Simple SummaryHigh-grade bone and soft tissue sarcomas are known for high recurrence and low survival rates. New drug development is costly and time-consuming. Instead, new combinations of existing drugs may offer novel anticancer strategies. We previously demonstrated that the combination of the mammalian target of rapamycin inhibitor everolimus and the proteasome inhibitor bortezomib is effective against osteosarcoma cells in a drug screening test. Building on previous research, we showed that the combination of everolimus and bortezomib exerted synergistic antiproliferative and apoptotic effects against fibrosarcoma and osteosarcoma, suppressing pulmonary metastases in osteosarcoma-bearing mice. Furthermore, we identified that everolimus and bortezomib inhibited tumor growth via the JNK/p38/ERK MAPK and AKT pathways. This combination treatment may be effective against bone and soft tissue sarcoma suppressing tumor growth and metastasis.AbstractThe combination of the mammalian target of rapamycin and proteasome inhibitors is a new treatment strategy for various tumors. Herein, we investigated the synergistic effect of everolimus and bortezomib on tumor growth and metastasis in bone and soft tissue sarcomas. The antitumor effects of everolimus and bortezomib were assessed in a human fibrosarcoma (FS) cell line (HT1080) and mouse osteosarcoma (OS) cell line (LM8) by MTS assays and Western blotting. The effects of everolimus and bortezomib on HT1080 and LM8 tumor growth in xenograft mouse models were evaluated using tumor volume and the number of metastatic nodes of the resected lungs. Immunohistochemistry was used to evaluate cleaved PARP expression. The combination therapy decreased FS and OS cell proliferation compared with either drug alone. This combination induced more intense p-p38, p-JNK, and p-ERK and activated apoptosis signals, such as caspase-3, compared with single-agent treatment. The combination treatment reduced p-AKT and MYC expression, decreased FS and OS tumor volumes, and suppressed lung metastases of OS. The combination therapy inhibited tumor growth in FS and OS and metastatic progression of OS via the JNK/p38/ERK MAPK and AKT pathways. These results could aid in the development of new therapeutic strategies for sarcomas.