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Small Interfering RNAs Induce Macrophage Migration Inhibitory Factor Production and Proliferation in Breast Cancer Cells via a Double-Stranded RNA-Dependent Protein Kinase-Dependent Mechanism¹

School of Medicine and Medical Science, College of Life Sciences, University College Dublin Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland

Small interfering RNAs (siRNAs) represent a novel tool to induce gene silencing in mammalian cells and clinical trials are currently ongoing to assess the therapeutic efficacy of siRNAs in various human diseases, including age-related macular degeneration and respiratory syncytial virus infection. However, previously reported off-target, nonspecific effects of siRNAs, including activation of type I IFNs and proinflammatory cytokines, remain an outstanding concern regarding use of these agents in vivo. Macrophage-migration inhibitory factor (MIF) is a pleiotropic cytokine with well-described roles in cell proliferation, tumorigenesis, and angiogenesis and represents a target gene for siRNA-based therapy in the treatment of breast cancer. However, in this study we describe an increase in MIF production from mammary adenocarcinoma (MCF-7) cells following transfection with MIF siRNA and various control siRNAs. This effect was shown to be dose-dependent and was attenuated in the presence of a double-stranded RNA-dependent protein kinase inhibitor, 2-aminopurine. Furthermore, treatment of MCF-7 cells with poly(I:C) also stimulated a PKR-dependent increase in MIF production from MCF-7 cells. The biological consequence of the siRNA-induced increase in MIF production from MCF-7 cells was a PKR-dependent increase in proliferation of breast cancer cells. Furthermore, in cDNAs prepared from a primary human breast cancer cohort, we demonstrated a significant correlation (Spearman rank correlation coefficient, r = 0.50, p < 0.0001, n = 63) between PKR- and MIF-mRNA expression. In conclusion, this study highlights the potential biological consequences of off-target, nonspecific effects of siRNAs and underlines the safety concerns regarding the use of siRNAs in the treatment of human diseases, such as cancer.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Science Foundation Ireland.

² Address correspondence and reprint requests to Dr. Seamas Donnelly, School of Medicine and Medical Science, College of Life Sciences, University College Dublin Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. E-mail address: seamas.donnelly{at}ucd.ie

³ Abbreviations used in this paper: siRNA, small interfering RNA; RIG, retinoic acid-inducible gene; MDA, melanoma differentiation-associated gene; pDC, plasmacytoid dendritic cell; PACT, PKR-activating protein; MIF, migration inhibitory factor; siGFP, fluorescein control siRNA; siLamin, human Lamin A/C siRNA; siControl, nontargeting siRNA control; QPCR, quantitative real-time PCR; MCF-7, mammary adenocarinoma; 2-AP, 2-aminopurine; RISC, RNA-induced silencing complex; PKR, double-stranded RNA-dependent protein kinase; ISRE, IFN-stimulated response element.