Introduction The activation of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is

Introduction The activation of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is one the most frequent genetic events in breast cancer, consequently the development of PI3K inhibitors has attracted much attention. most affected biological process after exposure to ETP-45658 (or our control PI3K inhibitor PI-103), that despite the multiple transcription factors that are regulated by the PI3K/AKT signalling cascade, only the binding sites for FOXO transcription factors were significantly enriched and only a subset of all FOXO-dependent genes were induced. This disparity in gene transcription was not due to differential FOXO promoter recruitment. Conclusions The constitutive activation of PI3Ks and thus the exclusion of FOXO transcription factors from the nucleus is a key feature of breast cancer. Our results presented here highlight that PI3K inhibition activates specific FOXO-dependent genes that mediate cell cycle arrest in breast cancer cells. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0482-y) contains supplementary material, which is available to authorized users. Introduction Breast cancer is the most common and the leading cause of cancer deaths in women [1]. Similar to other cancers, breast cancers are extremely heterogeneous with significant attention directed towards screening and targeting the epidermal growth factor HER2 and the estrogen receptor alpha. However, in addition to these two molecular targets, an extremely high percentage of Rabbit polyclonal to FOXQ1 breast cancers are characterized by the constitutive activation of phosphatidylinositol 3-kinases (PI3Ks) MK-0359 manufacture [2]. PI3Ks are a family of lipid and protein kinases that are divided into three classes based on their primary structure and substrate specificity [3]-[5]. The best studied class I PI3Ks are heterodimeric kinases that are composed of a catalytic subunit and a regulatory adaptor protein. The activation of PI3Ks can MK-0359 manufacture be triggered by growth factors and insulin that target the PI3K catalytic subunit to the plasma membrane placing it in close proximity with its substrate phosphatidylinositol 4,5-bisphosphate (PIP2) [6]. The class I PI3Ks preferentially phosphorylate PIP2 to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3) that as a secondary messenger activates the serine/threonine kinase AKT [7]. The PI3K signalling cascade is controlled by the dual lipid and protein phosphatase PTEN that negatively regulates the intracellular levels of PIP3 [8]. The constitutive activation of the PI3K/AKT signalling cascade is very common in cancer and occurs at different levels typically either activating mutations in the genes encoding the kinases PI3K (parameters of each compound, we analysed both agents using PhysChem Batch software (ACD20, Advanced Chemistry Development, Inc. Version 12 ACD/Labs, Toronto, ON, Canada) that is based on the quantitative structure-property relationship (QSPR) methodology (including pKa and LogD values). Boyden chamber matrigel invasion assay Cell-invasive capacity was determined using two-compartment Boyden chamber matrigel invasion assay (BD BioCoat? Matrigel? Invasion Chambers, BD Biosciences 354480, BD Biosciences, San Jose, CA, USA). MDA-MB231 cells were allowed to invade for 72?hours at 37C. All of the inserts and the liquid in each well of the companion plate were removed. 10% serum RPMI medium with 5?M calcein was added to each and incubated for 1?hour (in darkness). Fluorescence was measured with a MK-0359 manufacture luminometer (Envision, PerkinElmer, Inc, Waltham, MA, USA) at 485/535?nm and the results were analyzed in Activity Base (IDBS, Guildford, UK). Proliferation assays Cells were seeded in 96-well microtitre plates. Compounds (in DMSO) were added to each well (at a final concentration MK-0359 manufacture of 10?M). The medium was removed from the cells and replaced with 0.2?ml of medium containing either compound for 72?hours and then processed for MTT assay (Promega Corp, Madison, WI, USA) N?=?6. Cell cycle analysis The effect on the cell cycle following treatment with each compound was assessed by flow cytometry. Cells were grown to 70% confluence prior to drug treatment (1 to 10?M) for 24?hours. Cells were stained with 10?l of propidium iodide (Sigma-Aldrich). A total of 20,000 size gated cells were analysed by FACSCalibur (BD Biosciences). Immunoblotting Sub-confluent cells were incubated under the conditions indicated in each figure and washed twice with phosphate-buffered saline (PBS) prior to lysis. RIPA lysis buffer was added (50?mM TrisHCl, 150?mM NaCl, 1% NP-40, 2?mM Na3VO4, 100?mM NaF, 20?mM Na4P2O7 and 1x protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA). The membranes were incubated overnight for total AKT, phospho-serine-473-AKT, total p53 (DO1) (Santa Cruz Biotechnology, Dallas, TX, USA), phospho-threonine 32-FOXO3a MK-0359 manufacture (Merck Millipore, Darmstadt, Germany).

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