The rapamycin-regulated gene expression signature determines prognosis for breast cancer

Abstract

Background: Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes) may also be used to simulate a biologic process or effects of a drug treatment. In this study, we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer.

Results: Colony formation and sulforhodamine B (IC50 < 1 nM) assays, and xenograft animals showed that MDA-MB-468 cells were sensitive to treatment with rapamycin. The comparison of in vitro and in vivo gene expression data identified a signature, termed rapamycin metagene index (RMI), of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%). In the Miller dataset, RMI did not correlate with tumor size or lymph node status. High (>75th percentile) RMI was significantly associated with longer survival (P = 0.015). On multivariate analysis, RMI (P = 0.029), tumor size (P = 0.015) and lymph node status (P = 0.001) were prognostic. In van 't Veer study, RMI was not associated with the time to develop distant metastasis (P = 0.41). In the Wang dataset, RMI predicted time to disease relapse (P = 0.009).

Conclusion: Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment.

Figure 1

Rapamycin sensitivity of the MDA-MB-468 breast cancer cell line. (A) MDA-MB-468 cells were treated with rapamycin at various concentrations. SRB assay was performed 5 days later. The assay results shown are the mean (± standard deviation) of three independent experiments. (B) 2 × 10³ MDA-MB-468 cells were plated in 60-mm plates in triplicate and treated with DMSO or 100 nM rapamycin. Two weeks later, cell colonies were stained with crystal violet, and the plates were scanned and colonies quantitated. The results shown are the mean (± standard deviation) for three plates. (C) Mice with established MDA-MB-468 tumor xenografts received DMSO or rapamycin (15 mg/kg) intraperitoneally once a week for 3 weeks. The tumor volumes were then measured using calipers every other day and presented as the mean (± standard error of the mean). Solid line, rapamycin; dashed line, DMSO. * P < 0.05.

Figure 2

Lack of correlation of the RMI with prognostic factors for breast cancer in Miller et al. data set [27]. The nonparametric box plots show interquartile range, horizontal line is mean. The RMI is distributed according to (A) tumor size, (B) lymph node status, and (C) patient age. o, outlier.

Figure 3

Overall survival rate according to the RMI in patients with breast cancer in Miller et al. data set [27]. Overall survival rate based on high and low RMI values were calculated using Cox proportional hazards analysis.

Figure 4

Metastasis-free survival rate according to the RMI in patients with breast cancer in Wang et al. data set [28]. Metastasis-free survival rate based on high and low RMI values were calculated using of Cox proportional hazards analysis.