Genomic signatures of pregnancy-associated breast cancer epithelia and stroma and their regulation by estrogens and progesterone

Abstract

Pregnancy-associated breast cancers (PABC) generally present at advanced stages and have a poor prognosis. The reasons are unclear but we hypothesized that the continuous high levels of estrogens and progesterone were involved. We have now carried out a detailed analysis of PABC compared to tumors of age-matched nonpregnant (non-PABC) women. Malignant epithelia and tumor-associated stroma of PABC and non-PABC were isolated by laser capture microdissection and gene expression profiled. Additionally, normal breast epithelia and stroma adjacent to the two tumor types were analyzed. Lastly, subsets of previously identified E- and P-regulated genes were defined in all tissues. We find that PABC signatures cluster with established breast cancer subtypes. Major hormone-regulated genes whose expression correlated with epithelia of PABC dealt with regulation of cell proliferation, metabolism, and tumor aggressiveness, including genes used to predict tumor recurrence. Compared to normal epithelia, a significant number of genes associated with cell cycle processes were enriched in PABC, many of which are hormone regulated. Thus, compared to normal epithelia, many of the genes that were differentially expressed in epithelia of PABC were distinct from those differentially expressed in non-PABC. With regard to the tumor microenvironment, immune-related genes were enriched in tumor-associated stroma of PABC. Compared to normal stroma, PABC-associated stroma overexpressed immune response genes, while genes involved in angiogenesis and extracellular matrix deposition were more commonly downregulated. This suggests that the heightened aggressiveness of PABC may involve a predisposition to metastasis through extracellular matrix degradation, plus angiogenesis independence. Moreover, genes encoding cell proliferative factors, signaling, immunomodulators and cell death, were hormone regulated in stroma. In sum, these analyses demonstrate complex patterns of enrichment and hormonal regulation of genes in PABC and suggest that it may have a distinct biological nature.

Fig. 1

Unsupervised hierarchical clustering reveals samples cluster primarily on tumor status and cell type. a Unsupervised hierarchical clustering of human breast tumor samples analyzed using Affymetrix HG-U133 Plus 2.0 gene chips as described in the “Methods” section. To avoid redundancy, a single probe set was used for each gene. b Hierarchal clustering of 20,761 genes revealed three main groups: “normal”, “tumor associated stroma”, and “epithelial tumor”. A scaled-down representation of the entire cluster is shown. Branches are color coded according to the group with which the corresponding tumor sample showed the highest correlation. Samples are also color coded based on normal (pink) versus tumor (red), epithelial (blue) versus stroma (purple), PABC (orange) versus non-PABC (green), and ER-positive (gray) versus ER-negative status (white). c Dendogram showing PABC samples cluster with different breast cancer subtypes. Samples are color coded based on luminal A/B (blue), normal (light brown), basal (green), her2 (light pink)

Fig. 2

Subsets of E- and P-regulated genes clustered samples based on pregnancy status in epithelial tumors. Each column represents a single sample and each row a single gene. Red upregulated expression, green downregulated expression. a Hierarchal clustering of the most highly up- or downregulated differentially expressed genes (1,097) identified between PABC and non-PABC epithelium. b Hierarchal clustering of 113 E-regulated gene subset and c 78 P-regulated gene subset differentially expressed between PABC and non-PABC. E-and P-regulated genes were identified as described in the “Methods” section

Fig. 3

Venn diagram shows little overlap of hormone-regulated genes associated with tumor progression in the epithelium of PABC compared to non-PABC tumors. Venn diagrams display the number of genes associated with tumor progression in PABC (red) and non-PABC (blue) epithelium. The intersection indicates the number of probe sets that were changed by both experimental groups for a E-regulated genes and b P-regulated genes. Pie charts indicate the top functional categories represented among the hormone-regulated genes identified in each group. The bold lines indicate related functional categories

Fig. 4

Subsets of E- and P-regulated genes clustered samples based on pregnancy status in tumor associated stroma. Each column represents a single sample and each row a single gene. Red upregulated expression, green downregulated expression. a Hierarchal clustering of the most highly up- or downregulated differentially expressed genes (1,097) identified between PABC and non-PABC. b Hierarchal clustering of 25 E-regulated gene subset and c 20 P-regulated gene subset differentially expressed between PABC and non-PABC. E-and P-regulated genes were identified as described in the “Methods” section

Fig. 5

Venn diagram shows little overlap of hormone regulated genes associated with tumor progression in the stroma of PABC compared to non-PABC tumors. a Venn diagrams display the number of genes associated with tumor progression in the stroma of PABC (red) and non-PABC (blue). The intersection indicates the number of probe sets that were changed by both experimental groups for: a E-regulated genes and b P-regulated genes. Pie charts indicate the top functional categories represented among the hormone regulated genes identified in each group