Abstract P2-09-10: Antiprogestin CDB4124 suppressed progesterone receptor-mediated DNA binding activity, proliferation and gene expression in T47D cancer cells and normal human breast microstructures

Abstract

Abstract Introduction: The majority of breast cancers are estrogen and progesterone receptor positive (ER+PR+); hence targeting PRs with anti-progestins should be a useful strategy for breast cancer therapy. New generation anti-progestins lack the high anti-glucocorticoid activity of older agents; we evaluated one of these (CDB4124, telapristone) for specific inhibition of PR binding to the progesterone receptor element (PRE); effect on cell proliferation in T47D cells; and expression of PR target genes in normal human breast microstructures cultured in 3D conditions. Methods: To measure PRE activity in T47D cells dual luciferase reporter assay was performed, cells transfected with PRE-luc plasmid and treated for a 24h with vehicle, progesterone (P4), medroxyprogesterone (MPA), or a PR agonist (R5020), as well as three doses of CDB4124 (10nM, 100nM, 1μM) alone or in combination with P4 or MPA or R5020. Separately, T47D cells were simulated E2 plus P4 with and without CDB4124 for 72h and processed for cell cycle analysis by flow cytometer. For measurement of PR target genes, normal human breast microstructures were isolated after digestion of reduction mammoplasty samples. These microstructures were treated with E2 and P4 alone or in combination with CDB4124 for 24h. RNAs were collected after 24h and reverse-transcribed to cDNA for real time PCR. All experiments were performed in triplicate. Results: Increased in relative luciferase activity indicate an increase in PRE activity of T47D cells when treated with R5020 (2212.36±53.11 RLA) or P4 (1548.5 ± 158.84 RLA) or MPA (1422.63 ± 209.84 RLA) at 10nM alone compared to vehicle (33.08 ± 10.16 RLA). In contrast, 1μM CDB4124 alone have similar PRE activity (26.64 ± 3.94 RLA) in comparison to vehicle. The hormone-stimulated PRE activity was completely inhibited in T47D cells treated with 1μM CDB4124 combined with P4 (25.758 ± 2.32 RLA) or MPA (32.21 ± 2.46 RLA). In cells treated with R5020 plus CDB4124, 80% of PRE activity was inhibited (476.57± 5.72 RLA) in comparison to R5020 alone. Cell cycle data in T47D cells using flow cytometry showed significant arrest in G1 phase, with 12.6% decrease at 1nM E2+10nM P4+ 1μM CDB4124, and 14.9% decrease at 1nM E2+50nM P4+1μM CDB4124, relative to cells treated with hormone alone. The S-phase fraction also showed significant decrease when1μM CDB4124 was added to cells treated with 1nM E2+10nM P4 (12.4% decrease) and to 1nM E2+50nM P4 (10.5% decrease). CDB4124 treatment also led to decrease in expression of PR target genes i.e. RANKL, SKG1, FKB5 in normal breast microstructure in 3D culture in presence of P4 [50nM] alone or in combination with 1nM E2 plus P4 [50nM].Conclusion: CDB4124 is a potent antiprogestin; it abrogates PRE activity in the presence of R5020, or P4, or MPA. Furthermore, significant inhibition of hormone-induced cell proliferation is observed in T47D cells treated with CDB4124. In addition, PR target gene expressions were inhibited in normal breast microstructures, Based on these results, we expect that targeting PR by CDB4124 will inhibit PR regulated genes involved in cell proliferation and survival of breast cancer cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-10.