Apoptosis Induction by MEK Inhibition in Human Lung Cancer Cells Is Mediated by Bim

AZD6244 (ARRY-142886) is an inhibitor of MEK1/2 and can inhibit cell proliferation or induce apoptosis in a cell-type dependent manner. The precise molecular mechanism of AZD6244-induced apoptosis is not clear. To investigate mechanisms of AZD6244 induced apoptosis in human lung cancer, we determined the molecular changes of two subgroups of human lung cancer cell lines that are either sensitive or resistant to AZD6244 treatment. We found that AZD6244 elicited a large increase of Bim proteins and a smaller increase of PUMA and NOXA proteins, and induced cell death in sensitive lung cancer cell lines, but had no effect on other Bcl-2 related proteins in those cell lines. Knockdown of Bim by siRNA greatly increased the IC50 and reduced apoptosis for AZD6244 treated cells. We also found that levels of endogenous p-Thr32-FOXO3a and p-Ser253-FOXO3a were lower in AZD6244-sensitive cells than in AZD6244-resistant cells. In the sensitive cells, AZD6244 induced FOXO3a nuclear translocation required for Bim activation. Moreover, the silencing of FOXO3a by siRNA abrogated AZD6244-induced cell apoptosis. In addition, we found that transfection of constitutively active AKT up-regulated p-Thr32-FOXO3a and p-Ser253-FOXO3a expression and inhibited AZD6244-induced Bim expression in sensitive cells. These results show that Bim plays an important role in AZD6244-induced apoptosis in lung cancer cells and that the PI3K/AKT/FOXO3a pathway is involved in Bim regulation and susceptibility of lung cancer cells to AZD6244. These results have implications in the development of strategies to overcome resistance to MEK inhibitors

Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)

mTOR (mammalian target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (protein kinase A/protein kinase G/protein kinase C) family kinases such as Akt (protein kinase B), S6K (p70 ribosomal S6 kinase) and SGK (serum and glucocorticoid protein kinase). mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of ∼10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of cancers in which this pathway is inappropriately activated

Preclinical Pharmacology of AZD5363, an Inhibitor of AKT: Pharmacodynamics, Antitumor Activity, and Correlation of Monotherapy Activity with Genetic Background

AKT is a key node in the most frequently deregulated signaling network in human cancer. AZD5363, a novel pyrrolopyrimidine-derived compound, inhibited all AKT isoforms with a potency of 10 nmol/L or less and inhibited phosphorylation of AKT substrates in cells with a potency of approximately 0.3 to 0.8 mmol/L. AZD5363 monotherapy inhibited the proliferation of 41 of 182 solid and hematologic tumor cell lines with a potency of 3 mmol/L or less. Cell lines derived from breast cancers showed the highest frequency of sensitivity. There was a significant relationship between the presence of PIK3CA and/or PTEN mutations and sensitivity to AZD5363 and between RAS mutations and resistance. Oral dosing of AZD5363 to nude mice caused dose- and time-dependent reduction of PRAS40, GSK3β, and S6 phosphorylation in BT474c xenografts (PRAS40 phosphorylation EC 50 ∼ 0.1 μmol/L total plasma exposure), reversible increases in blood glucose concentrations, and dose-dependent decreases in 2[18F]fluoro-2-deoxy-D-glucose ( 18F-FDG) uptake in U87-MG xenografts. Chronic oral dosing of AZD5363 caused dose-dependent growth inhibition of xenografts derived from various tumor types, including HER2 + breast cancer models that are resistant to trastuzumab. AZD5363 also significantly enhanced the antitumor activity of docetaxel, lapatinib, and trastuzumab in breast cancer xenografts. It is concluded that AZD5363 is a potent inhibitor of AKT with pharmacodynamic activity in vivo, has potential to treat a range of solid and hematologic tumors as monotherapy or a combinatorial agent, and has potential for personalized medicine based on the genetic status of PIK3CA, PTEN, and RAS. AZD5363 is currently in phase I clinical trials. ©2012 AACR

Small molecule binding sites on the Ras:SOS complex can be exploited for inhibition of Ras activation.

Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras

The effect of Bim-specific small interfering RNA (siRNA) on AZD6244-induced apoptosis.

<p>(A) Calu-6 and H3122 cells were transfected with Bim-specific or control siRNA and then treated with 3 µM AZD6244 for 48 hours. Expression of Bim, PARP and Caspase-9 were analyzed by Western blotting. (B) Cells were cultured in medium containing various concentrations of AZD6244 for 96 hours. Cell viability was determined by sulforhodamine B, and relative cell viability was plotted as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013026#s2" target="_blank">Materials and Methods</a> section. Values represent mean ± SD of three independent triplicate assays. (C) Parallel cells were fixed with ethanol and stained with propidium iodide; DNA content was analyzed by flow cytometry. Numbers represent percentages of apoptotic sub-G₁–phase cells. Data represent one of three independent experiments with similar results. <i>Columns</i>, mean; <i>bar</i>, SD. * , <i>P</i><0.05, compared with the control siRNA transfected cells. (D) Parallel cells were also fixed for TUNEL and DAPI staining. Apoptotic cell nuclei in TUNEL staining were labeled with FITC and visualized under fluorescence microscopy. The relative apoptotic cells were determined by counting TUNEL positive cells in five random fields (at 100× magnification) for each sample. <i>Columns</i>, mean; <i>bar</i>, SD. *, <i>P</i><0.05, compared with the control siRNA transfected cells. The representative photographs of Calu-6 were shown in the upper panel and the percentage of apoptotic cells of both Calu-6 and H3122 were showed in the lower panel. (E) Calu-6 cells were transfected with BimEL expression vector and control vector for 48 hrs. Expression of Bim was analyzed by Western blotting and apoptotic cells were detected with TUNEL assay.</p

The expression of various Bcl-2 family proteins in lung cancer cells after AZD6244 treatment.

<p>Western blots of Bcl-2 family members after treatment with AZD6244. (A) Human lung cancer cell lines (Calu-6, H2347, and H3122) were treated with 3 µM AZD6244 for 4, 8, 24, 48, and 72 hours. (B) Human lung cancer cell lines (Calu-3, H196, H522, and HCC2450) were treated with 3 µM AZD6244 for 4, 24, and 72 hours. (C) Calu-6, H2347, H196 and H522 cells were treated with 0.03, 0.1, 0.3, 1 and 3 µM of AZD6244 for 24 hours. Data represent one of three independent experiments with similar results.</p

Model depicting the signaling pathways utilized by AZD6244 in lung cancer cells to induced Bim activation and subsequent apoptosis.

<p>Our results suggest that AZD6244-induced up-regulation of Bim is mediated by FOXO3a, which is regulated through both the MAPK/ERK and PI3K/AKT pathways. In sensitive cells, MEK inhibition is sufficient to induce expression of the downstream molecule, Bim, and to induce apoptosis. However, in resistant cells, in which the PI3K/AKT/FOXO3a pathway is constitutively activated, suppression of ERK is insufficient to induce apoptosis because of suppression of Bim expression.</p