BackgroundMifepristone (MF) has been largely used in reproductive medicine due to its capacity to modulate the progesterone receptor (PR). The study of MF has been expanded to the field of oncology; yet it remains unclear whether the expression of PR is required for MF to act as an anti-cancer agent. Our laboratory has shown that MF is a potent inhibitor of ovarian cancer cell growth. In this study we questioned whether the growth inhibitory properties of MF observed in ovarian cancer cells would translate to other cancers of reproductive and non-reproductive origin and, importantly, whether its efficacy is related to the expression of cognate PR.MethodsDose-response experiments were conducted with cancer cell lines of the nervous system, breast, prostate, ovary, and bone. Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and analysed for cell number and cell cycle traverse, and hypodiploid DNA content characteristic of apoptotic cell death. For all cell lines, expression of steroid hormone receptors upon treatment with vehicle or cytostatic doses of MF for 24 h was studied by Western blot, whereas the activity of the G1/S regulatory protein Cdk2 in both treatment groups was monitored in vitro by the capacity of Cdk2 to phosphorylate histone H1.ResultsMF growth inhibited all cancer cell lines regardless of tissue of origin and hormone responsiveness, and reduced the activity of Cdk2. Cancer cells in which MF induced G1 growth arrest were less susceptible to lethality in the presence of high concentrations of MF, when compared to cancer cells that did not accumulate in G1. While all cancer cell lines were growth inhibited by MF, only the breast cancer MCF-7 cells expressed cognate PR.ConclusionsAntiprogestin MF inhibits the growth of different cancer cell lines with a cytostatic effect at lower concentrations in association with a decline in the activity of the cell cycle regulatory protein Cdk2, and apoptotic lethality at higher doses in association with increased hypodiploid DNA content. Contrary to common opinion, growth inhibition of cancer cells by antiprogestin MF is not dependent upon expression of classical, nuclear PR.

BackgroundChanges in cell shape and plasticity in cytoskeletal dynamics are critically involved in cell adhesion, migration, invasion and the overall process of metastasis. Previous work in our laboratory demonstrated that the synthetic steroid mifepristone inhibited the growth of highly metastatic cancer cells, while simultaneously causing striking changes in cellular morphology. Here we assessed whether such morphological alterations developed in response to cytostatic concentrations of mifepristone are reversible or permanent, involve rearrangement of cytoskeletal proteins, and/or affect the adhesive capacity of the cells.MethodsCancer cell lines of the ovary (SKOV-3), breast (MDA-MB-231), prostate (LNCaP), and nervous system (U87MG) were exposed to cytostatic concentrations of mifepristone and studied by phase-contrast microscopy. The transient or permanent nature of the cytostasis and morphological changes caused by mifepristone was assessed, as well as the rearrangement of cytoskeletal proteins. De-adhesion and adhesion assays were utilized to determine if mifepristone-arrested and morphologically dysregulated cells had abnormal de-adhesion/adhesion dynamics when compared to vehicle-treated controls.ResultsMifepristone-treated cells displayed a long, thin, spindle-like shape with boundaries resembling those of loosely adhered cells. Growth arrest and morphology changes caused by mifepristone were reversible in SKOV-3, MDA-MB-231 and U87MG, but not in LNCaP cells that instead became senescent. All cancer cell types exposed to mifepristone displayed greatly increased actin ruffling in association with accelerated de-adhesion from the culture plate, and delayed adhesion capacity to various extracellular matrix components.ConclusionsCytostatic concentrations of mifepristone induced alterations in the cellular structure of a panel of aggressive, highly metastatic cancer cells of different tissues of origin. Such changes were associated with re-distribution of actin fibers that mainly form non-adhesive membrane ruffles, leading to dysregulated cellular adhesion capacity.

For most cancers, it is metastasis and burden of the secondary tumors that account for nearly 90% of cancer-related mortality. The process of metastatic dissemination of tumor cells is extremely complex. Among the critical parameters involved in metastatic spread are cellular detachment from the initial tumor, migration, invasion and adhesion to secondary sites, all processes associated with changes in cell morphology caused by cytoskeletal rearrangements. Our laboratory previously demonstrated that the synthetic antiprogestin mifepristone (MF) inhibits the growth of cancer cells of reproductive and non-reproductive origin. We also observed that in response to MF, the cells displayed drastic changes in morphology. Due the impact of cell structure on the metastatic process, this study questioned whether MF has anti-metastatic potential deregulating key processes required for metastatic dissemination. Cancer cell lines of the ovary (SK-OV-3), breast (MDA-MB-231), prostate (LNCaP), and brain (U87MG) were chosen from a panel of cell lines in which we previously observed cytostatic activity of MF associated with changes in cellular morphology. Cell cultures were exposed to vehicle or MF for 72 h at which point we examined the morphology of the cells, the distribution of cytoskeletal proteins actin and tubulin, the adhesion capacity to components of the extracellular matrix, and the cell migratory and invasive properties. MF induced distinct morphology changes after 48 h exposure, independent of the density of the cell culture population. Interestingly, MF induced similar morphology changes in all cell lines, which included shrinkage of the cell body, long, thin cellular extensions, and a loss of cell-to-cell connections. Morphology changes were associated with rearrangement of actin and tubulin filaments. Upon removal of MF, cell morphology and proliferation returned to that of untreated cultures in all cell lines expect for LNCaP. For all cell lines, MF caused accelerated trypsin-induced detachment and decrease in cell adhesion to various extracellular matrix substrates suggesting that MF may impair re-adhesion of cells en route to colonize a secondary location. Moreover, MF significantly inhibited the ability of cells to migrate in wound healing and Boyden chamber assays, as well as their capacity to invade through extracellular matrix-coated polycarbonate membranes. Overall this study suggests that antiprogestin MF could be of potential therapeutic use, not only as a cytostatic agent impairing tumor growth, but also to halt metastatic spread of tumor cells. (Supported by NCI grants K22CA121991 and K22CA121991S1). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2362. doi:10.1158/1538-7445.AM2011-2362

The synthetic steroid hormone mifepristone (MF) was originally developed as a glucocorticoid receptor (GR) antagonist, but the capacity of this compound to modulate the activity of the progesterone receptor (PR) led to an intensive study of its potential applications as an antiprogestin. The emphasis on the interaction between MF and PR has been translated to its application as a potential therapeutic agent in the field of oncology; however, it remains unclear whether the expression of PR is required for MF to act as anti-cancer agent. Our laboratory has previously shown that MF is a potent inhibitor of ovarian cancer (OvCa) cell growth, and that the cytostatic action of MF occurs through an arrest in the G1 phase of the cell cycle characterized by a decrease in cyclin dependent kinase (Cdk) 2 activity. In this study, we questioned whether the growth inhibitory properties of MF observed in OvCa cells would be effectively translated to other cancers of reproductive and non-reproductive origin, and, importantly, whether the efficacy of MF as an antiproliferative agent was dependent upon PR expression. Dose-response experiments were conducted with a panel of cancer cell lines of the brain (meningioma IOMM-Lee and glioblastoma U87MG cells), breast (estradiol-responsive MCF-7 and estradiol-unresponsive MDA-MB-231 cells), prostate (androgen-responsive LNCaP and androgen-unresponsive PC-3 cells), ovary (OVCAR-3 and SK-OV-3 cells), and bone (osteosarcoma U-2OS and SAOS-2 cells). Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and then analyzed for cell number and cell cycle traverse. For all cell lines, expression of steroid hormone receptors and cell cycle regulatory proteins controlling the G1-S phase transition in cells treated with vehicle or MF for 24 h was studied by Western blot analysis. The activity of Cdk2 in both treatment groups was monitored in vitro by the capacity of Cdk2 to phosphorylate its substrate histone H1. MF growth inhibited all cancer cell lines included in this study, regardless of tissue of origin and hormone responsiveness. Changes in the abundance of the cell cycle regulatory proteins p21cip1, p27kip1, cyclin E, and Cdk2 along with changes in Cdk2 activity following 24 h exposure to MF suggested that MF induces a G1 phase arrest in the majority of these cell lines, consistent with our previous results in OvCa cells. Importantly, studies of the basal expression of steroid hormone receptors in this panel of cell lines showed that while all cell lines were growth inhibited by MF, only MCF-7 cells expressed PR. This study supported our previous results with MF-treated OvCa cells suggesting a mechanism of action involving G1 arrest and a corresponding decrease in Cdk2 activity. Contrary to common opinion, growth inhibition of this panel of cancer cell lines by MF was not dependent upon PR expression. NCI K22CA121991. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4546. doi:10.1158/1538-7445.AM2011-4546