Influence of Tie2 on tumor vascular signaling, integrity, and response to therapy

Signaling through the Tie2 receptor in endothelial cells has been shown to play an important role in normal and pathological vascular development. Therapeutic studies using a decoy Tie2 receptor suggest blocking of ligand binding to Tie2 inhibits tumor angiogenesis. The mechanism by which Tie2 inhibition affects tumor growth remains unclear. We generated an inducible soluble Tie2 receptor (Tie2Ex) in K1735 murine melanoma tumor cells to study the effects of inhibiting Tie2 signaling on tumor vasculature. Tie2Ex induction decreased tumor endothelial cell (EC) AKT activation but did not affect ERK activation. Immunostaining for tumor EC fate revealed an increase in EC TUNEL staining but no change in the proliferative marker Ki-67 in ECs. Prolonged Tie2Ex induction resulted in delayed tumor growth when induction started at a smaller size but not at a larger size. Treated tumors had regions of tumor cell loss as revealed by histology, suggesting increased EC death may be responsible for the secondary effect of decreased tumor cell survival. The mechanism for decreased EC survival following Tie2 inhibition appears to be through the P13K-AKT pathway and not through the Raf-MEK-ERK pathway. Activated Raf-MEK-ERK pathway has also been shown to play a critical role in tumor angiogenesis. To determine whether inhibiting both the PI3K-AKT and Raf-MEK-ERK pathway in ECs affects tumor growth at a larger size, we treated Tie2Ex-induced K1735 tumors with sorafenib, previously shown to effectively inhibit tumor EC ERK activation and tumor angiogenesis. Dual treatment with Tie2Ex and sorafenib resulted in improved tumor control compared to single agent alone. Tumor ECs in dually treated tumors had decreased activated AKT and ERK signaling, along with decreased EC survival and proliferation. These studies suggest altering tumor EC fate by inhibiting EC signaling through multiple pathways can improve the control of tumor growth. We also examined the vascular effects of ionizing radiation (IR) in K1735 murine melanoma tumors. Single-fraction and fractionated radiation virtually arrested growth of these tumors for about a week, after which they resumed more rapid growth. Irradiated tumors following re-growth had decreased vascularity along with increased tumor cell hypoxia, indicating IR inhibited tumor neovascularization. However, EC proliferation as detected by Ki-67 was increased in irradiated tumors that had grown substantially, the fact that increased EC proliferative activity produced fewer vessels suggests that angiogenesis is defective or ineffective after radiation