Interferon regulatory factor-1 (IRF-1) exhibits tumor suppressor activities in breast cancer associated with caspase activation and induction of apoptosis.
We have directly assessed the ability of interferon regulatory factor-1 (IRF-1) to act as a tumor suppressor gene in human breast cancer cells and explored whether this suppressor function is mechanistically conferred by affecting cell cycle transition, apoptosis and/or caspase activation. We have used a dual approach, measuring whether overexpression of wild-type IRF-1 or a dominant negative IRF-1 (dnIRF-1) produce opposing effects on breast cancer cell proliferation in vitro or tumorigenicity in athymic nude mice. Mechanistic studies determined the effects of blocking endogenous IRF-1 expression on cell cycle transition by flow cytometry, on apoptosis by Annexin V staining, and on caspase activation by fluorescent substrate cleavage. IRF-1 mRNA (P < or = 0.001) and protein (P < or = 0.001) are highly expressed in non-tumorigenic, normal, mammary epithelial cells, with intermediate expression in tumorigenic, but non-metastatic, cells and very low expression in metastatic cell lines. In MCF-7 cells transfected with a wild-type IRF-1 (MCF-7/IRF-1), IRF-1 mRNA expression inversely correlates with the rate of cell proliferation (r = -0.91; P = 0.002). Conversely, expression of dnIRF-1 in both MCF-7 (MCF-7/dnIRF-1; p53 wild-type) and T47D cells (T47D/dnIRF-1; p53 mutant) increases cell proliferation (P < or = 0.001). In athymic nude mice, the incidence of MCF-7/IRF-1 xenografts is reduced (P = 0.045), whereas MCF-7/dnIRF-1 xenografts exhibit a significantly higher tumor incidence (P < or = 0.001). Effects of IRF-1/dnIRF-1 are mediated through changes in the rates of apoptosis and not through cell cycle regulation. MCF-7/dnIRF-1 cells exhibit a 50% decrease in basal apoptosis (P = 0.007) and a significant reduction in caspase 8 activity (P = 0.03); similar effects occur in T47D/dnIRF-1 cells, where the effects on apoptosis appear to be mediated through inhibition of caspases 3/7 (P < 0.001) and caspase 8 (P = 0.03). These data establish a functional role for IRF-1 in the growth suppression of breast cancer cells and strongly implicate IRF-1 as a tumor suppressor gene in breast cancer that acts, independent of p53, to control apoptosis.
Bouker
Kerrie B
KB
Lombardi Comprehensive Cancer Center and Department of Oncology, Georgetown University School of Medicine, 3970 Reservoir Road NW, Washington, DC 20057, USA.
Skaar
Todd C
TC
Riggins
Rebecca B
RB
Harburger
David S
DS
Fernandez
David R
DR
Zwart
Alan
A
Wang
Antai
A
Clarke
Robert
R
D017209:Apoptosis; D001940:Breast / Q000166:cytology / Q000502:physiology; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle; D004268:DNA-Binding Proteins / Q000235:genetics*; D004847:Epithelial Cells / Q000502:physiology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D050856:Interferon Regulatory Factor-1; D007700:Kinetics; D010750:Phosphoproteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D013489:Suppression, Genetic
D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D010750:Phosphoproteins; D012333:RNA, Messenger; D020169:Caspases