Constitutive MEK/MAPK activation leads to p27(Kip1) deregulation and antiestrogen resistance in human breast cancer cells.

Jeffrey C.H Donovan,Joyce M Slingerland,Andrea Milic

doi:10.1074/jbc.m106448200

Jeffrey C.H Donovan, Joyce M Slingerland + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m106448200

Copy DOI

Abstract

Antiestrogens, such as the drug tamoxifen, inhibit breast cancer growth by inducing cell cycle arrest. Antiestrogens require action of the cell cycle inhibitor p27(Kip1) to mediate G1 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.

Highlights

P27kip1 is a member of the KIP1 family of cdk inhibitors that regulate the cyclin-cdk complexes governing cell cycle transitions [1]
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
The importance of p27 as a G1-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 G0 and early G1 during which time p27 binds tightly and inhibits cyclin E1-cdk2. p27 translation rates decrease, and its proteolysis increases during G1-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 (Thr187) by cyclin E1-cdk2 [7, 8]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—MCF-7 cells [42] and LY-2 cells [43] were obtained from the laboratory of M. The effects of MEK inhibition on the cell cycle were assayed following addition of 0.1 ␮M U0126 (Promega) for 2 or 24 h prior to recovery for immunoblotting or flow cytometric analysis. The effects of MEK inhibition on antiestrogen sensitivity in the antiestrogenresistant lines, LY-2 or MCF-7/HER2–18, were assayed by treating cells with 0.1 ␮M U0126 for 2 h followed by an additional 48 h with either 1 ␮M 4-OH-TAM or 10 nM ICI 182780 prior to recovery of cells for protein or flow cytometric analysis. Assays of p27 Inhibitory Function—Cell lysates (250 ␮g) from asynchronously proliferating MCF-7, MCF-7/MEKEE (line M2), or LY-2 cells were immunoprecipitated with pAb5588 anti-p27 serum or control polyclonal rabbit IgG, and precipitates were collected on protein A-Sepharose beads. To confirm that the most positively charged p27 isoforms represented unphosphorylated p27, cells were labeled with [32P]orthophosphate (1 mCi/100-dish) for 3 h, and p27 immunoprecipitates were isolated and subjected to 2D-IEF. p27 immunoprecipitated from unlabeled cells was resolved by 2D-IEF in parallel with the labeled p27, and the resolution pattern of the cold p27 was compared with the phosphate-labeled p27

RESULTS

Findings

DISCUSSION