Estrogens Down-regulate p27 Kip1 in Breast Cancer Cells through Skp2 and through Nuclear Export Mediated by the ERK Pathway

James S Foster,Romaine I Fernando,Noriko Ishida,Keiichi I Nakayama,Jay Wimalasena

doi:10.1074/jbc.m302830200

James S Foster, Romaine I Fernando + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m302830200

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Abstract

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.

Highlights

Estrogenic steroids are essential for normal development and function of female reproductive tissues yet play a pivotal, causative role in the initiation and progression of breast cancer [1, 2]
We investigated the respective roles of Skp2 and subcellular localization of p27Kip1 in down-regulation of p27Kip1 induced in MCF-7 cells by estrogens. 17␤-Estradiol treatment increased Skp2 expression in MCF-7 cells; this increase was prevented by G1 blockade mediated by p16Ink4a or the cyclin-dependent kinase (CDK) inhibitor roscovitine, whereas down-regulation of p27Kip1 was maintained
Skp2 Expression in Response to E2 Is Prevented by G1 Blockade, but p27Kip1 Down-regulation Is Maintained—Estrogen stimulation of G0/G1-arrested MCF-7 cells leads to down-regulation of p27Kip1 protein expression corresponding with cyclin E-CDK2 activation and preceding entry into S phase [15, 16, 28]

Summary

Introduction

Estrogenic steroids are essential for normal development and function of female reproductive tissues yet play a pivotal, causative role in the initiation and progression of breast cancer [1, 2]. The results suggest that Skp2 participates in ubiquitination/degradation of p27Kip1 in the nucleus of estrogen-treated MCF-7 cells and further demonstrate that Skp2 overexpression prevents antiestrogen-mediated growth arrest of MCF-7 cells by downregulating p27Kip1 expression and maintaining CDK2 activity.

Results

Conclusion