Efficacy and mechanism of action of Proellex, an antiprogestin in aromatase overexpressing and Letrozole resistant T47D breast cancer cells

https://doi.org/10.1016/j.jsbmb.2012.08.004Get rights and content

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.

Highlights

Aromatase overexpressing and Letrozole resistant T47D cell lines were generated. ► Antiprogestin, Proellex, suppressed growth of these cells in vitro. ► Proellex action is mediated by inhibiting PR-B/p300 complex formation. ► Proellex suppresses EGFR and PR-B expression.

Introduction

The treatment of breast cancer patients has been revolutionized in part due to the use of diagnostic markers such as steroid receptors, Her2/neu and BRCA [1]. The clear demarcation of ER and PR positive, Her2/neu positive and triple negative (ER, PR, Her-2/neu negative) patients has streamlined treatment approaches [2]. Generally Her2/neu positive patients have benefited from herceptin treatment whereas the steroid receptor positive patients receive anti-hormone therapy [3]. Tamoxifen is routinely used with excellent outcome [4]. Yet another very successful approach is the use of aromatase inhibitors for steroid receptor positive patients [5]. Aromatase is a cytochrome P450 CYP19 A1 enzyme that is responsible for catalyzing the reaction for the conversion of C19 androgens to C18 estrogen [6]. Usually the expression of aromatase enzyme is reported to be higher in postmenopausal women diagnosed with breast cancer as compared to premenopausal females [7]. Therefore, in postmenopausal women the treatment strategy has included preventing the conversion of androstenedione or testosterone to estradiol and thereby preventing cell proliferation of ER positive breast cancer cells. The aromatase inhibitors have also undergone transformation from the first and second generation aromatase inhibitors such as aminoglutethimide and 4-OHA to the third generation inhibitors such as Letrozole, Exemestane and Anastrozole [8]. First and second generation compounds were discontinued due to the toxicity associated with them. Letrozole, a third generation AI, is relatively nontoxic and often prescribed for patients for five years. The lack of toxic effects and much better tolerability has made this treatment approach as a strategy of choice for ER and PR+ patients [9]. Tamoxifen is equally effective, however patients receiving Tamoxifen are at a higher risk of developing endometrial cancers [10]. Despite the fact that Letrozole is a very effective antiestrogenic agent, patients do develop acquired resistance to Letrozole (or any other aromatase inhibitors or antiestrogens) [11]. Therefore a search for more effective therapeutic agents that can be substituted in the case of acquired resistance, singly or in combination with current therapies, has been ongoing.

The role of PR in breast cancer diagnosis has been well established [12], [13]. However the efficacy of antiprogestins has not received much importance. In earlier studies, use of synthetic progesterone derivatives such as medroxyprogesterone acetate (MPA), and role of progesterone receptor in breast tumor progression have been evaluated for increased risk of developing breast cancer [14]. Likewise, an increase in hormonal replacement therapy associated with an increased risk of breast cancer. In similar fashion, in vivo studies have demonstrated the involvement of progesterone in the development of malignant tumors in mice [15]. Progestins are also known to activate growth stimulation pathways through production of EGF (epidermal growth factor) which leads to growth stimulation of breast cancer cells [16]. It has been reported that progesterone receptors, in the presence of ligands, can interact with the growth factor pathways such as EGFR/ERK/Src [17]. Cumulatively, these studies suggest a dominant role of progestin and progesterone receptor in development of aggressive hormone dependent breast cancer.

Progesterone receptor is an estrogen responsive target gene. Induction of estrogen receptor or estrogen function is most often associated with increased PR [18], [19]. Yet the strategies to target PR for possible breast cancer treatment have not been extensively explored. One of the reasons for the hampered use of progesterone receptor antagonists is their undesirable side effect of down regulating glucocorticoid functions [20], [21]. For this reason, only a few clinical studies have been conducted using an antiprogestin such as Mifepristone also known as RU486 [22], [23]. Mifepristone mediated cell death or decreased cell proliferation is accompanied by the upregulation of p21, p27 and down regulation of cyclin D and E associated kinases [24], [25], [26]. Antiprogestins have also been shown to decrease transcriptional activity of PR by preventing PR-B/p300 complex formation in T47D breast cancer cells [27]. Thus, if a PR antagonist devoid of unwanted side effects is identified, it could serve as a potential therapeutic drug alternative to the aromatase inhibitors or antiestrogens. One of the obstacles in evaluation of new aromatase inhibitors is a lack of suitable research tools. Most established cell lines that are ER/PR positive do not express aromatase enzyme. Over the past several years, Brodie and her team have established and investigated properties of genetically engineered breast cancer cell lines that overexpress aromatase [28], [29]. In this report, we have generated an aromatase overexpressing ER/PR positive cell line and examined the response of Proellex. Proellex is an anti-progestin which is FDA-approved for the investigation of the treatment of endometriosis under an IND and is under review for the investigation of the treatment of fibroids under another IND. Previously, we reported that Proellex inhibits development of MNU-induced mammary tumors in rats [30]. In this report, we determined the effects and possible mechanism of action of Proellex in genetically engineered breast cancer cell lines, which are either resistant to AI or overexpressing aromatase and responsive to Letrozole.

Section snippets

Chemicals

Testosterone and Tamoxifen were obtained from Sigma–Aldrich (St. Louis, MO). Proellex (17α-acetoxy-21 methoxy-11β[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione, also known as progenta, telapristone acetate, and CDB 4124) was provided by Repros Therapeutics (The Woodlands, TX). Letrozole, Anastrozole and Exemestane, were purchased from AvaChem Scientific (San Antonio, TX). G418 was obtained from Research Product International (RPI) Corp. (Palos Heights, IL). All steroids and

Characteristics of T47Dcon, T47Darom and T47Darom LR cells

In the present study, a human breast cancer cell line (T47D) was transfected with a mammalian expressing plasmid vector (pcDNA3.1) containing human aromatase gene (CYP19A1); clones were selected and expanded into stable aromatase expressing cell line, T47Darom. By maintaining T47Darom cells in the continuous presence of 10 μM Letrozole for 75 weeks, Letrozole resistant aromatase overexpressing cell line was generated and designated as T47DaromLR. Empty vector transfected T47D cells served as a

Discussion

Aromatase is a CYP19, P450 cytochrome that catalyzes the conversion of C19 androgens to C18 estrogens [6]. Since the breast cancers in nearly 60–70% pre and postmenopausal women are estrogen receptor positive, blocking estrogen function has resulted in a very effective therapy. These therapies include treatment with antiestrogens such as Tamoxifen or preventing the synthesis of estrogen mediated by aromatase with aromatase inhibitors. Progression from the first generation aromatase inhibitor

Conclusion

In summary, this report introduces Proellex as a potent antiprogestin with potential which translational significance in suppressing proliferation of ER/PR positive aromatase overexpressing cells as well as cells that have acquired resistance to aromatase inhibitors. Proellex could also be valuable as an alternative choice for currently used aromatase inhibitors in the clinics. Our results suggest that Proellex could serve as a unique chemotherapeutic agent for the treatment of subset of breast

Competing interests

RW is a Vice President and Scientific Officer for Repros Therapeutics and has financial interest in the company. Other authors have no interest in the antiprogestin used in the project.

Authors’ contributions

AG designed and performed all the experiments and prepared the draft of the manuscript. RM evaluated results on the immunofluorescence, helped with study design and wrote parts of the manuscript. FA conducted all the western blot analyses in the paper and discussed results in light of the imaging studies. XP designed primers and helped with cloning of aromatase, establishment of stably transfected cell lines and interpretation of QRT-PCR studies. GM participated in the design of the studies

Acknowledgements

We thank Dr. Guan Chen (Medical College of Wisconsin) for kindly providing the ERE reporter plasmid. The project was in part supported by the Repros Therapeutics, The Woodlands, TX 77380.

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