Abstract
The neu (c-erbB-2) proto-oncogene encodes a tyrosine kinase receptor that is overexpressed in 20 to 30% of human breast tumors. Herein, cyclin D1 protein levels were increased in mammary tumors induced by overexpression of wild-type Neu or activating mutants of Neu in transgenic mice and in MCF7 cells overexpressing transforming Neu. Analyses of 12 Neu mutants in MCF7 cells indicated important roles for specific C-terminal autophosphorylation sites and the extracellular domain in cyclin D1 promoter activation. Induction of cyclin D1 by NeuT involved Ras, Rac, Rho, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, but not phosphatidylinositol 3-kinase. NeuT induction of the cyclin D1 promoter required the E2F and Sp1 DNA binding sites and was inhibited by dominant negative E2F-1 or DP-1. Neu-induced transformation was inhibited by a cyclin D1 antisense or dominant negative E2F-1 construct in Rat-1 cells. Growth of NeuT-transformed mammary adenocarcinoma cells in nude mice was blocked by the cyclin D1 antisense construct. These results demonstrate that E2F-1 mediates a Neu-signaling cascade tocyclin D1 and identify cyclin D1 as a critical downstream target of neu-induced transformation.
ACKNOWLEDGMENTS
We are grateful to S. Cook, R. Davis, N. Dyson, G. Gill, M. Gilman, E. Harlow, W. Kaelin, J. Massague, F. McCormick, J. Nevins, S. Reeves, P. Farnham, J. Wells, and L. Zhu for plasmids, antibodies, and helpful discussions. We thank R. Russell for pathological assessment of tumors.
This work was supported in part by grants R29CA70897, RO1CA75503, and 5-P30-CA13330-26 (R.G.P.), CA536340 (J.M.H.), NIH training grant T32 DK 07513 (R.J.L.), and grant CA09475-12 (M.D.). W.J.M. is supported by research grants awarded by the Canadian Breast Cancer Initiative and is a recipient of an MRC of Canada Scientist award. R.G.P. is a recipient of the Irma T. Hirschl Award and an award from the Susan G. Komen Breast Cancer Foundation. Work conducted at the Albert Einstein College of Medicine was supported by Cancer Center Core National Institutes of Health grant 5-P30-CA13330-26 and the Mortimer Harrison Foundation (R.G.P.).