title: Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.
abstract: BACKGROUND
  Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown.
  
  
  METHODS
  MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively.
  
  
  RESULTS
  We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF.
  
  
  CONCLUSIONS
  E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.
authors.affiliation: Department of Surgery, Indiana University School of Medicine, 980 West Walnut Street, Indianapolis, IN 46202, USA.
authors.forename: Poornima
authors.initials: P
authors.lastname: Bhat-Nakshatri
authors.forename: Eun-Kyung
authors.initials: EK
authors.lastname: Song
authors.forename: Nikail R
authors.initials: NR
authors.lastname: Collins
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Uversky
authors.forename: A Keith
authors.initials: AK
authors.lastname: Dunker
authors.forename: Bert W
authors.initials: BW
authors.lastname: O'Malley
authors.forename: Tim R
authors.initials: TR
authors.lastname: Geistlinger
authors.forename: Jason S
authors.initials: JS
authors.lastname: Carroll
authors.forename: Myles
authors.initials: M
authors.lastname: Brown
authors.forename: Harikrishna
authors.initials: H
authors.lastname: Nakshatri
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects*; D001665:Binding Sites; D004958:Estradiol / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics; D061986:MCF-7 Cells; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000235:genetics / Q000378:metabolism; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism
chemical_list: D047628:Estrogen Receptor alpha; D018960:Hyaluronan Receptors; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019014:fas Receptor; D004958:Estradiol; D051497:Receptor, Fibroblast Growth Factor, Type 2; D051057:Proto-Oncogene Proteins c-akt