Abstract 5023: Down-regulating interferon regulatory factor-1 by siRNA promotes endurance to fulvestrant in sensitive breast cancer cells partially via Ras/Raf/MEK/ERK pathway

Abstract

Abstract AIM: Interferon Regulatory Factor 1 (IRF1) is a tumor suppressor gene that has an antitumor role in some cancers. In this study, we showed that reducing IRF1 expression by siRNA promotes resistance to Fulvestrant in MCF7/LCC1 cells at least partly by acting though the Ras/Raf/MEK/ERK pathway. METHODS: After transfection with IRF1 siRNA by Amaxa Nuclofection, the MCF7/LCC1 cells were treated with 100 nM Fulvestrant for 2 days for monitoring gene expression, and for 3 and 6 days for apoptosis and cell proliferation assays, respectively. Five μM U0126 or 10 μM PD98059, the inhibitors of MEK, were used when necessary. Transactivation of IRF1, Bcl-2, Cyclin D1, and ERE were detected by luciferase assays after transfection with the appropriate reporter plasmid. Caspase 7, 8 and 9 activities were detected by cleavage of their respective substrates. RESULTS: After transfected with IRF1 siRNA, the mRNA and protein levels of IRF1 expression were greatly reduced in MCF7/LCC1 cells. Knocking-down IRF1 expression resulted in increased proliferation, increased S-phase ratio, and decreased apoptosis of MCF7/LCC1 cells compared to mock or control siRNA groups. mRNA and protein levels of Bcl-2, Bcl-w, Bcl-xL, Survivin and CyclinD1 were increased by IRF1 siRNA transfection. Luciferase assays showed that the Bcl-2 P2 (but not P1) promoter is inhibited by IRF1 siRNA transfection. At same time, the activities of Caspase 7 and 8 cannot be increased by Fulvestrant treatment after IRF1 siRNA transfection. Knocking down IRF1 expression partially restores Fulvestrant's inhibition of ERE-drive transcription. Total and phosphor-ER α expression were reduced by IRF1 siRNA transfection. Further, we found that the phosphor-ERK1/2 expression levels were increased by IRF1 siRNA transfection. Inhibition MEK with inhibitors could partially reverse the effect of IRF1 siRNA transfection on proliferation via inhibiting the effect of cell cycle other than apoptosis. Using the inhibitors has no effect on the expression of ER α and the transcriptional activity of ERE. CONCLUSIONS: Knocking down IRF1 expression promotes resistance to Fulvestrant in MCF/LCC1 breast cancer cells partially by increasing the expression of phosphor-ERK1/2. The reduced of ERα expression and increased transcriptional activity of ERE has also involved in the mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5023.