Molecular and Cellular Endocrinology 229 (2005) 161–173 Estrogen and insulin/IGF-1 cooperatively stimulate cell cycle progression in MCF-7 breast cancer cells through differential regulation of c-Myc and cyclin D1 Amanda Mawson a , Angela Lai b , Jason S. Carroll c , C. Marcelo Sergio a , Christopher J. Mitchell d , Boris Sarcevic a,∗ a Cancer Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010, Australia b Johnson and Johnson Research Laboratories, Eveleigh, NSW 1430, Australia c Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA d Diabetes and Obesity Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010, Australia Received 11 May 2004; received in revised form 14 July 2004; accepted 11 August 2004 Abstract Estrogen and insulin/insulin-like growth factor-I (IGF-I) are major mitogens for breast epithelial cells and when co-administered, syner- gistically induce G 1 –S phase cell cycle progression. We investigated this cooperativity by evaluating if the key cell cycle regulators, c-Myc and cyclin D1, represent points of convergence in the action of these mitogens in MCF-7 breast cancer cells. These studies demonstrated that estrogen significantly increased both c-Myc and cyclin D1 protein, while insulin predominantly increased cyclin D1 levels. This cumulative increase in c-Myc and cyclin D1 contributes to the cooperativity of these mitogens, since ectopic expression of c-Myc or cyclin D1 cooperates with either the estrogen or insulin signaling pathways to increase cell cycle progression. Inhibition of the MAPK or PI3-kinase pathways significantly reduced c-Myc and cyclin D1 protein levels and cell cycle progression. Ectopic expression of cyclin D1 partially overcame this inhibition, while ectopic expression of c-Myc partially overcame MAPK but not PI3-kinase inhibition. Therefore, estrogen and insulin/IGF-1 differentially regulate c-Myc and cyclin D1 to cooperatively stimulate breast cancer cell proliferation. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Estrogen; Insulin; IGF-1; c-Myc; Cyclin D1 1. Introduction Estrogens elicit mitogenic activity which is important for the development of female reproductive tissues, particularly the uterus and mammary gland (Couse et al., 1999) and play a central role in the development and progression of human breast cancer (Colditz, 1998). The mechanisms through which estrogens stimulate cell proliferation are believed to be due to activation of estrogen receptor (ER) transcriptional activity (Couse and Korach, 1999) and potentially through non-transcriptional mechanisms by activation of intracellular signaling pathways such as the MAPK pathway (Migliaccio ∗ Corresponding author. Tel.: +61 2 9295 8330; fax: +61 2 9295 8321. E-mail address: b.sarcevic@garvan.org.au (B. Sarcevic). et al., 1996; Castoria et al., 1999). In addition to estrogen, polypeptide growth factors such as IGF-1 also display mitogenic activity on target cells, including human breast epithelial cells (Surmacz et al., 1998). Binding of IGF-1 to IGF-1-IR leads to dimerization of the receptor, activation of its tyrosine kinase activity and phosphorylation of key sub- strate proteins such as insulin receptor substrate proteins-1 (IRS-1) (Myers et al., 1994) and -2 (IRS-2) (He et al., 1996) and Src homolgy collagen (SHC) (Giorgetti et al., 1994). The phosphorylated IRS and SHC substrate proteins in turn recruit different SH2-containing proteins to activate specific intracellular signaling pathways. Thus, phosphorylated IRS-1 activates phosphatidylinositol 3-kinase (PI3-kinase) by recruiting the p85 regulatory subunit of this complex (Jackson et al., 1998), while phosphorylated SHC activates 0303-7207/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2004.08.002