ABSTRACT: The role of androgen in breast cancer development is not fully understood although androgen receptors (AR) have been identified in breast cancer clinical samples and cell lines. However the whole spectra of androgen actions cannot be accounted to the classic AR mode of action and the possible existence of a cell surface AR has been suggested. Indeed androgens like all steroids have been reported to trigger membrane initiated signaling activity and exert specific actions. Androgens acting on the membrane can rapidly activate kinase signaling pathways and ultimately could affect gene expression. However, the molecular nature of membrane androgen binding sites represents another major persisting question. In the present study, we investigated early transcriptional effects of testosterone and the impermeable testosterone-BSA conjugate, in two breast cancer cell lines, in an attempt to decipher specific genes modified in each case, providing evidences about specific membrane initiating actions. Our data indicate that the two agents tested affect the expression of several genes. A group of genes were commonly affected while others were uniquely modified by each agent. In MDA-MB-231 cells, that are AR negative, the majority of genes affected by testosterone were also affected by testosterone-BSA indicating a membrane action. Subsequent analysis revealed that the two agents trigger different molecular pathways and cellular/molecular functions, suggestive of a molecular heterogeneity of membrane and intracellular AR. In addition, the phenotypic interactions of membrane-acting androgen with growth factor were verified at the transcriptomic level. Finally an interesting interplay between membrane-acting androgen with inflammation-related molecules, with potential clinical implications was revealed. Experiment Overall Design: Cell lines and culture conditions Experiment Overall Design: The hormone-sensitive breast cancer cell line T47D (expressing ERα, ERβ and AR) and the hormone insensitive breast cancer cell line MDA-MB-231 (expressing only ERβ) were purchased from the European Collection of Cell Cultures (Salisbury, UK). T47D cells and MDA-MB-231 were cultured in RPMI-1640 medium, supplemented with 10% charcoal treated fetal bovine serum (FBS), in a humidified atmosphere of 5% CO2 in air, at 37°C. Culture media and serum were from Gibco BRL (Life Technologies, Paisley, UK). Experiment Overall Design: Treatment- Gene expression assay Experiment Overall Design: Cells were serum starved for 6 hours and then treated with 10-7M Testosterone or charcoal-treated Testosterone-BSA, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Düren, Germany), labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.
