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...

No abstract

Antiestrogens, such as the drug tamoxifen, inhibit breast cancer growth by inducing cell cycle arrest. Antiestrogens require action of the cell cycle inhibitor p27Kip1 to mediate G 1 arrest in estrogen receptor-positive breast cancer cells. We report that constitutive activation of the mitogen-activated protein kinase (MAPK) pathway alters p27 phosphorylation, reduces p27 protein levels, reduces the cdk2 inhibitory activity of the remaining p27, and contributes to antiestrogen resistance. In two antiestrogen-resistant cell lines that showed increased MAPK activation, inhibition of the MAPK kinase (MEK) by addition of U0126 changed p27 phosphorylation and restored p27 inhibitory function and sensitivity to antiestrogens. Using antisense p27 oligonucleotides, we demonstrated that this restoration of antiestrogen-mediated cell cycle arrest required p27 function. These data suggest that oncogene-mediated MAPK activation, frequently observed in human breast cancers, contributes to antiestrogen resistance through p27 deregulation. p27kip1 is a member of the KIP 1 (kinase inhibitory protein) family of cdk inhibitors that regulate the cyclin-cdk complexes governing cell cycle transitions (1). The importance of p27 as a G 1 -to-S phase regulator is highlighted by the finding that antisense-mediated inhibition of p27 expression is sufficient to induce cell cycle entry in quiescent fibroblasts (2) and in steroid-depleted breast cancer cells (3). p27 protein levels are high in G 0 and early G 1 during which time p27 binds tightly and inhibits cyclin E1-cdk2. p27 translation rates decrease, and its proteolysis increases during G 1 -to-S phase progression, leading to p27 protein loss as cells enter S phase (4 -6). p27 proteolysis is regulated by phosphorylation of p27 on threonine 187 (Thr-187) by cyclin E1-cdk2 (7,8). While mutations or deletions in the p27 gene are uncommon (9, 10), p27 degradation is increased in many cancers, including breast cancer (11,12).An increasing body of data suggests that p27 is regulated by mitogenic signal transduction pathways, including Rasdependent activation of the mitogen-activated protein kinase (MAPK) pathway (13-17). Many mitogens increase the cellular levels of GTP-bound Ras, leading to activation of the downstream target, Raf-1. The Raf-1 kinase can phosphorylate and activate the dual specificity kinases MEK1 and MEK2, which in turn activate MAPK (also known as p42 ERK2 and p44 ERK1 ). Once activated, MAPK can phosphorylate several nuclear transcription factors including Myc, Elk, and Rsk (for review, see Ref. 18). p27 itself has several MAPK consensus sites, and MAPK can phosphorylate p27 in vitro (16) and reduce the ability of recombinant p27 to bind and inhibit cdk2 in vitro (15). While constitutive activation of Ras-MAPK can reduce p27 inhibitory function in immortal and cancer-derived lines, it is not clear whether MAPK directly regulates p27 during cell cycle progression in normal cell types.Studies of p27 regulation by the Ras-MAPK pathway were initially carried out in fibrobla...