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

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

Affiliations

PMID: 18067675
PMCID: PMC2246188
DOI: 10.1186/bcr1834

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

Matthew J Smalley et al. Breast Cancer Res. 2007.

Abstract

Introduction: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

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Figures

Figure 1

Isolation and characterisation of mammary epithelial cell subpopulations. (a) Flow cytometric staining profiles (dead and CD45+ cells excluded) of anti-Sca-1 and 33A10 stained, freshly isolated mouse mammary cell preparations together with nonspecific IgG-stained control. (b) Graphical space representation of principal component analysis of mammary fibroblasts, Sca-1+ 33A10Low/-, Sca-1- 33A10High and Sca-1- 33A10Low/- cells. (c) Mean fold differences ± 95% confidence limits in RNA abundance measured by quantitative real-time PCR for estrogen receptor (Esr1) and prolactin receptor (Prlr) transcripts in Sca-1+33A10Low/- (n = 5 samples) and Sca-1- 33A10High (n = 3 samples) mammary subpopulations compared with bulk mammary cell preparations depleted for CD45+ cells (comparator; n = 3 samples). The dotted lines indicate the 95% confidence limits of the comparator sample. All samples show a significant difference to the comparator (**P < 0.01) [35].

Figure 2

Rgs2 is highly expressed in CD24Low Sca-1- 33A10- mouse mammary basal/myoepithelial and human breast myoepithelial cells. (a) Flow cytometric staining profile of mouse mammary cell preparations stained with anti-Sca-1 and anti-CD24 antibodies together with either a nonspecific rat IgG and anti-rat-FITC or 33A10 and anti-rat FITC. The nonspecific IgG and 33A10 staining profiles of the CD24Low Sca-1- basal/myoepithelial cells (33A10-), CD24High Sca-1- (Esr1-) luminal cells (33A10High) and CD24High Sca-1+ (Esr1+) luminal cells (33A10Low) [14] are indicated. (b) Mean fold differences ± 95% confidence limits in RNA abundance for the Rgs2 gene in CD24Low Sca-1- basal/myoepithelial, CD24High Sca-1- (Esr1-) luminal epithelial and CD24High Sca-1+ (Esr1+) luminal epithelial mouse mammary cells (n = 3 for all samples) [14]. The dotted lines indicate the 95% confidence limits of the comparator sample. All samples show a significant difference to the comparator (**P < 0.01). The basal myoepithelial cells also have a significantly higher level of Rgs2 expression than either of the two luminal populations. (c) Mean fold differences ± 95% confidence limits in expression levels for the RGS2 gene in myoepithelial and luminal epithelial human breast cells compared with human breast fibroblasts (comparator). See Materials and methods for details of the samples. The dotted lines indicate the 95% confidence limits of the comparator sample. Both samples show a significant difference to the comparator (**P < 0.01) and the myoepithelial cells have a significantly higher level of RGS2 expression than the luminal cells.

Figure 3

RGS2 overexpression attenuates oxytocin receptor signalling to p44/42 MAPK. (a) quantitative real-time PCR analysis of RGS2 gene expression in triplicate samples of stably transfected Hs578T-pcDNA (parental vector) compared with Hs578T-RGS2 cells. Mean expression levels ± 95% confidence limits are shown. Significant differences are indicated (**P < 0.01). RGS2 was fivefold overexpressed in the Hs578T-RGS2 cells compared with the control cell line. (b) Analysis of calcium flux in Hs578T-pcDNA and Hs578T-RGS2 cells stimulated with 5 × 10-7 mol/l oxytocin. Both cell lines showed an identical response to the stimulus. (c) Time course analysis of p44/42 mitogen-activated protein kinase (MAPK) phosphorylation in Hs578T-pcDNA and Hs578T-RGS2 cells stimulated with 5 × 10-7 mol/l oxytocin. (d) Quantitation of phosphorylation analysis. p44/42 MAPK phosphorylation was significantly reduced (P < 0.001) in the Hs578T-RGS2 cell line compared with the control Hs578T-pcDNA cells.

Figure 4

Silencing of endogenous RGS2 enhances oxytocin receptor signalling via p44/42 MAPK in Hs578T cells. (a) Hs578T cells transfected with either a scrambled control small interfering (si)RNA (SiCON) or an siRNA targeting RGS2 (siRGS) were serum and insulin starved for 2 hours then stimulated with 1 × 10-7 mol/l oxytocin. Cells were harvested at timepoints up to 120 minutes after oxytocin addition and lysates analysed for levels of phospho- and total p44/42 mitogen-activated protein kinase (MAPK) by immunoblotting. (b) Quantitation of siRNA knockdown from two independent transfections. The upper plot is the quantitation of the blots shown in panel a. siRGS2 caused a significant (P < 0.001) increase in phosphorylation in response to oxytocin. (c) Quantitative real-time PCR analysis of RGS2 expression in Hs578T cells transfected with the siRGS2. Each bar represents the mean ± 95% confidence limits of the fold difference in expression compared with the mean expression in the siCON transfected cells. Data from four samples harvested from two independent transfections is shown (one of which was also used in the lower oxytocin response experiment shown in panel b). Significant differences are indicated (**P < 0.01).

Figure 5

RGS2 is expressed in human myoepithelial and luminal cells and in breast cancers. (a) RNA isolated from normal primary breast cells, normal and breast cancer cell lines and primary breast cancers was analysed by semi-quantitative RT-PCR for expression at the transcriptional level of RGS2, OXR and a housekeeping gene B2M. (b) Quantitative real-time PCR analysis of RGS2 expression levels in a selection of primary human cells, human breast cancer cell lines, solid breast cancers and F19-depleted cancers. Data are mean relative expression levels ± 95% confidence limits (n = 3 analyses of each sample). For comparison, the primary myoepithelial and primary luminal cell data from Figure 2c have been included on this graph. Note that the y-axis is a log10 scale.

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