title: Insulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER+ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim.
abstract: BACKGROUND
  In this pre-clinical in vitro study conducted in estrogen receptor positive (ER+) breast cancer cells, we have characterized the effects of insulin-like growth factor I (IGF-1) on the cytostatic and cytotoxic action of antiestrogen treatment when used as a single agent or in combination with the antiprogestin mifepristone (MIF). Our goal was to identify new molecular targets to improve the efficacy of hormonal therapy in breast cancer patients that have a poor response to hormonal therapy, in part, due to high circulating levels of unbound insulinIGF-1.
  
  
  METHODS
  IGF-1-mediated effects on cytostasis and apoptotic cell death were determined with cell counts conducted in the presence and absence of trypan blue; enzyme-linked immunosorbent assays to determine the intracellular levels of cleaved cytokeratin 18, a marker of epithelial cancer cell apoptosis; and immunoblot analysis to determine the levels of cleaved poly-ADP ribose polymerase (PARP) and lamin A that result from caspase-dependent apoptosis. Cytotoxicity was further characterized by determination of the levels of reactive oxygen species (ROS) and the percent of mitochondrial membrane depolarization in cell populations treated with the different hormones in the presence and absence of IGF-1. Small molecule inhibitors of the dual-specificity protein kinase MEK1, MEK1 siRNA, Bim siRNA, and vectors overexpressing MEK1 wild type and mutant, dominant negative cDNA were used to identify key IGF-1 downstream prosurvival effectors.
  
  
  RESULTS
  IGF-1, at physiologically relevant levels, blocked the cytotoxic action(s) of the antiestrogens 4-hydroxytamoxifen (4-OHT) and tamoxifen (TAM) when used as single agents or in combination with the antiprogestin MIF. The antiapoptotic action of IGF-1 was mediated primarily through the action of MEK1. MEK1 expression reduced the levels of ROS and mitochondrial membrane depolarization induced by the hormonal treatments via a mechanism that involved the phosphorylation and proteasomal turnover of the proapoptotic BH3-only Bcl-2 family member Bim. Importantly, small-molecule inhibitors of MEK1 circumvented the prosurvival action of IGF-1 by restoring Bim to levels that more effectively mediated apoptosis in ER+ breast cancer cells.
  
  
  CONCLUSIONS
  his study provides strong support for the use of MEK1 inhibitors in combination with hormonal therapy to effectively affect cytostasis and activate a Bim-dependent apoptotic pathway in ER+ breast cancer cells. We discuss that MEK1 blockade may be a particularly effective treatment for women with high circulating levels of IGF-1, which have been correlated to a poor prognosis.
authors.affiliation: Department of Cellular Biology and Anatomy, Georgia Health Sciences University, 1459 Laney Walker Blvd,, Augusta, GA 30912, USA.
authors.forename: Sudharsan
authors.initials: S
authors.lastname: Periyasamy-Thandavan
authors.forename: Suchreet
authors.initials: S
authors.lastname: Takhar
authors.forename: Adam
authors.initials: A
authors.lastname: Singer
authors.forename: Michael Robert
authors.initials: MR
authors.lastname: Dohn
authors.forename: William Hutch
authors.initials: WH
authors.lastname: Jackson
authors.forename: April Eve
authors.initials: AE
authors.lastname: Welborn
authors.forename: Derek
authors.initials: D
authors.lastname: LeRoith
authors.forename: Mario
authors.initials: M
authors.lastname: Marrero
authors.forename: Muthusamy
authors.initials: M
authors.lastname: Thangaraju
authors.forename: Shuang
authors.initials: S
authors.lastname: Huang
authors.forename: Patricia Veronica
authors.initials: PV
authors.lastname: Schoenlein
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D020847:Estrogen Receptor Modulators / Q000494:pharmacology*; D005260:Female; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism* / Q000494:pharmacology; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D015735:Mifepristone / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D004791:Enzyme Inhibitors; D004965:Estrogen Antagonists; D020847:Estrogen Receptor Modulators; D006727:Hormone Antagonists; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D017382:Reactive Oxygen Species; D011960:Receptors, Estrogen; D013629:Tamoxifen; C016601:afimoxifene; D015735:Mifepristone; D007334:Insulin-Like Growth Factor I; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human