title: Targeting triple-negative breast cancer cells with the histone deacetylase inhibitor panobinostat.
abstract: BACKGROUND
  Of the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo.
  
  
  METHODS
  TNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology.
  
  
  RESULTS
  Panobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat up-regulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype.
  
  
  CONCLUSIONS
  This study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types.
authors.affiliation: Department of Medicine, Section of Hematology and Medical Oncology, Tulane University Health Sciences Center, 1430 Tulane Ave, New Orleans, LA 70112, USA.
authors.forename: Chandra R
authors.initials: CR
authors.lastname: Tate
authors.forename: Lyndsay V
authors.initials: LV
authors.lastname: Rhodes
authors.forename: H Chris
authors.initials: HC
authors.lastname: Segar
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Driver
authors.forename: F Nell
authors.initials: FN
authors.lastname: Pounder
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Burow
authors.forename: Bridgette M
authors.initials: BM
authors.lastname: Collins-Burow
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D015820:Cadherins / Q000378:metabolism; D064200:Cdh1 Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D005260:Female; D015870:Gene Expression; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006657:Histones / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology*; D051379:Mice; D016513:Mice, SCID; D000077767:Panobinostat; D011897:Random Allocation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism; D011980:Receptors, Progesterone / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation; D023041:Xenograft Model Antitumor Assays
chemical_list: D015820:Cadherins; D064200:Cdh1 Proteins; D018797:Cell Cycle Proteins; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D007211:Indoles; D000077767:Panobinostat; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; C577585:Fzr1 protein, mouse; C508053:ERBB2 protein, human