title: Fulvestrant induces resistance by modulating GPER and CDK6 expression: implication of methyltransferases, deacetylases and the hSWI/SNF chromatin remodelling complex.
abstract: BACKGROUND
  Breast cancer is the leading cause of cancer death in women living in the western hemisphere. Despite major advances in first-line endocrine therapy of advanced oestrogen receptor (ER)-positive breast cancer, the frequent recurrence of resistant cancer cells represents a serious obstacle to successful treatment. Understanding the mechanisms leading to acquired resistance, therefore, could pave the way to the development of second-line therapeutics. To this end, we generated an ER-positive breast cancer cell line (MCF-7) with resistance to the therapeutic anti-oestrogen fulvestrant (FUL) and studied the molecular changes involved in resistance.
  
  
  METHODS
  Naive MCF-7 cells were treated with increasing FUL concentrations and the gene expression profile of the resulting FUL-resistant strain (FR.MCF-7) was compared with that of naive cells using GeneChip arrays. After validation by real-time PCR and/or western blotting, selected resistance-associated genes were functionally studied by siRNA-mediated silencing or pharmacological inhibition. Furthermore, general mechanisms causing aberrant gene expression were investigated.
  
  
  RESULTS
  Fulvestrant resistance was associated with repression of GPER and the overexpression of CDK6, whereas ERBB2, ABCG2, ER and ER-related genes (GREB1, RERG) or genes expressed in resistant breast cancer (BCAR1, BCAR3) did not contribute to resistance. Aberrant GPER and CDK6 expression was most likely caused by modification of DNA methylation and histone acetylation, respectively. Therefore, part of the resistance mechanism was loss of RB1 control. The hSWI/SNF (human SWItch/Sucrose NonFermentable) chromatin remodelling complex, which is tightly linked to nucleosome acetylation and repositioning, was also affected, because as a stress response to FUL treatment-naive cells altered the expression of five subunits within a few hours (BRG1, BAF250A, BAF170, BAF155, BAF47). The aberrant constitutive expression of BAF250A, BAF170 and BAF155 and a deviant stress response of BRG1, BAF170 and BAF47 in FR.MCF-7 cells to FUL treatment accompanied acquired FUL resistance. The regular and aberrant expression profiles of BAF155 correlated directly with that of CDK6 in naive and in FR.MCF-7 cells corroborating the finding that CDK6 overexpression was due to nucleosome alterations.
  
  
  CONCLUSIONS
  The study revealed that FUL resistance is associated with the dysregulation of GPER and CDK6. A mechanism leading to aberrant gene expression was most likely unscheduled chromatin remodelling by hSWI/SNF. Hence, three targets should be conceptually addressed in a second-line adjuvant therapy: the catalytic centre of SWI/SNF (BRG1) to delay the development of FUL resistance, GPER to increase sensitivity to FUL and the reconstitution of the RB1 pathway to overcome resistance.
authors.affiliation: Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
authors.forename: B
authors.initials: B
authors.lastname: Giessrigl
authors.forename: W M
authors.initials: WM
authors.lastname: Schmidt
authors.forename: M
authors.initials: M
authors.lastname: Kalipciyan
authors.forename: M
authors.initials: M
authors.lastname: Jeitler
authors.forename: M
authors.initials: M
authors.lastname: Bilban
authors.forename: M
authors.initials: M
authors.lastname: Gollinger
authors.forename: S
authors.initials: S
authors.lastname: Krieger
authors.forename: W
authors.initials: W
authors.lastname: Jäger
authors.forename: R M
authors.initials: RM
authors.lastname: Mader
authors.forename: G
authors.initials: G
authors.lastname: Krupitza
mesh_terms: D018931:Antineoplastic Agents, Hormonal / Q000627:therapeutic use*; D001943:Breast Neoplasms / Q000188:drug therapy*; D017024:Chemotherapy, Adjuvant; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D051361:Cyclin-Dependent Kinase 6 / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004958:Estradiol / Q000031:analogs & derivatives* / Q000627:therapeutic use; D005260:Female; D000077267:Fulvestrant; D015972:Gene Expression Regulation, Neoplastic; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D008780:Methyltransferases / Q000378:metabolism; D010879:Piperazines / Q000627:therapeutic use; D011725:Pyridines / Q000627:therapeutic use; D011960:Receptors, Estrogen / Q000235:genetics*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D018931:Antineoplastic Agents, Hormonal; D002868:Chromosomal Proteins, Non-Histone; C109584:GPER1 protein, human; D010879:Piperazines; D011725:Pyridines; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled; C445179:SWI-SNF-B chromatin-remodeling complex; D014157:Transcription Factors; D000077267:Fulvestrant; D004958:Estradiol; D008780:Methyltransferases; C495911:CDK6 protein, human; D051361:Cyclin-Dependent Kinase 6; D006655:Histone Deacetylases; C500026:palbociclib