title: Nuclear factor-kappaB signature of inflammatory breast cancer by cDNA microarray validated by quantitative real-time reverse transcription-PCR, immunohistochemistry, and nuclear factor-kappaB DNA-binding.
abstract: OBJECTIVE
  Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer with high metastatic potential. In a previous study, we showed that IBC is a different form of breast cancer compared with non-IBC by cDNA microarray analysis. A list of 756 genes with significant expression differences between IBC and non-IBC was identified. In-depth functional analysis revealed the presence of a high number of nuclear factor-kappaB (NF-kappaB) target genes with elevated expression in IBC versus non-IBC. This led to the hypothesis that NF-kappaB contributes to the phenotype of IBC. The aim of the present study was to further investigate the role of NF-kappaB in IBC.
  
  
  METHODS
  Immunohistochemistry and NF-kappaB DNA-binding experiments were done for all NF-kappaB subunits (RelA, RelB, cRel, NFkB1, and NFkB2) using IBC and non-IBC specimens. Transcriptionally active NF-kappaB dimers were identified by means of coexpression analysis. In addition, quantitative real-time reverse transcription-PCR for eight NF-kappaB target genes, selected upon a significant, 3-fold gene expression difference between IBC and non-IBC by cDNA microarray analysis, was done.
  
  
  RESULTS
  We found a significant overexpression for all of eight selected NF-kappaB target genes in IBC compared with non-IBC by quantitative real-time reverse transcription-PCR. In addition, we found a statistically elevated number of immunostained nuclei in IBC compared with non-IBC for RelB (P = 0.038) and NFkB1 (P < 0.001). Immunohistochemical data were further validated by NF-kappaB DNA-binding experiments. Significant correlations between immunohistochemical data and NF-kappaB DNA binding for RelA, RelB, NFkB1, and NFkB2 were found. Transcriptionally active NF-kappaB dimers, composed of specific combinations of NF-kappaB family members, were found in 19 of 44 IBC specimens compared with 2 of 45 non-IBC specimens (P < 0.001). In addition, we found evidence for an estrogen receptor (ER)-mediated inhibition of the NF-kappaB signaling pathway. NF-kappaB target genes were significantly elevated in ER- versus ER+ breast tumors. Also, the amount of immunostained nuclei for RelB (P = 0.025) and NFkB1 (P = 0.031) was higher in ER- breast tumors versus ER+ breast tumors.
  
  
  CONCLUSIONS
  The NF-kappaB transcription factor pathway probably contributes to the phenotype of IBC and possibly offers new options for treatment of patients diagnosed with this aggressive form of breast cancer.
authors.affiliation: Translational Cancer Research Group (Laboratory of Pathology, University of Antwerp and Oncology Center, General Hospital Sint-Augustinus), Wilrijk, Belgium.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Van Laere
authors.forename: Ilse
authors.initials: I
authors.lastname: Van der Auwera
authors.forename: Gert G
authors.initials: GG
authors.lastname: Van den Eynden
authors.forename: Hilde J
authors.initials: HJ
authors.lastname: Elst
authors.forename: Joost
authors.initials: J
authors.lastname: Weyler
authors.forename: Adrian L
authors.initials: AL
authors.lastname: Harris
authors.forename: Peter
authors.initials: P
authors.lastname: van Dam
authors.forename: Eric A
authors.initials: EA
authors.lastname: Van Marck
authors.forename: Peter B
authors.initials: PB
authors.lastname: Vermeulen
authors.forename: Luc Y
authors.initials: LY
authors.lastname: Dirix
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D007249:Inflammation / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D016328:NF-kappa B