title: Distinct pathways regulated by RET and estrogen receptor in luminal breast cancer demonstrate the biological basis for combination therapy.
abstract: OBJECTIVE
  We investigated directed therapy based on TFAP2C-regulated pathways to inform new therapeutic approaches for treatment of luminal breast cancer.
  
  
  BACKGROUND
  TFAP2C regulates the expression of genes characterizing the luminal phenotype including ESR1 and RET, but pathway cross talk and potential for distinct elements have not been characterized.
  
  
  METHODS
  Activation of extracellular signal-regulated kinases (ERK) and AKT was assessed using phosphorylation-specific Western blot. Cell proliferation was measured with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] after siRNA (small interfering RNA) gene knockdown or drug treatment. Cell cycle, Ki-67, and cleaved caspase 3 were measured by fluorescence-activated cell sorting. Tumorigenesis was assessed in mice xenografts.
  
  
  RESULTS
  Knockdown of TFAP2C or RET inhibited GDNF (glial cell line-derived neurotrophic factor)-mediated activation of ERK and AKT in MCF-7 cells. Similarly, sunitinib, a small-molecule inhibitor of RET, blocked GDNF-mediated activation of ERK and AKT. Inhibition of RET either by gene knockdown or by treatment with sunitinib or vandetanib reduced RET-dependent growth of luminal breast cancer cells. Interestingly, knockdown of TFAP2C, which controls both ER (estrogen receptor) and RET, demonstrated a greater effect on cell growth than either RET or ER alone. Parallel experiments using treatment with tamoxifen and sunitinib confirmed the increased effectiveness of dual inhibition of the ER and RET pathways in regulating cell growth. Whereas targeting the ER pathway altered cell proliferation, as measured by Ki-67 and S-phase, anti-RET primarily increased apoptosis, as demonstrated by cleaved caspase 3 and increased TUNEL (terminal deoxyneucleotidyl transferase dUTP nick end labeling) expression in xenografts.
  
  
  CONCLUSIONS
  ER and RET primarily function through distinct pathways regulating proliferation and cell survival, respectively. The findings inform a therapeutic approach based on combination therapy with antiestrogen and anti-RET in luminal breast cancer.
authors.affiliation: From the Department of Surgery, University of Iowa, Iowa City.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Spanheimer
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Cyr
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Gillum
authors.forename: George W
authors.initials: GW
authors.lastname: Woodfield
authors.forename: Ryan W
authors.initials: RW
authors.lastname: Askeland
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Weigel
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D014408:Biomarkers, Tumor / Q000378:metabolism*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D047628:Estrogen Receptor alpha / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007211:Indoles / Q000008:administration & dosage; D020935:MAP Kinase Signaling System / Q000187:drug effects; D061986:MCF-7 Cells; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy* / Q000378:metabolism; D051379:Mice; D010880:Piperidines / Q000008:administration & dosage; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret / Q000378:metabolism*; D011758:Pyrroles / Q000008:administration & dosage; D011799:Quinazolines / Q000008:administration & dosage; D011897:Random Allocation; D015398:Signal Transduction / Q000187:drug effects; D000077210:Sunitinib; D013629:Tamoxifen / Q000008:administration & dosage; D050656:Transcription Factor AP-2 / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D007211:Indoles; D010880:Piperidines; D011758:Pyrroles; D011799:Quinazolines; C493679:TFAP2C protein, human; D050656:Transcription Factor AP-2; D013629:Tamoxifen; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D051057:Proto-Oncogene Proteins c-akt; D000077210:Sunitinib; C452423:vandetanib
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