title: A novel phosphatidylinositol 3-kinase (PI3K) inhibitor directs a potent FOXO-dependent, p53-independent cell cycle arrest phenotype characterized by the differential induction of a subset of FOXO-regulated genes.
abstract: BACKGROUND
  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. Here we evaluate the effect of PI3K inhibition on global gene expression in breast cancer cells.
  
  
  METHODS
  We used a range of methodologies that include in silico compound analysis, in vitro kinase assays, cell invasion assays, proliferation assays, genome-wide transcription studies (Agilent Technologies full genome arrays), gene set enrichment analysis, quantitative real-time PCR, immunoblotting in addition to chromatin immunoprecipitation.
  
  
  RESULTS
  We defined the physico-chemical and the biological properties of ETP-45658, a novel potent PI3K inhibitor. We demonstrated that ETP-45658 potently inhibited cell proliferation within a broad range of human cancer cells, most potently suppressing the growth of breast cancer cells via inhibiting cell cycle. We show that this response is Forkhead box O (FOXO) protein dependent and p53 independent. Our genome-wide microarray analysis revealed that the cell cycle was the 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.
authors.affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal. drrjhill@gmail.com.|Regenerative Medicine Program, Department of Biomedical Sciences and Medicine, University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal. drrjhill@gmail.com.
authors.forename: Richard
authors.initials: R
authors.lastname: Hill
authors.affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal. ravikiran.k@gmail.com.
authors.forename: Ravi Kiran Reddy
authors.initials: RK
authors.lastname: Kalathur
authors.affiliation: Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Calle de Melchor Fernández Almagro 3, 28029, Madrid, Spain. scallejas@cnic.es.
authors.forename: Sergio
authors.initials: S
authors.lastname: Callejas
authors.affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal. lauracolaco21@gmail.com.
authors.forename: Laura
authors.initials: L
authors.lastname: Colaço
authors.affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal. r.brandao13@gmail.com.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Brandão
authors.affiliation: Experimental Therapeutics Program, Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029, Madrid, Spain. bserelde@cnio.es.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Serelde
authors.affiliation: Experimental Therapeutics Program, Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029, Madrid, Spain. acebria@cnio.es.
authors.forename: Antonio
authors.initials: A
authors.lastname: Cebriá
authors.affiliation: Experimental Therapeutics Program, Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029, Madrid, Spain. cblanco@cnio.es.
authors.forename: Carmen
authors.initials: C
authors.lastname: Blanco-Aparicio
authors.affiliation: Experimental Therapeutics Program, Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029, Madrid, Spain. jpastor@cnio.es.
authors.forename: Joaquín
authors.initials: J
authors.lastname: Pastor
authors.affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal. mfutschik@ualg.pt.
authors.forename: Matthias
authors.initials: M
authors.lastname: Futschik
authors.affiliation: Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Calle de Melchor Fernández Almagro 3, 28029, Madrid, Spain. adopazo@cnic.es.
authors.forename: Ana
authors.initials: A
authors.lastname: Dopazo
authors.affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal. walink@ualg.pt.|Regenerative Medicine Program, Department of Biomedical Sciences and Medicine, University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal. walink@ualg.pt.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Link
mesh_terms: D000230:Adenocarcinoma; D001943:Breast Neoplasms; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D003198:Computer Simulation; D005260:Female; D051858:Forkhead Transcription Factors / Q000187:drug effects* / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D066298:In Vitro Techniques; D061986:MCF-7 Cells; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects* / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C544684:ETP 45658; D051858:Forkhead Transcription Factors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D051057:Proto-Oncogene Proteins c-akt