Gene expression signature of estrogen receptor alpha status in breast cancer

Abstract

Background: Estrogens are known to regulate the proliferation of breast cancer cells and to modify their phenotypic properties. Identification of estrogen-regulated genes in human breast tumors is an essential step toward understanding the molecular mechanisms of estrogen action in cancer. To this end we generated and compared the Serial Analysis of Gene Expression (SAGE) profiles of 26 human breast carcinomas based on their estrogen receptor alpha (ER) status. Thus, producing a breast cancer SAGE database of almost 2.5 million tags, representing over 50,000 transcripts.

Results: We identified 520 transcripts differentially expressed between ERalpha-positive (+) and ERalpha-negative (-) primary breast tumors (Fold change >or= 2; p < 0.05). Furthermore, we identified 220 high-affinity Estrogen Responsive Elements (EREs) distributed on the promoter regions of 163 out of the 473 up-modulated genes in ERalpha (+) breast tumors. In brief, we observed predominantly up-regulation of cell growth related genes, DNA binding and transcription factor activity related genes based on Gene Ontology (GO) biological functional annotation. GO terms over-representation analysis showed a statistically significant enrichment of various transcript families including: metal ion binding related transcripts (p = 0.011), calcium ion binding related transcripts (p = 0.033) and steroid hormone receptor activity related transcripts (p = 0.031). SAGE data associated with ERalpha status was compared with reported information from breast cancer DNA microarrays studies. A significant proportion of ERalpha associated gene expression changes was validated by this cross-platform comparison. However, our SAGE study also identified novel sets of genes as highly expressed in ERalpha (+) invasive breast tumors not previously reported. These observations were further validated in an independent set of human breast tumors by means of real time RT-PCR.

Conclusion: The integration of the breast cancer comparative transcriptome analysis based on ERalpha status coupled to the genome-wide identification of high-affinity EREs and GO over-representation analysis, provide useful information for validation and discovery of signaling networks related to estrogen response in this malignancy.

Figure 1

Real time RT-PCR validation of nine over-expressed genes in 36 invasive breast carcinomas. a) SCUBE2 (p = 0.0001); b) SYTL4 (p = 0.0005); c) KIAA0882 (p = 0.0005); d) TSPAN1 (p = 0.001); e) CMYB (p = 0.002); f) CELSR2 (p = 0.011); g) NR4A1 (p = 0.029); h) ENO2 (p = 0.033); i) LGALS3BP (p = 0.079). Mean ± 2 Standard Error based on Log2 transformation of real time RT-PCR values of the assayed gene relative to 18S rRNA used as normalizing control.

Figure 2

GO classification of the ERα associated genes identified by SAGE. Percent of coverage representing the percentage of genes annotated with a specific GO term related to Biological Processes (blue bars) and Molecular Function (yellow bars).

Figure 3

High-affinity EREs in ERα (+) up-modulated genes (n = 163). a) Percentage of genes according to number of EREs. b) Distribution of EREs in 5' (blue bars) and 3' (aquamarine bars) regions relative to the TSS (-10 to + 5 kb). Each bar represents an interval width of 500 bp.

Figure 4

Cross-platform comparisons of the up-modulated transcripts in ERα (+) breast carcinomas. One hundred and eighty-three genes were identified by more than one study, eleven of which were commonly identified across the three platforms. a) Comparison between SAGE and oligonucleotide microarray platforms [12] showing a highly significant number of overlapping genes (p < 0.001) (see table 2). b) Comparison between SAGE and cDNA microarray platforms [13] (p > 0.05). c) Statistically significant number of overlapping genes identified by both DNA microarrays platforms (p < 0.01).