title: Mcl-1 confers protection of Her2-positive breast cancer cells to hypoxia: therapeutic implications.
abstract: BACKGROUND
  Molecular mechanisms leading to the adaptation of breast cancer (BC) cells to hypoxia are largely unknown. The anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) is frequently amplified in BC; and elevated Mcl-1 levels have been correlated with poor prognosis. Here we investigated the pathophysiologic role of Mcl-1 in Her2-positive BC cells under hypoxic conditions.
  
  
  METHODS
  RNA interference and a novel small molecule inhibitor, EU-5346, were used to examine the role of Mcl-1 in Her2-positive BC cell lines and primary BC cells (sensitive or intrinsically resistant to Her2 inhibitors) under hypoxic conditions (using a hypoxic incubation chamber). Mechanisms-of-action were investigated by RT-PCR, mitochondrial isolation, as well as immunoprecipitation/blotting analysis, and microscopy. The specificity against Mcl-1 of the novel small molecule inhibitor EU5346 was verified in Mcl-1(Δ/null) versus Mcl-1(wt/wt) Murine Embryonic Fibroblasts (MEFs). Proliferation, survival, and spheroid formation were assessed in response to Mcl-1 and Her2 inhibition.
  
  
  RESULTS
  We demonstrate for a strong correlation between high Mcl-1 protein levels and hypoxia, predominantly in Her2-positive BC cells. Surprisingly, genetic depletion of Mcl-1 decreased Her2 and Hif-1α levels followed by inhibition of BC cell survival. In contrast, Mcl-1 protein levels were not downregulated after genetic depletion of Her2 indicating a regulatory role of Mcl-1 upstream of Her2. Indeed, Mcl-1 and Her2 co-localize within the mitochondrial fraction and form a Mcl-1/Her2- protein complex. Similar to genetically targeting Mcl-1 the novel small molecule Mcl-1 inhibitor EU-5346 induced cell death and decreased spheroid formation in Her2-positive BC cells. Of interest, EU-5346 induced ubiquitination of Mcl-1- bound Her2 demonstrating a previously unknown role for Mcl-1 to stabilize Her2 protein levels. Importantly, targeting Mcl-1 was also active in Her2-positive BC cells resistant to Her2 inhibitors, including a brain-primed Her2-positive cell line.
  
  
  CONCLUSIONS
  Our data demonstrate a critical role of Mcl-1 in Her2-positive BC cell survival under hypoxic conditions and provide the preclinical framework for the therapeutic use of novel Mcl-1- targeting agents to improve patient outcome in BC.
authors.affiliation: Department of Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg, Im Neuenheimer Feld #460, Heidelberg, 69120, Germany.|Department of Pharmacology and Therapy, Faculty of Medicine, Universitas Padjadjaran, Jl. Eijkman 38, Bandung, 02215, Indonesia.
authors.forename: Muhammad Hasan
authors.initials: MH
authors.lastname: Bashari
authors.affiliation: Department of Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg, Im Neuenheimer Feld #460, Heidelberg, 69120, Germany.
authors.forename: Fengjuan
authors.initials: F
authors.lastname: Fan
authors.affiliation: Department of Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg, Im Neuenheimer Feld #460, Heidelberg, 69120, Germany.
authors.forename: Sonia
authors.initials: S
authors.lastname: Vallet
authors.affiliation: Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA, 02215, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Sattler
authors.affiliation: Eutropics, Inc., 767C Concord Avenue, Cambridge, MA, 02138, USA.
authors.forename: Melissa
authors.initials: M
authors.lastname: Arn
authors.affiliation: Department of Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg, Im Neuenheimer Feld #460, Heidelberg, 69120, Germany.|German Cancer Research Center (DKFZ), Im Neuenheimer Feld 460, 69120, Heidelberg, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Luckner-Minden
authors.affiliation: Department of Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg, Im Neuenheimer Feld #460, Heidelberg, 69120, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Schulze-Bergkamen
authors.affiliation: Department of Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg, Im Neuenheimer Feld #460, Heidelberg, 69120, Germany.|German Cancer Research Center (DKFZ), Im Neuenheimer Feld 460, 69120, Heidelberg, Germany.
authors.forename: Inka
authors.initials: I
authors.lastname: Zörnig
authors.affiliation: Department of Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg, Im Neuenheimer Feld #460, Heidelberg, 69120, Germany.
authors.forename: Frederik
authors.initials: F
authors.lastname: Marme
authors.affiliation: Department of Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg, Im Neuenheimer Feld #460, Heidelberg, 69120, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schneeweiss
authors.affiliation: Eutropics, Inc., 767C Concord Avenue, Cambridge, MA, 02138, USA.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Cardone
authors.affiliation: St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN, 38105, USA.
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Opferman
authors.affiliation: Department of Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg, Im Neuenheimer Feld #460, Heidelberg, 69120, Germany.|German Cancer Research Center (DKFZ), Im Neuenheimer Feld 460, 69120, Heidelberg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Jäger
authors.affiliation: Department of Medical Oncology, National Center for Tumor Diseases (NCT), University of Heidelberg, Im Neuenheimer Feld #460, Heidelberg, 69120, Germany. klaus.podar@nct-heidelberg.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Podar
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D015687:Cell Hypoxia / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000096:biosynthesis / Q000235:genetics*; D034622:RNA Interference; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics
chemical_list: D064549:Myeloid Cell Leukemia Sequence 1 Protein; D018719:Erb-b2 Receptor Tyrosine Kinases; C579095:MCL1 protein, human; C508053:ERBB2 protein, human