The Association of RGS2 and Slug in the Androgen-induced Acquisition of Mesenchymal Features of Breast MDA-MB-453 Cancer Cells

ABSTRACT

Background

Epithelial-mesenchymal transition (EMT) of tumor cells is a prerequisite to cancer cell invasion and metastasis. This process involves a network of molecular alterations. Androgen receptor (AR) plays an important role in the biology of breast cancers, particularly those dependent on AR expression like luminal AR (LAR) breast cancer subtype. We have recently reported that the AR agonist, dihydrotestosterone (DHT), induces a mesenchymal transition of MDA-MB-453 cells, concomitant with transcriptional up-regulation of Slug and regulator of G protein signaling 2 (RGS2).

Objective

The role of Slug and RGS2 in mediating the DHT-induced effects in these cells was investigated.

Methods

MDA-MB-453 cells were used as a model system of LAR breast cancer. Immunofluorescence was used to examine cell morphology and protein localization. Protein expression was analyzed by immunoblotting. Protein localization was confirmed by cell fractionation followed by immunoblotting. Protein-protein interaction was confirmed by co-immunoprecipitation followed by immunoblotting. Transwell membranes were used to assess cell migration. Transfection of cells with siRNA molecules that target Slug and RGS2 mRNA was utilized to delineate the modes of action of these two molecules.

Results

Treatment of MDA-MB-453 cells with DHT induced the expression of both proteins. In addition, AR-Slug, AR-RGS2, and Slug-RGS2 interactions were observed shortly after AR activation. Knocking down Slug abrogated the basal, but not the DHT-induced, cell migration and blocked DHT-induced mesenchymal transition. On the other hand, RGS2 knocked-down cells had an increased level of Slug protein and assumed mesenchymal cell morphology with induced migration, and the addition of DHT further elongated cell morphology and stimulated their migration. Inhibition of AR or β-catenin reverted the RGS2 knocked-down cells to the epithelial phenotype, but only inhibition of AR blocked their DHT-induced migration.

Conclusions

These results suggest the involvement of RGS2 and Slug in a complex molecular network regulating the DHT-induced mesenchymal features in MDA-MB-453 cells. The study may offer a better understanding of the biological role of AR in breast cancer toward devising AR-based therapeutic strategies.

KEYWORDS:

• slug
• dihydrotestosterone
• androgen receptor
• mda-mb-453 cells
• partial emt
• Slug
• rgs2

Acknowledgments

We would like to thank the scientific staff of the Cell Therapy Center and the University of Jordan, for facilitating the use of their facility. The authors would also like to thank Professor Lubna Tahtamouni for her technical and scientific support.

Disclosure Statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The authors confirm that the primary data shown in the paper will be made available to qualified investigators upon request.

Supplementary Material

Supplemental data for this article can be accessed on the publisher’s website

Additional information

Funding

This research was funded by generous grants from the Deanship of Scientific Research, The University of Jordan (1554/2017/19), and the Scientific Research and Innovation Fund, Ministry of Higher Education and Research (MPH/2/7/2014).