AGTR1 overexpression defines a subset of breast cancer and confers sensitivity to losartan, an AGTR1 antagonist

Abstract

Breast cancer patients have benefited from the use of targeted therapies directed at specific molecular alterations. To identify additional opportunities for targeted therapy, we searched for genes with marked overexpression in subsets of tumors across a panel of breast cancer profiling studies comprising 3,200 microarray experiments. In addition to prioritizing ERBB2, we found AGTR1, the angiotensin II receptor type I, to be markedly overexpressed in 10-20% of breast cancer cases across multiple independent patient cohorts. Validation experiments confirmed that AGTR1 is highly overexpressed, in several cases more than 100-fold. AGTR1 overexpression was restricted to estrogen receptor-positive tumors and was mutually exclusive with ERBB2 overexpression across all samples. Ectopic overexpression of AGTR1 in primary mammary epithelial cells, combined with angiotensin II stimulation, led to a highly invasive phenotype that was attenuated by the AGTR1 antagonist losartan. Similarly, losartan reduced tumor growth by 30% in AGTR1-positive breast cancer xenografts. Taken together, these observations indicate that marked AGTR1 overexpression defines a subpopulation of ER-positive, ERBB2-negative breast cancer that may benefit from targeted therapy with AGTR1 antagonists, such as losartan.

Fig. 1.

MetaCOPA analysis of breast cancer gene expression data. (A) MetaCOPA map. Each column in the map represents a breast cancer gene expression dataset. The numbers at the base of the map correspond to dataset details (Table S1). Each row indicates a gene. A red cell indicates that the gene was deemed to have an outlier expression profile in the respective dataset because it scored in the top 1% of COPA values at 1 of 3 percentile cutoffs. The line graph along the y axis indicates the P value for a gene based on the number of datasets in which the gene was deemed an outlier. A total of 158 genes were called outliers in a significant fraction of datasets (P < 1E-5). The bar graph indicates the number of samples in the respective datasets and the contribution of the dataset to the meta-analysis. The black bar on the left of the map indicates the top 25 meta-outliers, which are detailed in B for 3 datasets marked with an asterisk. (B) Heatmaps of COPA-normalized values for top-scoring meta-outliers across 3 highly contributory datasets: Miller et al. (26), Hess et al. (27), and Wang et al. (28). Genes are ranked by their MetaCOPA P values. For each gene, samples are ordered from left to right by their COPA-normalized expression values. Highest intensity of red indicates a COPA-normalized value of 6 or greater. White indicates a value of zero or less.

Fig. 2.

AGTR1 outlier expression in breast cancer. (A) AGTR1 expression profile in the Perou et al. (29) cDNA microarray dataset (n = 55). (B) In the same dataset, AGTR1 expression vs. ERBB2 expression. (C) AGTR1 expression profile in the van de Vijver et al. (30) oligonucleotide dataset, segregated by ER status (n = 295). (D) AGTR1 expression vs. ERBB2 expression in the same dataset. (E) AGTR1 expression by quantitative RT-PCR in formalin-fixed, paraffin-embedded tissue. Expression of AGTR1 was assessed in 3 normal breast tissue specimens, 36 primary breast tumor specimens, and 9 metastatic breast cancer specimens. Expression levels were normalized to GAPDH expression and then scaled by the median AGTR/GADPH ratio.

Fig. 3.

Copy number analysis of the AGTR1 locus. (A) A schematic of probes used for FISH analysis. (B) Representative image from FISH analysis. Left is taken from a representative negative case. Middle and Right are images from a representative case with definitive copy number gain of AGTR1. Red signal is the AGTR1 locus probe, and green signal is the probe near the chromosome 3 centromere. (C) Association of AGTR1 overexpression with copy number gain. Three expression bins were defined based on AGTR1/GAPDH ratios: low (<1.0), moderate (1.0–2.0), and high (>2.0).

Fig. 4.

AGTR1 overexpression and analysis of angiotensin II (AT) and losartan effects on cell invasion. (A) Matrigel invasion assays of H16N2 cells infected with adenovirus expressing AGTR1 or LacZ. Cells were cultured in serum-free media and were pretreated with and without AT and losartan. Similar results were observed for HME cells. (B) Colorimetry readout of invasion assays from transfection experiments. LacZ- or AGTR1-expressing adenovirus was infected into H16N2 and HME immortalized mammary epithelial cells, and cells were treated with or without 1 μM AT and losartan. Because of absent baseline invasion, the optical density (OD) measurements were background subtracted, and values below 0.01 were set to 0.01. (C) Colorimetry readout of invasion assays from a panel of cancer cell lines. Seven breast cancer cell lines and a prostate cancer cell line, DU145, were examined for invasion after treatment with or without 1 μM AT and losartan. AGTR1 expression levels are indicated and were obtained from published microarray data and qRT-PCR analysis (Fig. S7). The quantification of invasion was done as described in B.

Fig. 5.

Effect of losartan treatment on AGTR1- or GUS-overexpressing MCF7 cell xenografts. Female BALB/C nu/nu mice were implanted with 2.5 × 10⁶ stable MCF7 cells overexpressing AGTR1 or GUS resuspended in 100 μL of saline with 20% Matrigel into the mammary fat pad of anesthetized mice. Mice from both groups: MCF7-AGTR1 or MCF7-GUS (n = 10 for each group) were treated every day with losartan (90 mg/kg body weight) or vehicle control. All animals were monitored at weekly intervals for tumor growth, and tumor sizes were recorded using the formula (π/6) (L × W²), where L = length of tumor and W = width. Box plots of log₂ tumor volumes are shown. P values from 2-sided Student's t tests indicate statistical significance. (A) Xenograft tumor size at 2 weeks. (B) Xenograft tumor size at 8 weeks.