Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers

Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers

Breast Cancer Research, December 2007

18067675

Matthew J Smalley, Marjan Iravani, Maria Leao, Anita Grigoriadis, Howard Kendrick, Tim Dexter, Kerry Fenwick, Joseph L Regan, Kara Britt, Sarah McDonald, Christopher J Lord, Alan MacKay, Alan Ashworth

To understand which signalling pathways become deregulated in breast cancer, it is necessary to identify functionally significant gene expression patterns in the stem, progenitor, transit amplifying and differentiated cells of the mammary epithelium. We have previously used the markers 33A10, CD24 and Sca-1 to identify mouse mammary epithelial cell subpopulations. We now investigate the relationship between cells expressing these markers and use gene expression microarray analysis to identify genes differentially expressed in the cell populations. Freshly isolated primary mouse mammary epithelial cells were separated on the basis of staining with the 33A10 antibody and an alpha-Sca-1 antibody. The populations identified were profiled using gene expression microarray analysis. Gene expression patterns were confirmed on normal mouse and human mammary epithelial subpopulations and were examined in a panel of breast cancer samples and cell lines. Analysis of the separated populations demonstrated that Sca-1- 33A10High stained cells were estrogen receptor alpha (Esr1)- luminal epithelial cells, whereas Sca-1+ 33A10Low/- stained cells were a mix of nonepithelial cells and Esr1+ epithelial cells. Analysis of the gene expression data identified the gene Rgs2 (regulator of G-protein signalling 2) as being highly expressed in the Sca-1- 33A10Low/- population, which included myoepithelial/basal cells. RGS2 has previously been described as a regulator of angiotensin II receptor signalling. Gene expression analysis by quantitative real-time RT-PCR of cells separated on the basis of CD24 and Sca-1 expression confirmed that Rgs2 was more highly expressed in mouse myoepithelial/basal mammary cells than luminal cells. This expression pattern was conserved in normal human breast cells. Functional analysis demonstrated RGS2 to be a modulator of oxytocin receptor signalling. The potential significance of RGS2 expression in breast cancer was demonstrated by semi-quantitative RT-PCR analysis, data mining and quantitative real-time RT-PCR approaches, which showed that RGS2 was expressed in the majority of solid breast cancers at much higher levels than in normal human mammary cells. Molecular analysis of prospectively isolated mammary epithelial cells identified RGS2 as a modulator of oxytocin receptor signalling, which is highly expressed in the myoepithelial cells. The RGS2 gene, but not the oxytocin receptor, was also shown to be over-expressed in the majority of breast cancers, identifying the product of this gene, or the pathway(s) it regulates, as potentially significant therapeutic targets.