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Inhibition of mTOR Radiosensitizes Soft Tissue Sarcoma and Tumor Vasculature
I. J. Radiation Oncology d Biology d Physics
S96
171
Volume 69, Number 3, Supplement, 2007
Inhibition of mTOR Radiosensitizes Soft Tissue Sarcoma and Tumor Vasculature
J. D. Murphy, A. C. Spalding, Y. R. Somnay, M. E. Ray, D. A. Hamstra University of Michigan, Ann Arbor, MI Purpose/Objective(s): Clinically, soft tissue sarcomas (STS) display radioresistance; while the tumor suppressor PTEN, an inhibitor of the PI3K/Akt pathway, is frequently down-regulated in STS, leading to dysregulation of mammalian target of rapapmycin (mTOR) signaling, thus promoting cell survival. mTOR inhibitors such as rapamycin (Rap) have recently shown clinical benefit as monotherapy in STS. Additionally, inhibition of mTOR has also been associated with radio-sensitization of carcinoma cells and endothelial cells. The purpose of this study was to test the hypothesis that Rap radiosensitizes STS and endothelial cells in vitro and in vivo through inhibition of mTOR. Materials/Methods: Colony forming assays were preformed to determine radiosensitizing properties of Rap on three human STS cell lines (SK-LMS-1, SW-872, and HT-1080). In vivo radiosensitizing activity of Rap was assessed with subcutaneous SK-LMS-1 nude mice xenografts. Tumor-bearing mice were treated with concurrent daily Rap (2 mg/kg IP), RT (2 Gy daily, 30 Gy total), or both for three weeks. Western blots were performed on both in vitro and in vivo samples to determine the effects of Rap and RT on mTOR and its downstream target p70 S6 kinase (p70s6k). Both colony formation assays and microvascular sprouting assays were performed using primary human dermal microvascular endothelial cells (HDMEC). Enhancement ratios (ERs) were calculated for colony forming and tumor growth delay experiments, with values greater than 1 indicating radiosensitization. Results: In vitro radiosensitization was demonstrated in all three STS cell lines (ERs 1.2–1.3) after 1 hr exposure to minimally cytotoxic doses of Rap (range: 3–30 nM, IC0–IC50). SK-LMS-1 xenografts displayed significant tumor growth delay with Rap and RT compared with either treatment alone (ER 4.6, see Figure). Toxicity was minimal in all treatment groups (\5% weight loss). RT resulted in transient phosphorylation of p70s6k, whereas Rap decreased phosphorylation of both mTOR and p70s6k in irradiated and unirradiated samples. RT reduced HDMEC microvessel sprout formation by 21 ± 6%, while Rap caused a 34 ± 12% inhibition and the combination of RT and Rap resulted in 73 ± 5% inhibition of sprout formation (p \ 0.05). Conclusions: Minimally cytotoxic concentrations of Rap inhibited the mTOR/p70s6k cascade in culture and in vivo while radiosensitizing STS, and produced synergistic effects with radiation on HDMEC microvessel formation. By targeting both tumor and endothelial compartments, Rap produced potent radiosensitization of STS xenografts (see Figure). Clinical trials combining Rap and RT in STS are warranted.
Author Disclosure: J.D. Murphy, None; A.C. Spalding, None; Y.R. Somnay, None; M.E. Ray, None; D.A. Hamstra, None.
172
Noninvasive Monitoring of Ligand-Dependent VEGF Receptor-2 Dimerization With Split Firefly Luciferase
1
P. Lee , C. Chan2, A. Hua1, R. Paulmurugan2, D. Chan1, S. Gambhir2, Q. Le1, A. Giaccia1 1
Department of Radiation Oncology, 2Department of Radiology, Stanford University, Stanford, CA
Purpose/Objective(s): Tumor angiogenesis is an essential step in tumor growth, progression, and metastasis. The angiogenic growth factor VEGF and its receptor VEGFR-2 play a central role in the regulation of tumor angiogenesis. Novel inhibitors such as bevacizumab (a monoclonal antibody to VEGF) and small molecule tyrosine kinase inhibitors have shown promise in the treatment of solid tumors in clinical trials. However, antibody-based therapies and small molecule inhibitors are hindered by poor tumor penetration and the lack of receptor tyrosine kinase specificity, respectively. The split Firefly luciferase (FL) complementation-based bioluminescence system allows for molecular imaging of protein-protein interaction in both cell culture and in living subjects. The goals of this work are 1) to develop a split luciferase system to monitor VEGFR-2 dimerization; 2) to validate the system in living cells and animals using known inhibitors of this interaction; 3) to discover novel VEGF inhibitors through highthroughput screening. Materials/Methods: 293T cells were engineered to stably express the N-terminal (NhFL) or C-terminal (ChFL) portion of FL fused to the C-terminus of a kinase-inactive VEGFR-2 mutant. Stable clones were tested with recombinant VEGF protein to induce VEGFR-2 dimerization and split-FL complementation. Complementation-based luciferase activity in intact cells and living mice bearing xenograft tumors was imaged using a Cooled Charge-Coupled Device (CCD) camera (Xenogen IVIS, Xenogen, Alameda, CA). Known ligand (VEGF) and a control (bFGF) as well as inhibitors (i.e. bevacizumab) and a control monoclonal antibody to EGF receptor (cetuximab) were used to validate the system. Pilot studies using a high-throughput format were performed to screen for novel inhibitors.
S96
171
Volume 69, Number 3, Supplement, 2007
Inhibition of mTOR Radiosensitizes Soft Tissue Sarcoma and Tumor Vasculature
J. D. Murphy, A. C. Spalding, Y. R. Somnay, M. E. Ray, D. A. Hamstra University of Michigan, Ann Arbor, MI Purpose/Objective(s): Clinically, soft tissue sarcomas (STS) display radioresistance; while the tumor suppressor PTEN, an inhibitor of the PI3K/Akt pathway, is frequently down-regulated in STS, leading to dysregulation of mammalian target of rapapmycin (mTOR) signaling, thus promoting cell survival. mTOR inhibitors such as rapamycin (Rap) have recently shown clinical benefit as monotherapy in STS. Additionally, inhibition of mTOR has also been associated with radio-sensitization of carcinoma cells and endothelial cells. The purpose of this study was to test the hypothesis that Rap radiosensitizes STS and endothelial cells in vitro and in vivo through inhibition of mTOR. Materials/Methods: Colony forming assays were preformed to determine radiosensitizing properties of Rap on three human STS cell lines (SK-LMS-1, SW-872, and HT-1080). In vivo radiosensitizing activity of Rap was assessed with subcutaneous SK-LMS-1 nude mice xenografts. Tumor-bearing mice were treated with concurrent daily Rap (2 mg/kg IP), RT (2 Gy daily, 30 Gy total), or both for three weeks. Western blots were performed on both in vitro and in vivo samples to determine the effects of Rap and RT on mTOR and its downstream target p70 S6 kinase (p70s6k). Both colony formation assays and microvascular sprouting assays were performed using primary human dermal microvascular endothelial cells (HDMEC). Enhancement ratios (ERs) were calculated for colony forming and tumor growth delay experiments, with values greater than 1 indicating radiosensitization. Results: In vitro radiosensitization was demonstrated in all three STS cell lines (ERs 1.2–1.3) after 1 hr exposure to minimally cytotoxic doses of Rap (range: 3–30 nM, IC0–IC50). SK-LMS-1 xenografts displayed significant tumor growth delay with Rap and RT compared with either treatment alone (ER 4.6, see Figure). Toxicity was minimal in all treatment groups (\5% weight loss). RT resulted in transient phosphorylation of p70s6k, whereas Rap decreased phosphorylation of both mTOR and p70s6k in irradiated and unirradiated samples. RT reduced HDMEC microvessel sprout formation by 21 ± 6%, while Rap caused a 34 ± 12% inhibition and the combination of RT and Rap resulted in 73 ± 5% inhibition of sprout formation (p \ 0.05). Conclusions: Minimally cytotoxic concentrations of Rap inhibited the mTOR/p70s6k cascade in culture and in vivo while radiosensitizing STS, and produced synergistic effects with radiation on HDMEC microvessel formation. By targeting both tumor and endothelial compartments, Rap produced potent radiosensitization of STS xenografts (see Figure). Clinical trials combining Rap and RT in STS are warranted.
Author Disclosure: J.D. Murphy, None; A.C. Spalding, None; Y.R. Somnay, None; M.E. Ray, None; D.A. Hamstra, None.
172
Noninvasive Monitoring of Ligand-Dependent VEGF Receptor-2 Dimerization With Split Firefly Luciferase
1
P. Lee , C. Chan2, A. Hua1, R. Paulmurugan2, D. Chan1, S. Gambhir2, Q. Le1, A. Giaccia1 1
Department of Radiation Oncology, 2Department of Radiology, Stanford University, Stanford, CA
Purpose/Objective(s): Tumor angiogenesis is an essential step in tumor growth, progression, and metastasis. The angiogenic growth factor VEGF and its receptor VEGFR-2 play a central role in the regulation of tumor angiogenesis. Novel inhibitors such as bevacizumab (a monoclonal antibody to VEGF) and small molecule tyrosine kinase inhibitors have shown promise in the treatment of solid tumors in clinical trials. However, antibody-based therapies and small molecule inhibitors are hindered by poor tumor penetration and the lack of receptor tyrosine kinase specificity, respectively. The split Firefly luciferase (FL) complementation-based bioluminescence system allows for molecular imaging of protein-protein interaction in both cell culture and in living subjects. The goals of this work are 1) to develop a split luciferase system to monitor VEGFR-2 dimerization; 2) to validate the system in living cells and animals using known inhibitors of this interaction; 3) to discover novel VEGF inhibitors through highthroughput screening. Materials/Methods: 293T cells were engineered to stably express the N-terminal (NhFL) or C-terminal (ChFL) portion of FL fused to the C-terminus of a kinase-inactive VEGFR-2 mutant. Stable clones were tested with recombinant VEGF protein to induce VEGFR-2 dimerization and split-FL complementation. Complementation-based luciferase activity in intact cells and living mice bearing xenograft tumors was imaged using a Cooled Charge-Coupled Device (CCD) camera (Xenogen IVIS, Xenogen, Alameda, CA). Known ligand (VEGF) and a control (bFGF) as well as inhibitors (i.e. bevacizumab) and a control monoclonal antibody to EGF receptor (cetuximab) were used to validate the system. Pilot studies using a high-throughput format were performed to screen for novel inhibitors.