression of Fas/Fas ligand (Fas/FasL), and the presence of dendritic cells by immunostaining for CD1a, clusterin, and S100. Following surgery, patients received 12 additional cycles of therapy on alternating weeks or until progression. Event-free survival and survival, and feasibility of therapy delivery were evaluated. RESULTS: Dose escalation to 1,750 microg twice daily was feasible with no dose-limiting toxicity. Mean scores for Fas/FasL in nodules from patients with bilateral recurrence who underwent unilateral thoracotomy pretreatment (using a scoring system of 0-3) were 1.3 and 0.88, respectively, compared with 0.78 and 0.62 in nodules resected following two cycles of therapy. Only 11 of 30 nodules postinhalation were positive for CD1a, 4 of 30 for S100, and 6 of 30 for clusterin. Event-free and overall survival at 3 years were 7.8% and 35.4%, respectively. CONCLUSIONS: Inhalation of GM-CSF at doses from 250 to 1,750 microg twice daily on alternate weeks was feasible with low toxicity. However, no detectable immunostimulatory effect in pulmonary metastases or improved outcome postrelapse was seen.

Aerosol gemcitabine: preclinical safety and in vivo antitumor activity in osteosarcoma-bearing dogs.

BACKGROUND: Osteosarcoma is the most common skeletal malignancy in the dog and in young humans. Although chemotherapy improves survival time, death continues to be attributed to metastases. Aerosol delivery can provide a strategy with which to improve the lung drug delivery while reducing systemic toxicity. The purpose of this study is to assess the safety of a regional aerosol approach to chemotherapy delivery in osteosarcoma-bearing dogs, and second, to evaluate the effect of gemcitabine on Fas expression in the pulmonary metastasis. METHODS: We examined the systemic and local effects of aerosol gemcitabine on lung and pulmonary metastasis in this relevant large-animal tumor model using serial laboratory and arterial blood gas analysis and histopathology and immunohistochemistry, respectively. RESULTS AND CONCLUSIONS: Six hundred seventy-two 1-h doses of aerosol gemcitabine were delivered. The treatment was well tolerated by these subjects with osteosarcoma (n = 20). Aerosol-treated subjects had metastatic foci that demonstrated extensive, predominately central, intratumoral necrosis. Fas expression was decreased in pulmonary metastases compared to the primary tumor (p = 0.008). After aerosol gemcitabine Fas expression in the metastatic foci was increased compared to lung metastases before treatment (p = 0.0075), and even was higher than the primary tumor (p = 0.025). Increased apoptosis (TUNEL) staining was also detected in aerosol gemcitabine treated metastasis compared to untreated controls (p = 0.028). The results from this pivotal translational study support the concept that aerosol gemcitabine may be useful against pulmonary metastases of osteosarcoma. Additional studies that evaluate the aerosol route of administration of gemcitabine in humans should be safe and are warranted.

Topotecan enhances immune clearance of gliomas.

Despite aggressive surgery, radiation therapy, and chemotherapy, glioblastoma multiforme (GBM) is refractory to therapy, recurs quickly, and results in a median survival time of only 14 months. The modulation of the apoptotic receptor Fas with cytotoxic agents could potentiate the response to therapy. However, Fas ligand (FasL) is not expressed in the brain and therefore this Fas-inducing cell death mechanism cannot be utilized. Vaccination of patients with gliomas has shown promising responses. In animal studies, brain tumors of vaccinated mice were infiltrated with activated T cells. Since activated immune cells express FasL, we hypothesized that combination of immunotherapy with chemotherapy can activate Fas signaling, which could be responsible for a synergistic or additive effect of the combination. When we treated the human glioma cell line U-87 and GBM tumor cells isolated from patients with TPT, Fas was up regulated. Subsequent administration of soluble Fas ligand (sFasL) to treated cells significantly increased their cell death indicating that these Fas receptors were functional. Similar effect was observed when CD3(+) T cells were used as a source of the FasL, indicating that the up regulated Fas expression on glioma cells increases their susceptibility to cytotoxic T cell killing. This additive effect was not observed when glioma cells were pre-treated with temozolomide, which was unable to increase Fas expression in tumor. Inhibition of FasL activity with the antagonistic antibody Nok-1 mitigated these effects confirming that these responses were specifically mediated by the Fas-FasL interaction. Furthermore, the CD3(+) T cells co-cultured with topotecan treated U-87 and autologous GBM tumor cells showed a significant increase in expression in IFN-gamma, a key cytokine produced by activated T cells, and accordingly enhanced tumor cytotoxicity. Based on our data we conclude that drugs, such as topotecan, which cause up regulation of Fas on glioma cells can be potentially exploited with immunotherapy to enhance immune clearance of tumors via Fas signaling.