Abstract
Osteosarcoma (OS) patients with metastasis or recurrent tumors still suffer from poor prognosis. Studies have indicated the efficacy of DNA demethylation therapy for OS, but the underlying mechanism is still unclear. Here, we aimed to clarify the mechanism of how epigenetic therapy has therapeutic efficacy in OS. Treatment of four OS cell lines with a DNA demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC) treatment, markedly suppressed their growth, and in vivo efficacy was further confirmed using two OS xenografts. Genome-wide DNA methylation analysis showed that 10 of 28 primary OS had large numbers of methylated CpG islands while the remaining 18 OS did not, clustering together with normal tissue samples and Ewing sarcoma samples. Among the genes aberrantly methylated in primary OS, genes involved in skeletal system morphogenesis were present. Searching for methylation-silenced genes by expression microarray screening of two OS cell lines after 5-aza-dC treatment revealed that multiple tumor-suppressor and osteo/chondrogenesis-related genes were re-activated by 5-aza-dC treatment of OS cells. Simultaneous activation of multiple genes related to osteogenesis and cell proliferation, namely epigenetic reprogramming, was considered to underlie the efficacy of DNA demethylation therapy in OS.
Highlights
Osteosarcoma (OS) patients with metastasis or recurrent tumors still suffer from poor prognosis
While treatment of OS without metastases was advanced by introduction of multimodal therapies, including surgery combined with chemotherapy[1,14,15], treatment of OS with metastasis or recurrent tumors has not changed over the past 30 years, with a poor survival rate of less than 30%1,14,16–18
Necrosis in tumor tissues was increased by the 5-aza-dC treatment (Fig. 1E,F, Supplementary Fig. 1B,C), and the numbers of lung metastases tended to decrease to 97.4 and 57.1% of mock-treated mice by administration of 1 and 2 mg/kg, respectively, of 5-aza-dC, but without statistical significance (Supplementary Fig. 1D–F). These results showed that DNA demethylation therapy is effective for treatment of OS, in line with previous reports[7,9,10,32]
Summary
Osteosarcoma (OS) patients with metastasis or recurrent tumors still suffer from poor prognosis. Searching for methylation-silenced genes by expression microarray screening of two OS cell lines after 5-aza-dC treatment revealed that multiple tumor-suppressor and osteo/chondrogenesis-related genes were re-activated by 5-aza-dC treatment of OS cells. Simultaneous activation of multiple genes related to osteogenesis and cell proliferation, namely epigenetic reprogramming, was considered to underlie the efficacy of DNA demethylation therapy in OS. As a mechanism of action of DNA demethylating drugs, reactivation of specific tumor-suppressor genes used to be hypothesized, and this was proposed for OS5,6,11,12,19–21. Recent studies in other types of cancers highlighted the importance of simultaneous re-activation of multiple tumor-suppressor genes, namely epigenetic reprogramming[22,23,24], and emphasized the importance of low dose and prolonged exposure[25,26,27]. We aimed i) to confirm the efficacy of DNA demethylation therapy in OS using a low dose and prolonged exposure, and ii) to reveal the mechanism of how DNA demethylation therapy exerts its efficacy in OS
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