In this work, we demonstrate the therapeutic role of mTOR inhibition in chondrosarcoma in localized and advanced phase. Everolimus was tested in an orthotopic rat grade II chondrosarcoma model in macroscopic and “adjuvant” phase both reaching the same conclusion. As a single agent, the mTOR inhibitor everolimus did not cause tumor regression but induced a significant inhibition of tumor growth. Both the size and tumor growth rate were smaller in the everolimus treated groups than in other groups, as observed in other tumor models. Doxorubicin was inactive as single agent; when combined with everolimus, an antagonistic effect was actually observed in the combination group compared to the everolimus treated group. When compared to doxorubicin alone, the combination treatment showed however an increased therapeutic efficiency. Although these data are strongly contrasting with those observed in breast cancer models with paclitaxel and prostate cancer with doxorubicin, a similar effect was recently reported. In human cervical carcinoma xenograft models the addition of everolimus to doxorubicin showed an antitumor effect that was not significantly different from doxorubicin monotherapy. The mechanisms underlying this lack of synergism between the two drugs are unclear. One of the side effects of doxorubicin treatment is the induction of reactive oxygen species which in turn can activate the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways. This activation of the mTOR/Akt pathway induced by doxorubicin is reflected by slight increase in Akt phosphorylation in the doxorubicin treated group of our study. In the case of combined treatment this doxorubicin-induced Akt phosphorylation may not be overcome by everolimus at the concentration used and may counteract the antitumor activity of everolimus, as suggested by the higher expression of phospho Akt of the combination group compared to the everolimus-treated one. In the chondrosarcoma model the activity of the mTOR pathway in response to the different treatments was monitored by following activation levels of 4EBP1, S6K as potential surrogate markers of tumor response. Measurement of the phosphorylation status of ph-p70S6K1 and ph-4EBP1 in the tumor itself, confirmed that everolimus resulted in a downregulation of mTOR downstream effectors, whereas doxorubicin had no effect on its phosphorylation status. Everolimus exposure alone did not result in the activation of Akt, a phenomenon already ICI 182780 129453-61-8 reported in other studies. It is known that mTOR inhibitor- can induce a feedback activation of Akt thus contributing to a lesser therapeutic efficiency. This was not observed here with everolimus alone. The data obtained in these experiments indicate that everolimus may affect cell proliferation and metabolism as shown by the down regulation of Ki67 and Glut1 immunostaining. Such an antiproliferative effect has already been reported. The significantly decreased GLUT1 expression observed in the everolimus treated groups appears to be the result of mTOR inhibition and is a consequence of the cross-talk of mTOR downstream effectors with metabolic and hypoxic pathways. Inhibition of mTOR signaling may have direct effect on cell proliferation and also an indirect inhibitor effect on glucose metabolism through the inhibition of HIF1a which expression is (+)-JQ1 1268524-70-4 dependent upon mTOR. The decrease in HIF1a expression seen by immunofluorescence and in the levels of HIF1 a transcript seen by RT-qPCR in tumors of the everolimus treated groups support this bifunctional action of everolimus. Importantly, the present study also investigated the effects of everolimus on residual disease after intralesional curettage in the rat model of chondrosarcoma.