title: NOTCH-1 and NOTCH-4 are novel gene targets of PEA3 in breast cancer: novel therapeutic implications.
abstract: BACKGROUND
  Women with triple-negative breast cancer have the worst prognosis, frequently present with metastatic tumors and have few targeted therapy options. Notch-1 and Notch-4 are potent breast oncogenes that are overexpressed in triple-negative and other subtypes of breast cancer. PEA3, an ETS transcription factor, is also overexpressed in triple-negative and other breast cancer subtypes. We investigated whether PEA3 could be the critical transcriptional activator of Notch receptors in MDA-MB-231 and other breast cancer cells.
  
  
  METHODS
  Real-time PCR and Western blot analysis were performed to detect Notch-1, Notch-2, Notch-3 and Notch-4 receptor expression in breast cancer cells when PEA3 was knocked down by siRNA. Chromatin immunoprecipitation was performed to identify promoter regions for Notch genes that recruited PEA3. TAM-67 and c-Jun siRNA were used to identify that c-Jun was necessary for PEA3 enrichment on the Notch-4 promoter. A Notch-4 luciferase reporter was used to confirm that endogenous PEA3 or AP-1 activated the Notch-4 promoter region. Cell cycle analysis, trypan blue exclusion, annexin V flow cytometry, colony formation assay and an in vivo xenograft study were performed to determine the biological significance of targeting PEA3 via siRNA, Notch signaling via a γ-secretase inhibitor, or both.
  
  
  RESULTS
  Herein we provide new evidence for transcriptional regulation of Notch by PEA3 in breast cancer. PEA3 activates Notch-1 transcription in MCF-7, MDA-MB-231 and SKBr3 breast cancer cells. PEA3 activates Notch-4 transcription in MDA-MB-231 cells where PEA3 levels are endogenously high. In SKBr3 and BT474 breast cancer cells where PEA3 levels are low, overexpression of PEA3 increases Notch-4 transcripts. Chromatin immunoprecipitation confirmed the enrichment of PEA3 on Notch-1 and Notch-4 promoters in MDA-MB-231 cells. PEA3 recruitment to Notch-1 was AP-1-independent, whereas PEA3 recruitment to Notch-4 was c-JUN-dependent. Importantly, the combined inhibition of Notch signaling via a γ-secretase inhibitor (MRK-003 GSI) and knockdown of PEA3 arrested growth in the G1 phase, decreased both anchorage-dependent and anchorage-independent growth and significantly increased apoptotic cells in vitro. Moreover, either PEA3 knockdown or MRK-003 GSI treatment significantly reduced tumor growth of MDA-MB-231 xenografts in vivo.
  
  
  CONCLUSIONS
  Taken together, the results from this study demonstrate for the first time that Notch-1 and Notch-4 are novel transcriptional targets of PEA3 in breast cancer cells. Targeting of PEA3 and/or Notch pathways might provide a new therapeutic strategy for triple-negative and possibly other breast cancer subtypes.
authors.affiliation: Molecular and Cellular Biochemistry Program, Loyola University Medical Center, Maywood, IL 60153, USA.
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Clementz
authors.forename: Allison
authors.initials: A
authors.lastname: Rogowski
authors.forename: Kinnari
authors.initials: K
authors.lastname: Pandya
authors.forename: Lucio
authors.initials: L
authors.lastname: Miele
authors.forename: Clodia
authors.initials: C
authors.lastname: Osipo
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D051883:Receptor, Notch2 / Q000235:genetics / Q000378:metabolism*; D000071656:Receptor, Notch3; D000074562:Receptor, Notch4; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: C497761:NOTCH1 protein, human; C497764:NOTCH2 protein, human; C499374:NOTCH3 protein, human; C106075:NOTCH4 protein, human; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D016755:Proto-Oncogene Proteins c-jun; D034741:RNA, Small Interfering; D051881:Receptor, Notch1; D051883:Receptor, Notch2; D000071656:Receptor, Notch3; D000074562:Receptor, Notch4; D051880:Receptors, Notch; C439528:TAM67 peptide; D018808:Transcription Factor AP-1; D014157:Transcription Factors; C065788:transcription factor PEA3; D048031:JNK Mitogen-Activated Protein Kinases; D053829:Amyloid Precursor Protein Secretases