Radiation-Triggered NF-κB Activation is Responsible for the Angiogenic Signaling Pathway and Neovascularization for Breast Cancer Cell Proliferation and Growth

Abstract

Tumors require blood supply to survive, grow, and metastasize. This involves the process of angiogenesis signaling for new blood vessel growth into a growing tumor mass. Understanding the mechanism of the angiogenic signaling pathway and neovascularization for breast cancer cell proliferation and growth would help to develop molecular interventions and achieve disease free survival. Our hypothesis is that the surviving cancer cell(s) after radiotherapy can initiate angiogenic signaling pathway in the neighboring endothelial cells resulting in neovascularization for breast cancer cell growth. The angiogenic signaling pathway is initiated by angiogenic factors, VEGF and FGF-2, through activation of a transcriptional regulator NF-κB, which in turn is triggered by therapeutic doses of radiation exposure Human breast adenocarcinoma cells (MCF-7 cells) were exposed to Cesium-137 (137Cs) γ rays to a total dose of 2 Gy at a dose rate of 1.03 Gy/min. The results of mobility shift assay showed that radiation at clinical doses (2 Gy) could induce NF-κB DNA-binding activity. Then, we examined the communication of angiogenic signals from irradiated MCF-7 cells to vascular endothelial cells. At the protein level, the western blot showed induction of angiogenic factors VEGF and FGF-2 in MCF-7 cells irradiated with 2 Gy. Inhibition of NF-κB activation attenuated VEGF and FGF-2 levels. These factors are secreted into the medium. The levels of VEGF and FGF-2 in the extra cellular medium were both increased, after 2 Gy exposures. We also observed corresponding expression of VEGFR2 and FGFR1 in non-irradiated endothelial cells that were co-cultured with irradiated MCF-7 cells. In support of this, in vitro tube formation assays provided evidence that irradiated MCF-7 cells transmit signals to potentiate cultured non-irradiated endothelial cells to form tube networks, which is the hallmark of neovascularization. Inhibition of NF-κB activation attenuated irradiated MCF-7-induced tube network formation. The data provide evidence that the radiation exposure is responsible for tumor growth and maintenance by inducing an angiogenic signaling pathway through activation of NF-κB.