title: Differential impact of EGFR-targeted therapies on hypoxia responses: implications for treatment sensitivity in triple-negative metastatic breast cancer.
abstract: BACKGROUND
  In solid tumors, such as breast cancer, cells are exposed to hypoxia. Cancer cells adapt their metabolism by activating hypoxia-inducible factors (HIFs) that promote the transcription of genes involved in processes such as cell survival, drug resistance and metastasis. HIF-1 is also induced in an oxygen-independent manner through the activation of epidermal growth factor receptor tyrosine kinase (EGFR-TK). Triple-negative breast cancer (TNBC) is a subtype of invasive breast cancer characterized by negative expression of hormonal and HER2 receptors, and this subtype generally overexpresses EGFR. Sensitivity to three EGFR inhibitors (cetuximab, gefitinib and lapatinib, an HER2/EGFR-TK inhibitor) was evaluated in a metastatic TNBC cell model (MDA-MB-231), and the impact of these drugs on the activity and stability of HIF was assessed.
  
  
  RESULTS
  MDA-MB-231 cells were genetically modified to stably express an enhanced green fluorescent protein (EGFP) induced by hypoxia; the Ca9-GFP cell model reports HIF activity, whereas GFP-P564 reports HIF stability. The reporter signal was monitored by flow cytometry. HIF-1 DNA-binding activity, cell migration and viability were also evaluated in response to EGFR inhibitors. Cell fluorescence signals strongly increased under hypoxic conditions (> 30-fold). Cetuximab and lapatinib did not affect the signal induced by hypoxia, whereas gefitinib sharply reduced its intensity in both cell models and also diminished HIF-1 alpha levels and HIF-1 DNA-binding activity in MDA-MB-231 cells. This gefitinib feature was associated with its ability to inhibit MDA-MB-231 cell migration and to induce cell mortality in a dose-dependent manner. Cetuximab and lapatinib had no effect on cell migration or cell viability.
  
  
  CONCLUSIONS
  Resistance to cetuximab and lapatinib and sensitivity to gefitinib were associated with their ability to modulate HIF activity and stability. In conclusion, downregulation of HIF-1 through EGFR signaling seems to be required for the induction of a positive response to EGFR-targeted therapies in TNBC.
authors.affiliation: Department of Oncogenetic, Centre Jean Perrin, Clermont-Ferrand, France.
authors.forename: Abderrahim
authors.initials: A
authors.lastname: El Guerrab
authors.forename: Rabah
authors.initials: R
authors.lastname: Zegrour
authors.forename: Carine-Christiane
authors.initials: CC
authors.lastname: Nemlin
authors.forename: Flavie
authors.initials: F
authors.lastname: Vigier
authors.forename: Anne
authors.initials: A
authors.lastname: Cayre
authors.forename: Frederique
authors.initials: F
authors.lastname: Penault-Llorca
authors.forename: Fabrice
authors.initials: F
authors.lastname: Rossignol
authors.forename: Yves-Jean
authors.initials: YJ
authors.lastname: Bignon
mesh_terms: D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement; D002470:Cell Survival; D000068818:Cetuximab; D004247:DNA / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D066246:ErbB Receptors / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D000077156:Gefitinib; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D000860:Hypoxia; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism*; D000077341:Lapatinib; D008969:Molecular Sequence Data; D009362:Neoplasm Metastasis; D011799:Quinazolines / Q000627:therapeutic use; D014158:Transcription, Genetic
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D051793:Hypoxia-Inducible Factor 1; D011799:Quinazolines; D000077341:Lapatinib; D049452:Green Fluorescent Proteins; D004247:DNA; D066246:ErbB Receptors; D000068818:Cetuximab; D000077156:Gefitinib