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A2218 - Aberrant Resistance of IPF Fibroblasts to Ionizing Radiation Induced Apoptosis Via FoxM1 Alteration
Author Block: R. Nho1, J. Im2, J. Lawrence3; 1Medicine, Univ of Minnesota, Minneapolis, MN, United States, 2Medicine, University of Minnesota, Minneapolis, MN, United States, 3Univ of Minnesota, Minneapolis, MN, United States.
1. Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic and irreversible lung disease caused by aberrantly activated lung (myo)fibroblasts, yet the precise etiology has not been understood. Rates of pulmonary fibrosis are known to be increased in response to the high doses of radiation, suggesting that irradiation can potentially accelerate lung fibrosis due to the presence of persistent fibrotic fibroblasts. However, the pathological process by which irradiation affects an IPF fibroblast phenotype is not understood. We recently found that unlike non-IPF fibroblasts, IPF fibroblasts become resistant to γ-radiation induced cell death on collagen rich matrix. This finding indicates that IPF fibroblasts have acquired a pathological mechanism to repair DNA damages caused by ionizing radiation, thereby protecting them from radiation-induced apoptosis. Forkhead box M1 (FoxM1) is known to up-regulate DNA repair enzymes such as BRACA2 and Rad51, which subsequently protects cells from ionizing radiation-induced apoptosis. Therefore, we hypothesize that IPF fibroblasts utilize FoxM1-dependent Rad51 and BRACA2 axis to become desensitized to radiation-induced cell death via abnormally enhanced DNA damage repair process.
2. Methods: Fibroblast viability following radiation (9 Gy) in lung fibroblasts (n=8) from patients with IPF and non-IPF was measured using Cell Titer Blue reagent. TUNEL assay was conducted to measure apoptotic fibroblasts by DNA damages. Western analysis was performed to measure DNA damages using γ-H2AX and FoxM1 target antibodies.
3. Results: Unlike non-IPF fibroblasts, significant radiation resistance was present when IPF fibroblasts were cultured on collagen-rich matrix at 24, 48 and 72 h. γ-H2AX assay demonstrated that DNA damages after irradiation is reduced in IPF fibroblasts as a
function of time, and that this alteration is caused by the up-regulation of DNA repair enzyme, Rad51 and BRACA2 as a result of FoxM1 activation. TUNEL assay also confirmed the reduced apoptotic IPF fibroblasts compared to that of non-IPF fibroblasts after radiation. To support our findings, upon FoxM1 silencing, IPF cells were increasingly sensitive to radiation-induced cell death via Rad51 and BRACA2 suppression. In contrast, FoxM1 reconstitution clearly protected control fibroblasts from radiation-induced apoptosis via up-regulation of Rad51 and BRACA2.
4. Conclusion: Our results suggest that the alteration of FoxM1-dependent DNA repair systems may be a key instigator in the selection of pro-fibrotic fibroblasts, promoting lung fibrosis. Targeting radiation insensitive fibroblasts by FoxM1 inhibition may therefore abrogate radiation induced lung fibrosis (RILF) and facilitate approaches for more effective radiation protocols for the treatment or management of IPF.