Androgen receptor (AR) and its splicing variant 7 (ARv7) play vital roles in the pathobiology of breast cancer (BC) but their role in the estrogen receptor-positive (ER+) type is controversial, hence, we studied the influence of the blockers of AR (Enzalutamide) and ARv7 (EPI-001) on tumorigenesis processes using T47D, an ER+ BC cell line. We showed that although both inhibitors failed to reduce cell growth and affect AR content, only Enzalutamide reduced the ARv7. Mechanistically, the drugs successfully arrested the cell cycle at S-phase and downregulated the protein expression of cyclins A, E, & C. Additionally, they inhibited the cell proliferation stimulator nuclear factor kappa B (NF-ĸB), whereas only EPI-001 reduced the cell regulatory marker c-Myc. They also opposed the endothelial-to-mesenchymal transition (EMT) process, by boosting the epithelial marker E-cadherin and reducing the protein expression of the mesenchymal marker fibronectin. Their anti-metastatic potential was evidenced by the hindrance of cell migration using the wound healing assay and further confirmed by the downregulation of metalloproteinase (MMP) 2 and 9 protein expression, and protein content of Rho kinase (ROCK)1 and 2. Besides, by downregulating the protein expression of vascular endothelial growth factor (VEGF) the drugs point to their anti-angiogenic aptitude. In conclusion, this in-vitro study is the first to prove the importance of blocking AR/ARv7 using Enzalutamide and EPI-001 in decreasing cancer cell survival, EMT, and metastasis in ER+ BC cells, findings that still need further studies to unveil the role of these inhibitors in BC.

Purpose: Androgen receptor (AR) is often expressed in breast cancer, but its role in estrogen receptor positive (ER+) type is controversial. Although AR and its splicing variant 7 (ARV7) play a role in the pathobiology of breast cancer, the precise mechanisms are not fully understood. Therefore, the aim of the current study is to determine the influence of the blockers of AR (Enzalutamide) and ARV7 (EPI-001) on metastasis and epithelial to mesenchymal transition (EMT) in T47D, an ER+ breast cancer cell line. Methods: T47D cells were cultured/treated with Enzalutamide or EPI-001; cytotoxicity and cell cycle analysis were determined using Sulphorhodamine-B (SRB) assay and Flowcytometry analysis, respectively, whereas metastasis was assessed by the Scratch wound healing assay. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of AR, ARV7, C-myc, N-Cadherin, E-Cadherin, NF-κB, ROCK1 and 2 and Western blot was used for evaluating the protein expression of cyclin dependent kinases (CDK4, CDK6, Cyclin E), Fibronectin, vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP2 and MMP9). Results: Our results indicated that treatment with Enzalutamide or EPI-001 didn't significantly affect cell proliferation of T47D. However, cells were arrested at S-phase accompanied by a decline in CDK4, CDK6, Cyclin E and metastasis. Fibronectin, VEGF, MMP2, MMP9, ROCK1 and ROCK2 proteins were downregulated. C-myc and NF-κB levels were declined by EPI-001, but not Enzalutamide.Conclusion: Blocking AR/ARV7 has no effect on cell proliferation, but decreases metastasis by regulating key markers and processes involved in EMT in ER+ breast cancer cells.