Down-regulation of p21 ^WAF1/CIP1 or p27 ^Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells

Abstract

Estrogens and antiestrogens influence the G1 phase of the cell cycle. In MCF-7 breast cancer cells, estrogen stimulated cell cycle progression through loss of the kinase inhibitor proteins (KIPs) p27 and p21 and through G1 cyclin-dependent kinase (cdk) activation. Treatment with antiestrogen drugs, Tamoxifen or ICI 182780, caused cell cycle arrest, with up-regulation of both p21 and p27 levels, an increase in their binding to cyclin E-cdk2, and kinase inhibition. The requirement for these KIPs in the arrests induced by estradiol depletion or by antiestrogens was investigated with antisense. Antisense inhibition of p21 or p27 expression in estradiol-depleted or antiestrogenarrested MCF-7 led to abrogation of cell cycle arrest, with loss of cyclin E-associated KIPs, activation of cyclin E-cdk2, and S phase entrance. These data demonstrate that depletion of either p21 or p27 can mimic estrogen-stimulated cell cycle activation and indicate that both of these KIPs are critical mediators of the therapeutic effects of antiestrogens in breast cancer. E stradiol is mitogenic in up to 50% of de novo breast cancers, causing recruitment of quiescent cells into G 1 and shortening the G 1 -to-S phase interval (1, 2). Although 70% of breast cancers express the estrogen receptor (ER), only two-thirds of these will respond to antiestrogens, of which, Tamoxifen is the most widely used (3, 4). Antiestrogens, such as Tamoxifen, its active metabolite, 4-hydroxytamoxifen (4-OH TAM), and the more potent steroidal antiestrogen ICI 182780 (Faslodex) lead to a G 0 ͞G 1 arrest in susceptible ER-positive breast cancer cells (5-8). Unfortunately, hormonally responsive breast cancers invariably develop resistance to antiestrogens despite the continued expression of wild-type ER in most cases (9-12). Estrogens induce conformational changes in the ER, which promote its nuclear localization, dimerization, and function as a ligand-activated transcription factor (13-15). In addition, ligand binding to the ER can rapidly and transiently activate signal transduction pathways, notably the mitogen-activated protein kinase in breast cancer and in other cell types (16,17). Because antiestrogen resistance usually develops in the presence of an intact ER, the mechanisms by which ER modulates the cell cycle may be altered during breast cancer progression. The evolution of prostate cancer to hormone independence also occurs without loss of the androgen receptor (18,19) and may reflect a common mechanism of cell cycle misregulation.Progression through the cell cycle is governed by a family of cyclin-dependent kinases (cdks), whose activity is regulated by phosphorylation (20), activated by cyclin binding (21,22), and inhibited by the cdk inhibitors of the inhibitor of cdk4 (INK4) family (p16 INK4A , p15 INK4B , p18, and p19) and kinase inhibitor protein (KIP) family (p21 WAF-1͞CIP-1 , p27 Kip1 , and p57 KIP2 ;. Passage through G 1 into S phase is regulated by the activities of cyclin D-, cyclin E-, and cyclin A-associated kinases. Although p27 protein is st...

CRM1/Ran-Mediated Nuclear Export of p27Kip1Involves a Nuclear Export Signal and Links p27 Export and Proteolysis

“…The onset of this transient binding of p27 to CRM1 occurs before the increase in Skp2 protein levels observed 12 h after the addition of estradiol ( Figure 1B). In addition, the onset of p27-CRM1 binding occurs before activation of cyclin E-Cdk2 in this cell line (Cariou et al, 2000). …”

“…Cells were synchronized in G0 by estradiol depletion as described by Cariou et al (2000). Cells were released from quiescence by the addition of 10 Ϫ8 M 17␤-estradiol (Sigma, St. Louis, MO).…”

“…p27 localization was assayed at intervals across the cell cycle in synchronized MCF-7 cells (Cariou et al, 2000). p27 levels CRM1-Mediated Export of p27…”

“…p27 localization was assayed at intervals across the cell cycle in synchronized MCF-7 cells (Cariou et al, 2000). p27 levels and BrdU uptake were monitored by indirect immunofluorescence labeling and confocal microscopy.…”

Cyclin A is a prognostic indicator in early stage breast cancer with and without tamoxifen treatment

“…Both of these modulations affect the association between OR-a and co-activators of ORa (Rogatsky et al, 1999;Zwijsen et al, 1999) and may thereby affect the ability of anti-oestrogens to inhibit OR-a activity. It has, therefore, been hypothesized that tamoxifen treatment of breast cancer may be inefficient in tumour cases that harbour genetic lesions which result in elevated levels of cyclin D1 or A, or that lead to reduced levels of cdk inhibitors p21 or p27 (Michalides, 1999;Cariou et al, 2000). We have addressed this hypothesis by examining the effect of an altered expression of these cell cycle regulators on the outcome of tamoxifen treatment of patients with early stage of breast cancer.…”