title: Efficacy and mechanism of action of Proellex, an antiprogestin in aromatase overexpressing and Letrozole resistant T47D breast cancer cells.
abstract: Aromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI. In this study, we determined the response of antiprogestin CDB-4124 (Proellex) on the aromatase overexpressing and Letrozole resistant cell lines and also studies its mechanism of action in inhibition of breast cancer cell proliferation. For these studies we generated aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines by stable transfection with plasmid containing CYP19A1 gene, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10 μM Letrozole. Cell proliferation was determined by MTT or crystal violet assays. Gene expressions were quantified by QRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on the anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student's 't' test or ANOVA followed by Bonferroni's post hoc test. Results showed that T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA; 80-90 fold and protein) and as a result exhibited increased aromatization of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone (50-60%). In T47DaromLR cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex and other known aromatase inhibitors (Letrozole, Anastrozole, and Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. Cell growth inhibition was significantly greater when cells were treated with Proellex alone or in combination with other AIs as compared to AIs alone. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex is probably due to PR-B/EGFR modulation in ER+PR+, aromatase expressing cells. Overall these results suggest that antiprogestin, Proellex can be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for aromatase inhibitor resistant breast cancer patients.
authors.affiliation: Cancer Biology Division, IIT Research Institute, Chicago, IL 60616, USA. guptaka@iit.edu
authors.forename: Akash
authors.initials: A
authors.lastname: Gupta
authors.forename: Rajeshwari
authors.initials: R
authors.lastname: Mehta
authors.forename: Fatouma
authors.initials: F
authors.lastname: Alimirah
authors.forename: Xinjian
authors.initials: X
authors.lastname: Peng
authors.forename: Genoveva
authors.initials: G
authors.lastname: Murillo
authors.forename: Ronald
authors.initials: R
authors.lastname: Wiehle
authors.forename: Rajendra G
authors.initials: RG
authors.lastname: Mehta
mesh_terms: D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D001141:Aromatase / Q000235:genetics / Q000378:metabolism*; D047072:Aromatase Inhibitors / Q000494:pharmacology; D001483:Base Sequence; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D005260:Female; D015870:Gene Expression; D018773:Genes, erbB-1 / Q000187:drug effects; D006801:Humans; D000077289:Letrozole; D009570:Nitriles / Q000494:pharmacology; D009649:Norpregnadienes / Q000494:pharmacology*; D011372:Progestins / Q000037:antagonists & inhibitors*; D011397:Promegestone / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism; D013629:Tamoxifen / Q000494:pharmacology; D013739:Testosterone / Q000494:pharmacology; D014230:Triazoles / Q000494:pharmacology; D014410:Tumor Stem Cell Assay
chemical_list: D018931:Antineoplastic Agents, Hormonal; D047072:Aromatase Inhibitors; D009570:Nitriles; D009649:Norpregnadienes; D011372:Progestins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011980:Receptors, Progesterone; D014230:Triazoles; C097314:progesterone receptor B; D013629:Tamoxifen; C461063:telapristone acetate; D013739:Testosterone; D000077289:Letrozole; D011397:Promegestone; D001141:Aromatase