title: During hormone depletion or tamoxifen treatment of breast cancer cells the estrogen receptor apoprotein supports cell cycling through the retinoic acid receptor α1 apoprotein.
abstract: BACKGROUND
  Current hormonal adjuvant therapies for breast cancer including tamoxifen treatment and estrogen depletion are overall tumoristatic and are severely limited by the frequent recurrence of the tumors. Regardless of the resistance mechanism, development and progression of the resistant tumors requires the persistence of a basal level of cycling cells during the treatment for which the underlying causes are unclear.
  
  
  METHODS
  In estrogen-sensitive breast cancer cells the effects of hormone depletion and treatment with estrogen, tamoxifen, all-trans retinoic acid (ATRA), fulvestrant, estrogen receptor α (ER) siRNA or retinoic acid receptor α (RARα) siRNA were studied by examining cell growth and cycling, apoptosis, various mRNA and protein expression levels, mRNA profiles and known chromatin associations of RAR. RARα subtype expression was also examined in breast cancer cell lines and tumors by competitive PCR.
  
  
  RESULTS
  Basal proliferation persisted in estrogen-sensitive breast cancer cells grown in hormone depleted conditioned media without or with 4-hydroxytamoxifen (OH-Tam). Downregulating ER using either siRNA or fulvestrant inhibited basal proliferation by promoting cell cycle arrest, without enrichment for ErbB2/3+ overexpressing cells. The basal expression of RARα1, the only RARα isoform that was expressed in breast cancer cell lines and in most breast tumors, was supported by apo-ER but was unaffected by OH-Tam; RAR-β and -γ were not regulated by apo-ER. Depleting basal RARα1 reproduced the antiproliferative effect of depleting ER whereas its restoration in the ER depleted cells partially rescued the basal cycling. The overlapping tamoxifen-insensitive gene regulation by apo-ER and apo-RARα1 comprised activation of mainly genes promoting cell cycle and mitosis and suppression of genes involved in growth inhibition; these target genes were generally insensitive to ATRA but were enriched in RAR binding sites in associated chromatin regions.
  
  
  CONCLUSIONS
  In hormone-sensitive breast cancer, ER can support a basal fraction of S-phase cells (i) without obvious association with ErbB2/3 expression, (ii) by mechanisms unaffected by hormone depletion or OH-Tam and (iii) through maintenance of the basal expression of apo-RARα1 to regulate a set of ATRA-insensitive genes. Since isoform 1 of RARα is genetically redundant, its targeted inactivation or downregulation should be further investigated as a potential means of enhancing hormonal adjuvant therapy.
authors.affiliation: Department of Biochemistry and Cancer Biology, Medical University of Ohio, 3000 Arlington Avenue, Toledo, OH 43614, USA.
authors.forename: Marcela D
authors.initials: MD
authors.lastname: Salazar
authors.forename: Maya
authors.initials: M
authors.lastname: Ratnam
authors.forename: Mugdha
authors.initials: M
authors.lastname: Patki
authors.forename: Ivana
authors.initials: I
authors.lastname: Kisovic
authors.forename: Robert
authors.initials: R
authors.lastname: Trumbly
authors.forename: Mohamed
authors.initials: M
authors.lastname: Iman
authors.forename: Manohar
authors.initials: M
authors.lastname: Ratnam
mesh_terms: D001059:Apoproteins / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D020033:Protein Isoforms / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D020893:Receptor, ErbB-3 / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D000072482:Retinoic Acid Receptor alpha; D013629:Tamoxifen / Q000494:pharmacology / Q000627:therapeutic use*
chemical_list: D001059:Apoproteins; D002843:Chromatin; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D011960:Receptors, Estrogen; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D013629:Tamoxifen; C000607071:RARA protein, human