Inflammatory Breast Cancer (IBC) is an aggressive form of locally advanced breast cancer. At the moment of diagnosis, almost all patients have lymph node involvement and 1/3 has metastasis in distant organs. In order to characterize IBC at a molecular level, genome-wide gene expression analysis has been carried out.
For that purpose, RNA has been purified from 16 IBC tumour specimen and 18 non-IBC tumour specimen. This RNA was then reverse transcribed, amplified and labeled for subsequent analysis using cDNA microarrays. This technique is a miniaturized hybridization assay for studying the expression of thousands of nucleic acid fragments simultaneously. Microarrays consist of a collection of nucleic acid sequences, mostly Expressed Sequence Tags, immobilized onto a solid support so that each unique sequence forms a tiny feature, called a “spot” or “target”. A glass slide acts as the solid support onto which up to tens of thousands of spots, each spot representing a single gene, can be arrayed in a total area of a few square centimeters. During hybridization, labeled RNA, derived from the experimental samples, forms duplexes with their immobilized complementary targets. The number of duplexes formed, in every spot, reflects the relative amount of RNA, specific for a single gene, present in the sample. If a high concentration of RNA from a certain gene is present in the sample, this will result in the formation of many duplexes and hence a strong signal at the spot representing this gene, whereas low concentrations of RNA from a certain gene will result in a weak signal. Microarrays are then scanned using lasers and the intensity of the signal in every spot is quantified. Gene-expression data are generated and analyzed using a range of statistical techniques such as hierarchical clustering, supervised statistical analysis, self organizing maps, principle component analysis and others.
Using a two-dimensional hierarchical clustering method we demonstrated that IBC and non-IBC are characterized by a distinct gene expression signature, which was shown by the fact that all IBC tumour specimen clustered together in one group, separated from another cluster containing the non-IBC tumour specimen. Detailed analysis of the genes with different expression between the two groups under study revealed some biological processes more active in IBC compared to non-IBC: inflammatory response, immune response, cell-matrix adhesion, chemotaxis and activation of the NF-kappaB transcription factors. The latter was demonstrated by an increase of NF-kappaB target genes in IBC as well as an increase of upstream activators of the transcription factor.
NF-kappaB is a known mediator of the inflammatory process and also induces genes involved in the regulation of cellular motility and chemoattraction. This fits the picture of IBC, being an aggressive cancer with high metastatic potential and inflammatory symptoms at the time of diagnosis. Our genome-wide expression analysis also indicated that IBC probably belongs to the breast basal-cell phenotype, described by Perou et al (Nature, 2002). We found several markers, characterizing the breast basal-cell phenotype, upregulated in IBC. On the other hand, non-IBC was characterized by a strong expression of markers belonging to the ER positive/ luminal cell phenotype.
Previous findings have been validated using quantitative Real Time RT-PCR, a more sensitive technique for gene expression analysis of single genes. Using this technique, we additionally demonstrated that Caveolin-1 and -2 expression is upregulated in IBC. Both genes encode membrane bound proteins, Caveolin-1 being an NF-kappaB target gene and Caveolin-2 being a marker for the breast basal-cell phenotype. Both genes are possibly involved in the regulation of cellular motility, again fitting the picture of IBC. Further validation of the involvement of NF-kappaB in IBC is currently under study.
Relevant publications:
- Van Laere SJ, Van der Auwera I, Van den Eynden GG, Elst HJ, Weyler J, Harris AL, van Dam P, Van Marck EA, Vermeulen PB, Dirix LY. 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.Clin Cancer Res. 2006 Jun 1;12(11 Pt 1):3249-56.
- Van Laere SJ, Van den Eynden GG, Van der Auwera I, Vandenberghe M, Van Dam P, Van Marck EA, Van Golen KL, Vermeulen PB and Dirix LY. Identification of cell-of-origin breast tumor subtypes in Inflammatory Breast Cancer by gene expression profiling. Breast Cancer Res Treat. 2005 Oct 27;1-13 Epub ahead of print.
- Van Laere S, Van der Auwera I, Van den Eynden GG, Fox SB, Bianchi F, Harris AL, van Dam P, Van Marck EA, Vermeulen PB and Dirix LY. Distinct molecular signature of inflammatory breast cancer by cDNA microarray analysis. Breast Cancer Res. Treat. 2005 Aug 22; [Epub ahead of print].
