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 p27Kip1 plays a key role in growth and development of the mammary epithelium and in breast cancer. p27Kip1 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 p27Kip1 in down-regulation of p27Kip1 induced in MCF-7 cells by estrogens. 17beta-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G1 blockade mediated by p16Ink4a or the CDK inhibitor roscovitine, whereas down-regulation of p27Kip1 was maintained. Exogenous Skp2 prevented growth arrest of MCF-7 cells by antiestrogen, coinciding with decreased p27Kip1 expression. Under conditions of G1 blockade, p27Kip1 was stabilized by inhibition of CRM1-dependent nuclear export with leptomycin B or by mutation of p27Kip1 (Ser10 --> Ala; S10A) interfering with CRM1/p27Kip1 interaction. Antisense Skp2 oligonucleotides and a dominant-interfering Cul-1(1-452) mutant prevented down-regulation of p27Kip1S10A, whereas Skp2 overexpression elicited its destruction in mitogen-deprived cells. Active mediators of the extracellular signal-regulated kinase (ERK) pathway including Raf-1caax induced cytoplasmic localization of p27Kip1 in antiestrogen-treated cells and prevented accumulation of p27Kip1 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 p27Kip1 in response to estrogen. Our studies indicate that estrogens elicit down-regulation of p27Kip1 in MCF-7 cells through Skp2-dependent and -independent mechanisms that depend upon subcellular localization of p27Kip1 and require the participation of mediators of the Ras/Raf-1/ERK signaling pathway.
Foster
James S
JS
Department of Obstetrics and Gynecology, Graduate School of Medicine, Program in Comparative and Experimental Medicine, University of Tennessee Medical Center, Knoxville, Tennessee 37920, USA.
Fernando
Romaine I
RI
Ishida
Noriko
N
Nakayama
Keiichi I
KI
Wimalasena
Jay
J
D021581:Active Transport, Cell Nucleus; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000096:biosynthesis*; D002455:Cell Division; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003593:Cytoplasm / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation; D004967:Estrogens / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005434:Flow Cytometry; D016193:G1 Phase; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006801:Humans; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis*
D018797:Cell Cycle Proteins; D004967:Estrogens; D005231:Fatty Acids, Unsaturated; D016376:Oligonucleotides, Antisense; D044786:S-Phase Kinase-Associated Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D020928:Mitogen-Activated Protein Kinases; C038753:leptomycin B