Reevaluating the Role of Progesterone in Ovarian Cancer: Is Progesterone Always Protective?

Ovarian cancer (OC) represents a collection of rare but lethal gynecologic cancers where the difficulty of early detection due to an often-subtle range of abdominal symptoms contributes to high fatality rates. With the exception of BRCA1/2 mutation carriers, OC most often manifests as a post-menopausal disease, a time in which the ovaries regress and circulating reproductive hormones diminish. Progesterone is thought to be a "protective" hormone that counters the proliferative actions of estrogen, as can be observed in the uterus or breast. Like other steroid hormone receptor family members, the transcriptional activity of the nuclear progesterone receptor (nPR) may be ligand dependent or independent and is fully integrated with other ubiquitous cell signaling pathways often altered in cancers. Emerging evidence in OC models challenges the singular protective role of progesterone/nPR. Herein, we integrate the historical perspective of progesterone on OC development and progression with exciting new research findings and critical interpretations to help paint a broader picture of the role of progesterone and nPR signaling in OC. We hope to alleviate some of the controversy around the role of progesterone and give insight into the importance of nPR actions in disease progression. A new perspective on the role of progesterone and nPR signaling integration will raise awareness to the complexity of nPRs and nPR-driven gene regulation in OC, help to reveal novel biomarkers, and lend critical knowledge for the development of better therapeutic strategies.

Irilone, a Red Clover Isoflavone, Combined with Progesterone Enhances PR Signaling through the Estrogen and Glucocorticoid Receptors.

Trifolium pratense L. (red clover) is a popular botanical supplement used for women's health. Irilone isolated from red clover previously demonstrated progestogenic potentiation activity. In this study, irilone enhanced progesterone signaling was determined to not occur due to post-translational phosphorylation or by reducing progesterone receptor (PR) protein levels but instead increased PR protein levels in T47D breast cancer cells, which could be blocked by estrogen receptor (ER) antagonists, suggesting an ER dependent effect. Further, irilone increased luciferase activity from a hormone responsive element in a cell line that lacked ER and PR but expressed the glucocorticoid receptor (GR). A siRNA knockdown of GR in Ishikawa PR-B endometrial cancer cells reduced irilone's ability to enhance progesterone signaling. In an ovariectomized CD-1 mouse model, irilone did not induce uterine epithelial cell proliferation. The mechanism of action of irilone gives insight into PR crosstalk with other steroid hormone receptors, which can be important for understanding botanicals that are used for women's health.

Secoiridoids from Dogwood (Cornus officinalis) Potentiate Progesterone Signaling.

The use of botanical dietary supplements for the alleviation of conditions such as hot flashes, premenstrual syndrome, and fertility is prolific worldwide. Estrogen and progesterone receptors (ER and PR) and their corresponding steroid hormones are critical for the relief of hot flashes and the treatment of patients who develop endometriosis, and these pathways can influence the development of endometrial, ovarian, and breast cancers. However, few studies have investigated or identified the natural product components in herbal supplements that act on the PR. In the current study, a new secoiridoid, demethoxy-cornuside (1), along with six known secoiridoids (2-7) were isolated from the twigs of dogwood (Cornus officinalis) by bioassay-guided isolation with a progesterone response element (PRE)/luciferase (Luc) reporter assay in Ishikawa cells. Four phytoprogestins (1, 2, 6, 7) potentiated the effect of progesterone in the PRE/Luc assay. This study demonstrates that C. officinalis components might potentiate progesterone signaling in the presence of progesterone, which could modify progesterone receptor action in hormone-responsive tissues such as the uterus and mammary gland.

Baicalein Is a Phytohormone that Signals Through the Progesterone and Glucocorticoid Receptors.

While flavonoids have been studied extensively for estrogen receptor activity, they have not been well studied for their ability to modify progesterone receptor (PR) and glucocorticoid receptor (GR) signaling. Three flavonoid compounds, tangeretin, wogonin, and baicalein, were selected for testing for PR and GR activity based on their structural similarity to known phytoprogesterone-like compounds. Each compound was docked in the binding pocket of PR and GR. Of these compounds, baicalein was predicted to be most likely to bind to both receptors. A fluorescence polarization competitive binding assay for PR and GR confirmed that baicalein binds to both the PR and GR with IC50 values of 15.30 µM and 19.26 µM, respectively. In Ishikawa PR-B and T47D cells, baicalein acted as a PR antagonist in a hormone response element (HRE) luciferase (Luc) assay. In OVCAR5 cells, which only express GR, baicalein was a GR agonist via an HRE/Luc assay and induced GR target genes, FKBP5 and GILZ. RU486, a PR and GR antagonist, abrogated baicalein's activity in OVCAR5 cells, confirming baicalein's activity is mediated through the GR. In vivo, baicalein administered intraperitoneally to female mice twice a week for 4 weeks at a dose of 25 mg/kg induced the GR target gene GILZ in the reproductive tract, which was blocked by RU486. In summary, baicalein has PR antagonist and GR agonist activity in vitro and demonstrates GR agonist activity in the uterus in vivo.

Verticillin A Causes Apoptosis and Reduces Tumor Burden in High-Grade Serous Ovarian Cancer by Inducing DNA Damage.

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy in women worldwide and the fifth most common cause of cancer-related deaths among U.S. women. New therapies are needed to treat HGSOC, particularly because most patients develop resistance to current first-line therapies. Many natural product and fungal metabolites exhibit anticancer activity and represent an untapped reservoir of potential new agents with unique mechanism(s) of action. Verticillin A, an epipolythiodioxopiperazine alkaloid, is one such compound, and our recent advances in fermentation and isolation are now enabling evaluation of its anticancer activity. Verticillin A demonstrated cytotoxicity in HGSOC cell lines in a dose-dependent manner with a low nmol/L IC50 Furthermore, treatment with verticillin A induced DNA damage and caused apoptosis in HGSOC cell lines OVCAR4 and OVCAR8. RNA-Seq analysis of verticillin A-treated OVCAR8 cells revealed an enrichment of transcripts in the apoptosis signaling and the oxidative stress response pathways. Mass spectrometry histone profiling confirmed reports that verticillin A caused epigenetic modifications with global changes in histone methylation and acetylation marks. To facilitate in vivo delivery of verticillin A and to monitor its ability to reduce HGSOC tumor burden, verticillin A was encapsulated into an expansile nanoparticle (verticillin A-eNP) delivery system. In an in vivo human ovarian cancer xenograft model, verticillin A-eNPs decreased tumor growth and exhibited reduced liver toxicity compared with verticillin A administered alone. This study confirmed that verticillin A has therapeutic potential for treatment of HGSOC and that encapsulation into expansile nanoparticles reduced liver toxicity.

Loss of PTEN in Fallopian Tube Epithelium Results in Multicellular Tumor Spheroid Formation and Metastasis to the Ovary.

High-grade serous ovarian cancer (HGSOC) can originate in the fallopian tube and then spread to the ovary. Our objective was to evaluate the role of multicellular tumor spheroids (MTS) in ovarian metastasis. By testing a panel of murine oviductal epithelial (MOE) cells with genetic alterations mimicking those seen in HGSOC, we found that loss of PTEN allowed MTS formation under ultra-low adhesion conditions. Confirming these results in vivo, MTS-like structures were observed in the oviducts of PAX8Cre/+ PTENflox/flox mice. MOE PTENshRNA cells could incorporate up to 25% wild type cells into MTS, while higher percentages of wild type cells resulted in a loss of MTS formation. MTS formation allowed MOE PTENshRNA cells to survive better under ultra-low adhesion conditions than control cells. MTS also attached to the ovarian stroma, as would be exposed during ovulation. Interestingly, MTS more robustly cleared monolayers of murine ovarian surface epithelia than murine ovarian fibroblasts. When xenografted into the ovarian bursa, OVCAR8 MTS were able to form tumors in the ovary at a similar rate as an equal number of OVCAR8 cells grown on traditional cell culture plastic. In conclusion, loss of a single gene (PTEN) allows the fallopian tube epithelia to form MTS, which survive better under ultra-low adhesion conditions, attach to the extracellular matrix exposed during ovulation, and colonize the ovary. These results suggest that MTS may contribute to seeding of the ovary in HGSOC patients.

Irilone from Red Clover ( Trifolium pratense) Potentiates Progesterone Signaling.

The use of botanical dietary supplements is becoming increasingly popular for the alleviation of hormonal-based conditions such as hot flashes, premenstrual syndrome, and fertility. Estrogen and progesterone receptors (ER and PR) play an essential role in these processes. However, despite the fact that many therapies used to alleviate gynecological conditions act through PR-mediated mechanisms, few studies have investigated or identified any herbal natural product components that act on this receptor. In the current study, we used a progesterone response element (PRE)-luciferase (Luc) reporter assay to identify four phytoprogestins present in a standardized red clover ( Trifolium pratense) extract. We found that the component irilone (1) potentiated the effect of progesterone in both endometrial and ovarian cancer cell lines. In these cancers, progesterone action is generally associated with positive outcomes; thus the potentiating effect of 1 may provide entirely new strategies for enhancing progesterone signaling as a means of mitigating conditions such as fibroids and endometriosis. Formononetin (3) and biochanin A (4) exhibited mixed agonist activity, while prunetin (2) acted only as an antagonist. Collectively, these results suggest that the effects of red clover extract repeatedly observed in cultured cells and the inverse correlation between risk of various cancers and flavonoid intake may be due, in part, to altered progesterone signaling.