The histone deacetylase inhibitor, MS-275 (entinostat), downregulates c-FLIP, sensitizes osteosarcoma cells to FasL, and induces the regression of osteosarcoma lung metastases.

The purpose of this study was to determine the effects of the histone deacetylase inhibitor, MS-275, on the Fas signaling pathway and susceptibility of osteosarcoma (OS) to Fas ligand (FasL)-induced cell death. OS metastasizes almost exclusively to the lungs. We have shown that Fas expression in OS cells is inversely correlated with their metastatic potential. Fas(+) cells are rapidly eliminated when they enter the lungs via interaction with FasL, which is constitutively expressed in the lungs. Fas(-) OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Moreover, upregulation of Fas in established OS lung metastases results in tumor regression. Therefore, agents that upregulate Fas expression or activate the Fas signaling pathway may have therapeutic potential. Treatment of Fas(-) metastatic OS cell lines with 2 µM MS-275 sensitized cells to FasL-induced cell death in vitro. We found that MS-275 did not alter the expression of Fas on the cell surface; rather it resulted in the downregulation of the anti-apoptotic protein, c-FLIP (cellular FLICE-inhibitory protein), by inhibiting c-FLIP mRNA. Downregulation of c- FLIP correlated with caspase activation and apoptosis induction. Treatment of nu/nu-mice with established OS lung metastases with oral MS-275 resulted in tumor regression, increased apoptosis and a significant inhibition of c-FLIP expression in tumors. Histopathological examination of mice showed no evidence of significant toxicity. Overall, these results suggest that the mechanism by which MS-275 sensitizes OS cells and lung metastases to FasL-induced cell death may be by a direct reduction in the expression of c-FLIP.

Expression of c-FLIP in pulmonary metastases in osteosarcoma patients and human xenografts.

OBJECTIVE(S): We have previously shown that Fas expression inversely correlates with the metastatic potential of osteosarcoma (OS) to the lung. FasL is constitutively expressed in the lung microenvironment and eliminates Fas(+) OS cells leaving Fas(-) cells to form metastases. Absence of FasL in the lung epithelium or blocking the Fas-signaling pathway interfered with this clearance mechanism allowing Fas(+) cells to remain and form lung metastases. We also demonstrated that while the majority of patient OS lung metastases were Fas(-), 10-20% of the lesions contained Fas(+) cells, suggesting that these cells were not sensitive to FasL-induced apoptosis. The expression of c-FLIP, an inhibitor of the Fas pathway, has been associated with tumor development, progression, and resistance to chemotherapy. We therefore evaluated the expression of c-FLIP in OS patient tumor specimens and human xenograft lung metastases. METHODS: OS patient tissues, which included both primary and metastatic lesions, were evaluated for the expression of c-FLIP. In addition, tumors from human OS xenografts were examined for c-FLIP expression. RESULTS: c-FLIP expression was significantly higher in the lung metastases than in the primary tumors. CONCLUSION(S): c-FLIP may play an important role in the metastatic potential of OS to the lung. Inhibition of c-FLIP may be a future therapeutic target.

Effect of the histone deacetylase inhibitor SNDX-275 on Fas signaling in osteosarcoma cells and the feasibility of its topical application for the treatment of osteosarcoma lung metastases.

BACKGROUND: Patients with lung metastases from osteosarcoma (OS) have poor response to salvage therapy. Understanding the mechanisms involved in the metastatic process of OS may lead to new effective therapeutic approaches. The authors reported previously that up-regulation of the Fas receptor by transfecting OS cells with Fas plasmid inhibited the in vivo growth of metastases in the lungs. METHODS: In the current study, the authors treated OS cells with the histone deacetylase inhibitor SNDX-275 and studied its cytotoxicity and its effect on Fas signaling in vitro and in vivo. RESULTS: Subtoxic doses of SNDX-275 were able to activate the Fas pathway in OS cells by increasing the expression of Fas messenger RNA; however, the increased expression was not always followed by increased levels of Fas receptor expression on the cell surface. The treatment of cells with a combination of SNDX-275 and Fas ligand (FasL) had a stronger cytotoxic effect on tested OS cells than either agent alone. Inhibition of the Fas pathway in cells by inhibition of the Fas-associated death domain (FADD) molecule eliminated this combination effect, indicating that activity of FADD is important for the efficacy of this agent in the FasL-expressing environment of the lungs. Intranasal administration of SNDX-275 in mice with OS lung metastases revealed that SNDX-275 may inhibit metastatic growth at a dose of 0.13 mg/kg, which is approximately 200-fold lower than the therapeutically effective oral dose reported previously. CONCLUSIONS: The current findings indicated that SNDX-275 can activate Fas signaling in OS cells in vitro and in vivo and that the administration of SDNX-275 by inhalation is feasible as a treatment for OS metastases and warrants its further investigation.

Epigenetic regulation of apoptosis and cell cycle in osteosarcoma.

The role of genetic mutations in the development of osteosarcoma, such as alterations in p53 and Rb, is well understood. However, the significance of epigenetic mechanisms in the progression of osteosarcoma remains unclear and is increasingly being investigated. Recent evidence suggests that epigenetic alterations such as methylation and histone modifications of genes involved in cell cycle regulation and apoptosis may contribute to the pathogenesis of this tumor. Importantly, understanding the molecular mechanisms of regulation of these pathways may give insight into novel therapeutic strategies for patients with osteosarcoma. This paper serves to summarize the described epigenetic mechanisms in the tumorigenesis of osteosarcoma, specifically those pertaining to apoptosis and cell cycle regulation.