Abstract A186: Low dose paclitaxel treatment increase the stability of p27Kip1

Abstract

Abstract The purpose of our study is to better understand how taxanes (paclitaxel, docetaxel) induce cell death. Taxanes play a critical role in combination chemotherapy for many types of solid tumors, especially in advanced disease states. These drugs bind to tubulin in interphase and mitotic microtubules altering their dynamics. This leads to G2/M arrest that is thought to contribute to taxane-induced apoptosis, although the exact mechanisms remain elusive. We found that after treating MDA-MB-231 breast cancer cells with low dose (10X IC50 = 40 nM) paclitaxel over 4-48 h, the cyclin-dependent kinase inhibitor p27Kip1 progressively increases more than 2-fold. p27Kip1 is an important regulator of cell cycle, found in both the nucleus and cytoplasm, and its accumulation is associated with G1 arrest in most cells. Its tumor suppressor activity is associated with its nuclear localization, where it forms complexes with Cdk2 and cyclin E (or other cyclins) preventing DNA replication and cell cycle advance. In the cytoplasm, p27Kip1 was shown to enhance cell motility. When p27Kip1 becomes phosphorylated on Thr 187 leading to its ubiquitination and degradation, the cell cycle can progress. We pretreated cells with 10 μM cycloheximide for 1 h to prevent translational peptide elongation and found when cells were subsequently treated with 40 nM paclitaxel for up to 4 hours, quantitative Western blotting showed that p27Kip1 levels steadily increased, indicating that paclitaxel treatment stabilized p27Kip1 protein. Quantitative real-time PCR demonstrated a parallel increase in p27Kip1 mRNA over 8 hours and then a gradual decline in mRNA over 24-48 h, while the protein remained stable over this time period. Since paclitaxel binds to microtubules, we wanted to know whether association of p27Kip1 with microtubules might play a role in its stabilization. We isolated the intact microtubule cytoskeleton from cells using a 2M glycerol buffer (100 mM Tris pH 6.8, 0.5% Nonidet P-40, 1 mM MgCl2 and 2 mM EGTA) at 37°C and found that 20% of the total intracellular p27Kip1 is associated with the microtubule pellet in control cells and 40 nM paclitaxel treatment for 48 h reduces this amount by about half, demonstrating that paclitaxel treatment leads to an increased amount of p27Kip1 in the non-microtubule fraction of the cell lysate. Because the tumor suppressor function of p27Kip1 is associated with its nuclear localization, we isolated nuclear and cytoplasmic fractions from MDA-MB-231 cell lysates and found a 2-fold change in p27Kip1 with 40 nM paclitaxel treatment for 48 h, compared to cells treated with DMSO. The 2-fold increase in p27kip1 was found entirely in the cytoplasmic fraction. Western blotting with an antibody that recognizes p27Kip1 phosphorylated at Thr 187 surprisingly showed a nearly 2-fold increase in phospho- p27Kip1 with paclitaxel treatment in the cytoplasmic fractions. In summary, paclitaxel treatment of MDA-MB-231 breast cancer cells leads to a 2-fold increase in total p27Kip1. This increase is primarily in the cytoplasmic fraction and is associated with an increase in phospho-The 187-p27Kip1. Our data together suggest that paclitaxel treatment may block ubiquitination of phospho -Thr 187-p27Kip1 or its proteosomal degradation. These novel observations are important for understanding the regulation of p27Kip1 and the mode of action for paclitaxel. Citation Format: Sharon Lobert, Mary E. Graichen. Low dose paclitaxel treatment increase the stability of p27Kip1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A186.