Transcriptional responses to estrogen and progesterone in mammary gland identify a role for extracellular matrix in modulating p53.

B121

Combined treatment with estrogen and progesterone mimics the effect of parity in preventing carcinogen-induced mammary tumors and acts through p53-dependent pathways. Acute treatment with these hormones enhances p53-dependent responses to ionizing radiation in the mammary epithelium in vivo. Analysis of transcriptional profiles was undertaken to identify the mechanisms by which estrogen and progesterone combine to sensitize p53 function in mammary epithelium. Methods: Mature BALB/c-Trp53^+/+ mice were ovariectomized. One week later, the mice were treated daily with either 2 μg 17-β-estradiol (E), 1 mg progesterone (P), both (E+P), or vehicle alone (Vehicle) for 4 days. An additional group, the mammary epithelium was surgically cleared and treated with E+P (CFP) to control for effects in the stroma. RNA was isolated from lymph node-free mammary glands. Expression profiles were determined using oligonucleotide microarrays. Primary cultures of mammary epithelial organoids were prepared by collagenase digestion of mammary tissues from BALB/c-Trp53^+/+ or BALB/c-Trp53^-/- mice. The organoids were embedded in a 3-D matrix of either (i) agarose (does not support epithelial attachments), (ii) Matrigel (supports epithelial attachments); or (iii) Matrigel with antibodies blocking β1-integrin. Results: A classification approach was used to define regulatory networks. Using 1.5-fold differences from Vehicle-treated as the threshold, a total of 3803 probe sets were observed differentially regulated by E and/or P and were distributed among 15 classes defining distinct transcriptional responses in the mammary gland. The most abundant classes of genes were those regulated by either E-alone or E+P. Expression patterns for representative genes from these classes were confirmed by Northern blot. Analysis of Gene Ontology annotations identified an over-representation of extracellular matrix (ECM) genes following treatment with E+P suggesting that signals from the ECM may modulate p53 responsiveness. Primary cultures of mammary epithelial organoids showed decreased p53-dependent apoptosis when embedded in Matrigel. The effect of Matrigel was blocked by β1-integrins antibodies. Conclusions: These results define the set of transcriptional responses in mammary tissue induced by estrogen and progesterone in vivo and which lead to enhanced responsiveness of p53 to ionizing radiation. The combined treatment with E+P resulted in significant changes in expression of genes associated with the ECM. Therefore, remodeling of the ECM and alterations in β1-integrin signaling appear to be mechanisms by which E+P enhance p53 activity in the mammary epithelium and contribute to hormone-mediated protection from mammary tumors.