Inhibition of Akt increases p27Kip1 levels and induces cell cycle arrest in anaplastic large cell lymphoma

Abstract

Anaplastic large cell lymphoma (ALCL) is a highly proliferative neoplasm that frequently carries the t(2;5)(p23;q35) and aberrantly expresses nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). Previously, NPM-ALK had been shown to activate the phosphatidylinositol 3 kinase (PI3K)/Akt pathway. As the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) is usually not expressed in ALCL, we hypothesized that activated Akt (pAkt) phosphorylates p27 resulting in increased p27 proteolysis and cell cycle progression. Here we demonstrate that inhibition of pAkt activity in ALCL decreases p27 phosphorylation and degradation, resulting in increased p27 levels and cell cycle arrest. Using immunohistochemistry, pAkt was detected in 24 (57%) of 42 ALCL tumors, including 8 (44%) of 18 ALK-positive tumors and 16 (67%) of 24 ALK-negative tumors, and was inversely correlated with p27 levels. The mean percentage of p27-positive tumor cells was 5% in the pAkt-positive group compared with 26% in the pAkt-negative group (P = .0076). These findings implicate that Akt activation promotes cell cycle progression through inactivation of p27 in ALCL.

Figure 1. Inhibition of Akt increases total p27 levels and induces cell-cycle arrest in ALCL cells

(A) Akt-II inhibitor induced gradual decrease of pAkt (serine 473) levels. At a concentration of 10 μM, Akt-II induced almost complete absence of pAkt at 12 hours. Total Akt was also probed using the same membrane. No substantial changes were noticed in Akt levels. Top panel, SU-DHL1; bottom panel, Karpas 299. (B) Immunoprecipitation studies revealed a decrease in threonine phosphorylation of p27 (top panel) and an increase in total p27 levels in Karpas 299 cells treated with Akt-II inhibitor at 12 hours. WB indicates Western blot; and IP, immunoprecipitation. Densitometry of the immunoblot bands showed a substantial decrease in the threonine-phosphorylated p27/immunoglobulin G (IgG) ratio that was associated with increased total p27/IgG ratio. (C) Cell cycle analysis using BrdU uptake and flow cytometry in Karpas 299 cells 24 hours after treatment with Akt-II inhibitor. The S-phase fraction was 9% in cells treated with 5 μM of the Akt-II inhibitor compared with 39% in untreated (control) cells. Similar results were obtained for SU-DHL1 cells. (D) Total p27 levels after proteasome inhibition in ALCL cells. Treatment of Karpas 299 cells with LLnL and MG132 proteasome inhibitors for 16 hours resulted in a significant increase of total p27 levels (lanes 2 and 4 compared with lane 1), due to decreased p27 degradation through the ubiquitin-proteasome system. LLnL and MG132 were used at a concentration of 35 μM each and were previously shown to adequately block proteasome activity (data not shown). Pretreatment of ALCL cells with proteasome inhibitors for 4 hours followed by treatment of cells with both proteasome inhibitors and Akt-II for 12 hours resulted in no additional increase of total p27 levels (lanes 3 and 5), which demonstrates complete blockade of proteasome-mediated degradation.

Figure 2. Expression of Akt and p27 in ALCL primary tumors

(A) Subcellular fractionation followed by Western blot analysis demonstrated that Akt and pAkt are predominantly localized in the cytoplasm of Karpas 299 and SU-DHL1 cells. Retinoblastoma protein (Rb) was detected exclusively in the nuclear fraction and served as a control for nuclear localization of proteins in these cells. (B) Box-plot showing the significant difference in the percentage of p27-positive tumor cells between pAkt-positive and pAkt-negative ALCL tumors (P = .0076). Only nuclear expression of total p27 was considered positive in this analysis. Error bars indicate 5%–95%. (C–D) Immunohistochemical detection of pAkt in ALCL tumors showed strong cytoplasmic expression of pAkt in a subset of ALK-positive and ALK-negative ALCL tumors, shown in panels C and D, respectively. (Immunohistochemical staining, 600 × original magnification, obtained with a BX51 Olympus microscope [Melville, NY] and a DP12 Olympus camera.) Images were viewed through a universal semi-apochromat objective lens (UPlan Fl, Olympus America Inc, Woodbury, NY); original magnification × 400; numerical aperture 0.75.