Estrogens Down-regulate p27^Kip1 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 G₁/S transition, and independent of Skp2 in mid-G₁. 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. 17β-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G₁ 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 (Ser¹⁰ → 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^Kip1S10A, 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.