Inhibition of mTOR Radiosensitizes Soft Tissue Sarcoma and Tumor Vascualture
Reviewer: Eric Shinohara MD, MSCI
Abramson Cancer Center of the University of Pennsylvania
Last Modified: November 2, 2007
Presenter: James Murphy, MD Presenter's Affiliation: University of Michigan Type of Session: Scientific
Rapamycin is an FDA approved immunosuppressant used in organ transplant, especially renal. It was derived from a soil sample from the island of Rapa Nui.
Rapamycin was originally used as an antifungal but rejected due to immunosupressive and antiproliferative effects
Rapamycin is a known inhibitor of mTOR (Molecular Target of Rapamycin) and has been shown to have antiproliferative activity. Soft tissue sarcomas tend to be radioresistant and it is thought that this may be due to dysregualtion of the PI3K/Akt pathway, possibly due to loss of PTEN tumor suppressor activity. mTOR is a downstream target of Akt and inhibition of mTOR may be able to sensitize sarcoma cells to radiation.
Prior studies have demonstrated that mTOR inhibitors have antitumoral effects on sarcomas and can act as a radiosensitizer in some other cancers. Studies have also demonstrated that in some cells lines mTOR has no direct antitumoral effects but in both in vivo and in vitro models has antivascular effects.
The present preclinical study examines whether rapamycin can be used as a radiosensitizer in soft tissue sarcomas.
Materials and Methods
In vitro studies were performed in three sarcoma cell lines, SK-LMS-1, SW-872, and HT-1080, using clonogenic assays to determine rapamycin’s direct sensitizing effects on tumor. Cells were treated with a minimally cytotoxic dose of rapamycin (3-30 nM).
Inhibition of mTOR was assayed for using western blot analysis on both in vivo and in vitro specimens. Levels of phosphorylated mTOR were studied in response to radiation, rapamcyin or both. Phosphorylation levels of the downstream target of mTOR, p70 S6 kinase was also assayed for after radiation, rapamycin treatments, or both.
The effects of rapamycin on vasculature were studied in vitro using human dermal microvascular endothelial cells. Both colony forming assays and microvascular sprouting assays were used.
In vivo analysis of mTOR’s antitumoral effects was performed in nude mice (athymic). Mice were implanted with SK-LMS-1 soft tissue sarcoma cells and tumors were allowed to grow. Mice were divided into four groups, a control group, radiation alone, rapamycin alone, and radiation and rapamycin. Mice were treated with rapamycin, 2 mg/kg intraperitoneally. Radiation was given in 2 Gy fractions daily to a total of 30 Gy over the course of three weeks.
Enhancement ratios (ER) were used to calculate radiosensitization of tumor and vascular cells. An ER of greater than 1 indicated radiosensitization.
Results from the clonogenic assays for all three sarcoma cell lines demonstrated radiosensitization with rapamycin in vitro. The ER was 1.2-1.3 for these cell lines.
Western blot analysis demonstrated that radiation induced a transient increase in the phosphorylation of both mTOR and p70 S6K implying activation of the Akt pathway in response to radiation. This increased activity was inhibited with treatment with rapamycin, with decreased phosphorylation of both mTOR and p70 S6K.
Results from In vivo studies in nude mice demonstrated that rapamycin was minimally toxic with mice losing less than 5% of their weight. The tumor xenografts exhibited increased radiosensitization compared with the clonogenic results, with an ER of 4.6 in the combined radiation and rapamcyin group.
Clonogenic assays examining rapamycin’s radiosensitizing effects on the vaculature demonstrated no effects. However, the vascular sprout assay demonstrated reduced sprout formation in the combined treatment arm. Radiation alone decrease sprout formation by 21 ± 6%. Rapamycin alone reduced sprout formation by 34 ± 12%. The combination of both reduced sprout formation by 73 ± 5% (p<0.05).
This study demonstrated that radiation increased phosphorylation of mTOR and that this effect could be blocked with minimally toxic doses of rapamycin. They found that rapamycin could radiosensitize sarcoma cells in vitro and in vivo. In vitro studies demonstrated that there was also a vascular radiosensitizing effect with rapamycin treatment.
Clinical trials with combined radiation and rapamycin in soft tissue sarcomas are warranted.
It is interesting that rapamycin induced radiosensitization in the sprout assay but not in the clonogenic assay of vascular cells. Prior studies have found that vascular endothelial cells are radiosensitized in clonogenic assays (Shinohara ET et al. Oncogene, 2005).
It would be interesting to see what factors are contributing to this discrepancy. There are several studies which suggest that mTOR inhibition destroys tumor vaculature in vivo. Furthermore, additional studies have demonstrated that there is selective tumor vascular thrombosis with mTOR treatment (Guba, M. et al. Blood 2005). It is thought that VEGF levels may be directly related to these effects. Perhaps this discrepancy is due to the growth factors in the media used in the two assays. Though no specific vascular toxicity has been seen thus far, due to rapamycin’s antivascular effects this may be a toxicity to be cautious about.
Prior studies have shown similarly encouraging in vivo results in other cancers and there are currently several clinical trials underway. This includes a Phase I study of CCI-779 (an mTOR inhibitor) combined with temozolamide and radiation in glioblastoma multiforme. There is a Phase I/II study of preoperative rapamycin and radiation in rectal cancer. A Phase I study of concurrent rapamycin, cisplatin and radiation demonstrated that this treatment could be well tolerated.
Rapamycin as a monoagent in the treatment of advanced soft tissue sarcomas suggests that it is safe and effective (Chawla SP. et al. ASCO Annual Meeting 2007).
These results combined with the results from this study suggest that a Phase I study is warranted. Soft tissue sarcomas can be heterogeneous and the antivascular effects of rapamycin may allow greater effect even with a diverse population of cancer cells.
Partially funded by an unrestricted educational grant from Bristol-Myers Squibb.