title: Selective regain of egfr gene copies in CD44+/CD24-/low breast cancer cellular model MDA-MB-468.
abstract: BACKGROUND
  Increased transcription of oncogenes like the epidermal growth factor receptor (EGFR) is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies.
  
  
  METHODS
  The basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique.
  
  
  RESULTS
  Low and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain of egfr copies.
  
  
  CONCLUSIONS
  Progressive genome modulation in the CD44+/CD24-/low subpopulation of the breast cancer cell line MDA-MB-468 leads to different coexisting subclones. In isolated low-copy cells asymmetric chromosomal segregation leads to new cells with regained solely egfr gene copies. Furthermore, egfr regain resulted in enhanced signal transduction of the MAP-kinase and PI3-kinase pathway. We show here for the first time a dynamic copy number regain in basal-like/stemness cell type breast cancer subpopulations which might explain genetic heterogeneity. Moreover, this process might also be involved in adaptive growth factor receptor intracellular signaling which support survival and migration during cancer development and progression.
authors.affiliation: Department of Medicine, Hematology and Oncology, University of Muenster, Muenster, Germany. agelopoulos@uni-muenster.de
authors.forename: Konstantin
authors.initials: K
authors.lastname: Agelopoulos
authors.forename: Burkhard
authors.initials: B
authors.lastname: Greve
authors.forename: Hartmut
authors.initials: H
authors.lastname: Schmidt
authors.forename: Heike
authors.initials: H
authors.lastname: Pospisil
authors.forename: Stefan
authors.initials: S
authors.lastname: Kurtz
authors.forename: Kai
authors.initials: K
authors.lastname: Bartkowiak
authors.forename: Antje
authors.initials: A
authors.lastname: Andreas
authors.forename: Marek
authors.initials: M
authors.lastname: Wieczorek
authors.forename: Eberhard
authors.initials: E
authors.lastname: Korsching
authors.forename: Horst
authors.initials: H
authors.lastname: Buerger
authors.forename: Burkhard
authors.initials: B
authors.lastname: Brandt
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism*; D051927:CD24 Antigen / Q000096:biosynthesis*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D018628:Gene Dosage; D020869:Gene Expression Profiling; D014644:Genetic Variation; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis*; D007700:Kinetics; D020641:Polymorphism, Single Nucleotide; D015398:Signal Transduction
chemical_list: D051927:CD24 Antigen; D018960:Hyaluronan Receptors; D066246:ErbB Receptors