Abstract
Preoperative progesterone intervention has been shown to confer a survival benefit to breast cancer patients independently of their progesterone receptor (PR) status. This observation raises the question how progesterone affects the outcome of PR-negative cancer. Here, using microarray and RNA-Seq–based gene expression profiling and ChIP-Seq analyses of breast cancer cells, we observed that the serum- and glucocorticoid-regulated kinase gene (SGK1) and the tumor metastasis–suppressor gene N-Myc downstream regulated gene 1 (NDRG1) are up-regulated and that the microRNAs miR-29a and miR-101-1 targeting the 3′-UTR of SGK1 are down-regulated in response to progesterone. We further demonstrate a dual-phase transcriptional and post-transcriptional regulation of SGK1 in response to progesterone, leading to an up-regulation of NDRG1 that is mediated by a set of genes regulated by the transcription factor AP-1. We found that NDRG1, in turn, inactivates a set of kinases, impeding the invasion and migration of breast cancer cells. In summary, we propose a model for the mode of action of progesterone in breast cancer. This model helps decipher the molecular basis of observations in a randomized clinical trial of the effect of progesterone on breast cancer and has therefore the potential to improve the prognosis of breast cancer patients receiving preoperative progesterone treatment.
Highlights
Preoperative progesterone intervention has been shown to confer a survival benefit to breast cancer patients independently of their progesterone receptor (PR) status
The up-regulation of serum- and glucocorticoid-regulated kinase gene 1 (SGK1), known to harbor multiple progesterone response elements (PREs) [20, 21], and N-Myc downstream regulated gene 1 (NDRG1) were observed to be relatively higher among the PR-positive cells, whereas miR-29a and miR101-1 were lower in PR-negative cells in response to progesterone (Fig. 1, A–D)
We present an intricate convergence model indicating a dual-phase regulation downstream to progesterone treatment to regulate the expression of serum- and glucocorticoid-regulated kinase gene 1 (SGK1), predominantly driven as a direct transcriptional target, consistent with earlier reports [20, 21], in PR-positive breast cancer cells and down-regulation of miR29a and miR-101-1 targeting SGK1 with a relatively distinct effect in PR-negative breast cells in response to progesterone
Summary
An integrated analysis of microarray-based mRNA expression profile and deep sequencing of noncoding small RNA of breast cancer cells (as described under “Experimental procedures”) led us to identify up-regulation of a serum- and glucocorticoid-regulated kinase gene (SGK1) and N-Myc downstream regulated gene 1 (NDRG1), along with down-regulation of miR-29a and miR-101-1, predicted to bind the 3Ј-UTR of SGK1, independent of the hormonal receptor status of the cells (Fig. S1A and Tables S1 and S2). SGK1, when overexpressed in PR-positive T47D and PRnegative MDA-MB-231 breast cancer cells, mimicked the effect of progesterone by up-regulating the expression of NDRG1 (Fig. 3A), which in turn led to a significant reduction in cell migration and cell invasion (Fig. 3, B and C). Depletion of SGK1 in T47D and MDA-MB-231 cells led to a decrease in expression of NDRG1 (Fig. 4A) with an inverse effect observed on migration and invasion of the breast cancer cells (Fig. 4, B and C), regardless of progesterone treatment (Fig. 5, A and B). Our results suggest that the SGK1/ NDRG1 axis mediates regulation of activation of kinases involved in breast cancer cell migration, independent of their hormonal receptor status
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