title: Autocrine IGF-I/insulin receptor axis compensates for inhibition of AKT in ER-positive breast cancer cells with resistance to estrogen deprivation.
abstract: BACKGROUND
  Estrogen receptor α-positive (ER+) breast cancers adapt to hormone deprivation and acquire resistance to antiestrogen therapies. Upon acquisition of hormone independence, ER+ breast cancer cells increase their dependence on the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. We examined the effects of AKT inhibition and its compensatory upregulation of insulin-like growth factor (IGF)-I/InsR signaling in ER+ breast cancer cells with acquired resistance to estrogen deprivation.
  
  
  METHODS
  Inhibition of AKT using the catalytic inhibitor AZD5363 was examined in four ER+ breast cancer cell lines resistant to long-term estrogen deprivation (LTED) by western blotting and proliferation assays. Feedback upregulation and activation of receptor tyrosine kinases (RTKs) was examined by western blotting, real-time qPCR, ELISAs, membrane localization of AKT PH-GFP by immunofluorescence and phospho-RTK arrays. For studies in vivo, athymic mice with MCF-7 xenografts were treated with AZD5363 and fulvestrant with either the ATP-competitive IGF-IR/InsR inhibitor AZD9362 or the fibroblast growth factor receptor (FGFR) inhibitor AZD4547.
  
  
  RESULTS
  Treatment with AZD5363 reduced phosphorylation of the AKT/mTOR substrates PRAS40, GSK3α/β and S6K while inducing hyperphosphorylation of AKT at T308 and S473. Inhibition of AKT with AZD5363 suppressed growth of three of four ER+ LTED lines and prevented emergence of hormone-independent MCF-7, ZR75-1 and MDA-361 cells. AZD5363 suppressed growth of MCF-7 xenografts in ovariectomized mice and a patient-derived luminal B xenograft unresponsive to tamoxifen or fulvestrant. Combined treatment with AZD5363 and fulvestrant suppressed MCF-7 xenograft growth better than either drug alone. Inhibition of AKT with AZD5363 resulted in upregulation and activation of RTKs, including IGF-IR and InsR, upregulation of FoxO3a and ERα mRNAs as well as FoxO- and ER-dependent transcription of IGF-I and IGF-II ligands. Inhibition of IGF-IR/InsR or PI3K abrogated AKT PH-GFP membrane localization and T308 P-AKT following treatment with AZD5363. Treatment with IGFBP-3 blocked AZD5363-induced P-IGF-IR/InsR and T308 P-AKT, suggesting that receptor phosphorylation was dependent on increased autocrine ligands. Finally, treatment with the dual IGF-IR/InsR inhibitor AZD9362 enhanced the anti-tumor effect of AZD5363 in MCF-7/LTED cells and MCF-7 xenografts in ovariectomized mice devoid of estrogen supplementation.
  
  
  CONCLUSIONS
  These data suggest combinations of AKT and IGF-IR/InsR inhibitors would be an effective treatment strategy against hormone-independent ER+ breast cancer.
authors.forename: Emily M
authors.initials: EM
authors.lastname: Fox
authors.forename: María Gabriela
authors.initials: MG
authors.lastname: Kuba
authors.forename: Todd W
authors.initials: TW
authors.lastname: Miller
authors.forename: Barry R
authors.initials: BR
authors.lastname: Davies
authors.forename: Carlos L
authors.initials: CL
authors.lastname: Arteaga
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D004195:Disease Models, Animal; D019008:Drug Resistance, Neoplasm; D020847:Estrogen Receptor Modulators / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D008024:Ligands; D061986:MCF-7 Cells; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors*; D011743:Pyrimidines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D011972:Receptor, Insulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047368:Tumor Burden / Q000187:drug effects; D015854:Up-Regulation; D023041:Xenograft Model Antitumor Assays
chemical_list: D020847:Estrogen Receptor Modulators; D007334:Insulin-Like Growth Factor I; D008024:Ligands; D019869:Phosphatidylinositol 3-Kinases; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D011743:Pyrimidines; D011758:Pyrroles; D020794:Receptor Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin; D011960:Receptors, Estrogen; C575618:capivasertib