title: Maintenance of S-nitrosothiol homeostasis plays an important role in growth suppression of estrogen receptor-positive breast tumors.
abstract: BACKGROUND
  Protein denitrosylation by thioredoxin reductase (TrxR) is key for maintaining S-nitrosothiol (SNO) homeostasis, although its role in tumor progression is unknown. Therefore, the present study aimed to assess the role of altered SNO homeostasis in breast cancer cells.
  
  
  METHODS
  The impairment of SNO homeostasis in breast cancer cells was achieved with the highly specific TrxR inhibitor auranofin and/or exposure to S-nitroso-L-cysteine. S-nitrosylated proteins were detected using the biotin switch assay. Estrogen receptor (ER) alpha knockdown was achieved using RNA silencing technologies and subcellular localization of ERα was analyzed by confocal microscopy. The Oncomine database was explored for TrxR1 (TXNRD1) expression in breast tumors and TrxR1, ER and p53 expression was analyzed by immunohistochemistry in a panel of breast tumors.
  
  
  RESULTS
  The impairment of SNO homeostasis enhanced cell proliferation and survival of ER+ MCF-7 cells, but not of MDA-MB-231 (ER-, mut p53) or BT-474 (ER+, mut p53) cells. This enhanced cell growth and survival was associated with Akt, Erk1/2 phosphorylation, and augmented cyclin D1 expression and was abolished by the ER antagonist fulvestrant or the p53 specific inhibitor pifithrin-α. The specific silencing of ERα expression in MCF-7 cells also abrogated the growth effect of TrxR inhibition. Estrogenic deprivation in MCF-7 cells potentiated the pro-proliferative effect of impaired SNO homeostasis. Moreover, the subcellular distribution of ERα was altered, with a predominant nuclear localization associated with phosphorylation at Thr311 in those cells with impaired SNO homeostasis. The impairment of SNO homeostasis also expanded a cancer stem cell-like subpopulation in MCF-7 cells, as indicated by the increase of percentage of CD44+ cells and the augmented capability to form mammospheres in vitro. Notably, ER+ status in breast tumors was significantly associated with lower TXNDR1 mRNA expression and immunohistochemical studies confirmed this association, particularly when p53 abnormalities were absent.
  
  
  CONCLUSIONS
  The ER status in breast cancer may dictate tumor response to different nitrosative environments. Impairment of SNO homeostasis confers survival advantages to ER+ breast tumors, and these molecular mechanisms may also participate in the development of resistance against hormonal therapies that arise in this type of mammary tumors.
authors.forename: Amanda
authors.initials: A
authors.lastname: Cañas
authors.forename: Laura M
authors.initials: LM
authors.lastname: López-Sánchez
authors.forename: Araceli
authors.initials: A
authors.lastname: Valverde-Estepa
authors.forename: Vanessa
authors.initials: V
authors.lastname: Hernández
authors.forename: Elena
authors.initials: E
authors.lastname: Fuentes
authors.forename: Juan R
authors.initials: JR
authors.lastname: Muñoz-Castañeda
authors.forename: Chary
authors.initials: C
authors.lastname: López-Pedrera
authors.forename: Juan R
authors.initials: JR
authors.lastname: De La Haba-Rodríguez
authors.forename: Enrique
authors.initials: E
authors.lastname: Aranda
authors.forename: Antonio
authors.initials: A
authors.lastname: Rodríguez-Ariza
mesh_terms: D018931:Antineoplastic Agents, Hormonal / Q000627:therapeutic use*; D018501:Antirheumatic Agents / Q000494:pharmacology; D001310:Auranofin / Q000494:pharmacology; D052160:Benzothiazoles / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy / Q000473:pathology*; D051927:CD24 Antigen / Q000096:biosynthesis; D049109:Cell Proliferation; D002470:Cell Survival; D019938:Cyclin D1 / Q000096:biosynthesis; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D000077267:Fulvestrant; D006706:Homeostasis; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D061986:MCF-7 Cells; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering; D026403:S-Nitrosothiols / Q000737:chemistry* / Q000494:pharmacology; D018874:Spheroids, Cellular; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014050:Toluene / Q000031:analogs & derivatives / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000096:biosynthesis
chemical_list: D018931:Antineoplastic Agents, Hormonal; D018501:Antirheumatic Agents; D052160:Benzothiazoles; C530104:CCND1 protein, human; D051927:CD24 Antigen; C497889:CD24 protein, human; C497483:CD44 protein, human; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D018960:Hyaluronan Receptors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D026403:S-Nitrosothiols; D016159:Tumor Suppressor Protein p53; D019938:Cyclin D1; D000077267:Fulvestrant; D014050:Toluene; D001310:Auranofin; D004958:Estradiol; C021315:S-nitrosocysteine; C121565:pifithrin; D013880:Thioredoxin-Disulfide Reductase; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D003545:Cysteine