Abstract B52: Mechanisms of rapalogues' anti-vascular effects in head and neck cancer
Authors: Ekshyyan Oleksandr , Akbarian-Tefaghi Hesam , Green Adam , Rong Xiaohua , Alexander Jonathan Steven , Nathan Cherie-Ann O. 
Published: October 2015

Abstract
Background: Head and neck squamous cell carcinoma (HNSCC) is characterized by up-regulation of the mTOR pathway leading to uncontrolled cell growth. We demonstrated that mTOR inhibitors have potent antiangiogenic and anti-lymphangiogenic effects in addition to growth inhibitory effects on HNSCC. A previous study of the effects of rapamycin on HNSCC xenografts established with FaDu cells transfected with either a control vector (VT) or rapamycin resistant mTOR (RR-mTOR), showed that rapamycin reduced tumor growth in HNSCC xenograft tumors regardless of whether they contained the control or rapamycin resistant mTOR-expressing vector. This suggests that the anti-tumor activity of the drug is a result of its effects on stromal cells or interaction between stromal and tumor cells. The goal of this study is to elucidate the mechanisms underlying the anti-vascular effects of mTOR inhibitors and to identify potential candidate biomarkers for their use in clinical trials.
Methods: FaDu tumor cells (TCs) and endothelial cells (ECs) were transfected with either a control vector (VT) or RR-mTOR (RR). To evaluate the interaction between normal ECs and TCs and to test the effects of rapamycin we employed co-culture systems. Transfected ECs and FaDu cells were co-cultured in various combinations using transwell cell culture inserts and were treated with rapamycin. Cell proliferation was assessed using CellTiter 96 AQueous Cell Proliferation Assay (Promega) and transwell inserts with a 0.4-μm pore size. Levels of various growth factors and cytokines in cell culture media were measured using Milliplex bead immunoassay (EMD Millipore).
Results: There was no significant effect of TCs on proliferation of ECs. Co-culture of rapamycin-resistant TCs with ECs sensitive to rapamycin had no effect on the growth-inhibitory effect of rapamycin against ECs (e.g. there was no “protective” effect of rapamycin-resistant TCs against growth-inhibitory effect of rapamycin in ECs). However, surprisingly there was an obvious difference in the effect of rapamycin on the growth of rapamycin-sensitive TCs co-cultured with either vector-transfected ECs or with RR mTOR-transfected ECs. Co-culture of VT ECs with VT TCs had no effect on the sensitivity of TCs to rapamycin, while rapamycin-resistant ECs reduced growth-inhibitory effect of rapamycin against rapamycin-sensitive TCs. Preliminary results indicate that this effect may be associated with levels of soluble IL-6. When vector-transfected ECs were co-cultured with any of the FaDu tumor cells (VT, RR), rapamycin caused a significant decrease in the level of sIL-6. On the contrary, rapamycin treatment led to an upregulation in the level of sIL-6 when rapamycin-resistant ECs were co-cultured with VT or RR TCs.
Conclusions: Our preliminary results suggest that anti-vascular properties of rapamycin are primarily a result of drug's direct effects on endothelial cells, and not cross-talk mediated, effects. Soluble IL-6 can be a potential biomarker of response to mTOR inhibitor therapy.
Citation Format: Oleksandr Ekshyyan, Hesam Akbarian-Tefaghi, Adam Green, Xiaohua Rong, Jonathan Steven Alexander, Cherie-Ann O. Nathan. Mechanisms of rapalogues' anti-vascular effects in head and neck cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B52.