Estrogens down-regulate p27Kip1 in breast cancer cells through Skp2 and through nuclear export mediated by the ERK pathway

The cyclin-dependent kinase (CDK) inhibitor p27(Kip1) plays a key role in growth and development of the mammary epithelium and in breast cancer. p27(Kip1) levels are regulated through ubiquitin/proteasome-mediated proteolysis, promoted by CDK2 and the F box protein Skp2 at the G(1)/S transition, and independent of Skp2 in mid-G(1). We investigated the respective roles of Skp2 and subcellular localization of p27(Kip1) in down-regulation of p27(Kip1) induced in MCF-7 cells by estrogens. 17beta-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G(1) blockade mediated by p16(Ink4a) or the CDK inhibitor roscovitine, whereas down-regulation of p27(Kip1) was maintained. Exogenous Skp2 prevented growth arrest of MCF-7 cells by antiestrogen, coinciding with decreased p27(Kip1) expression. Under conditions of G1 blockade, p27(Kip1) was stabilized by inhibition of CRM1-dependent nuclear export with leptomycin B or by mutation of p27(Kip1) (Ser(10) --> Ala; S10A) interfering with CRM1/ p27(Kip1) interaction. Antisense Skp2 oligonucleotides and a dominant-interfering Cul-1(1-452) mutant prevented down-regulation of p27(Kip1)S10A, whereas Skp2 overexpression elicited its destruction in mitogen- deprived cells. Active mediators of the extracellular signal-regulated kinase (ERK) pathway including Raf-1(caax) induced cytoplasmic localization of p27(Kip1) in antiestrogen-treated cells and prevented accumulation of p27(Kip1) in these cells independent of Skp2 expression and coinciding with ERK activation. Genetic or chemical blockade of the ERK pathway prevented down-regulation and cytoplasmic localization of p27(Kip1) in response to estrogen. Our studies indicate that estrogens elicit down-regulation of p27(Kip1) in MCF-7 cells through Skp2-dependent and -independent mechanisms that depend upon subcellular localization of p27(Kip1) and require the participation of mediators of the Ras/Raf-1/ERK signaling pathway.