Downregulation of retinoblastoma protein is involved in the enhanced cytotoxicity of 4-hydroxytamoxifen plus mifepristone combination therapy versus antiestrogen monotherapy of human breast cancer

Abstract

Abstract. In this study, human MCF-7 breast cancer cells, which express functional estrogen and progesterone receptors, were used to compare the efficacy of combined antiestrogen plus antiprogestin therapy to antiestrogen monotherapy. Cells were treated with the antiestrogen 4-hydroxytamoxifen (4-OHT) and/or the antiprogestin mifepristone (MIF) and effects on cell proliferation (cytostatic action), cell cycle phase, the phosphorylation state of the tumor suppressor retinoblastoma protein (Rb), and induction of active cell death (cytotoxic action) were determined. Combination hormonal therapy showed both increased cytostatic and cytotoxic activity as compared to either monotherapy. The increased cytostatic action was mediated by Rb activation; whereas, the cytotoxic (pro-apoptotic) action of combined hormonal therapy correlated to a significant reduction in Rb protein levels. To test the apparent role of Rb protein loss in the pro-apoptotic action of combined hormonal therapy, Rb was downregulated in MCF-7 cells using siRNA-targeting. The siRNA-mediated knockdown of Rb combined with 4-OHT therapy resulted in a pro-apoptotic action similar to that resulting from 4-OHT and MIF combination treatment, which included increased cell detachment from the monolayer, high-molecular-weight genomic DNA fragmentation, and cleavage of poly ADP-ribose polymerase (PARP) and lamin A. From these studies, we conclude that Rb protein downregulation is required for 4-OHT-treated, estrogen receptor positive (ER + ) breast cancer cells to undergo active cell death. We discuss the potential of using an antiprogestin such as MIF plus antiestrogen treatment to more effectively downregulate Rb in ER + breast cancer cells to increase the overall cytotoxic action of hormonal therapy.

A role for macroautophagy in protection against 4-hydroxytamoxifen–induced cell death and the development of antiestrogen resistance

“…27]. However, our past studies have determined that only a small percentage of the population of ER + breast cancer cells die in response to antiestrogen therapy; most of the cell population is growth arrested in the G 1 phase of the cell cycle and viable (28,29). In this study, we tested the hypothesis that 4-OHT induces macroautophagy in ER + breast cancer cells that do not die and facilitates the development of antiestrogen resistance.…”

“…Thus, our working model is that macroautophagosome function contributes to ER + breast cancer cell survival and facilitates the development of acquired antiestrogen resistance, whereas blockade of autophagosome formation/function during antiestrogen therapy induces a caspase-9 -dependent cell death. From our previous studies, we know that caspase-9 is activated by antiestrogen treatment in MCF-7 cells (28,29). Thus, we used the caspase-9 inhibitor z-LEHD-fmk to block caspase-9 activation in 4-OHT -treated MCF-7 cells.…”

“…Our past studies have further determined that antiestrogen (4-OHT and ICI 182,780) treatment of MCF-7 cells typically induces cell death in only 20% to 30% of the population (6,28,29). The dying cells characteristically detach from the monolayer and show increased levels of high molecular weight DNA fragmentation, cleaved PARP, and cleaved lamin A compared with the adherent, viable monolayer cells (28,29). Because f70% of the MCF-7 cell population is not killed by antiestrogen therapy, this study sought to determine if 4-OHT -induced macroautophagy occurs in the surviving, adherent cells as well as the detached, dying 4-OHT -treated cells.…”

“…Size-separated proteins were transferred onto polyvinylidene difluoride membrane (Bio-Rad) by electroblotting. Immunoblotting was done as previously described (28,29). Primary antibodies were used according to the manufacturer's protocol and included Beclin 1 (Santa Cruz Biotechnology), cleaved poly(ADP-ribose) polymerase (PARP; Cell Signaling), cleaved lamin A (Cell Signaling), ERa (Santa Cruz Biotechnology), progesterone receptor (Santa Cruz Biotechnology), LC3 II (Abcam), caspase-3 (Cell Signaling), caspase-9 (Cell Signaling), and h-actin (Sigma).…”

Antiprogestins in Ovarian Cancer

“…In prostate cancer, the antiprogestin blocked growth of androgen-sensitive and androgen-insensitive LNCaP and PC-3 cells (El Etreby et al,2000a;El Etreby et al,2000b;Liang et al,2002;Tieszen et al,2011). In breast cancer, mifepristone inhibited the growth of T-47D (Musgrove et al,1997), MCF-7, and MDA-MB-231 cells (Tieszen et al,2011); particularly in MCF-7 cells, mifepristone had an additive lethal effect when associated with the antiestrogen tamoxifen (El Etreby et al,1998), as well as a synergistic lethal interaction with the Chk-1 inhibitor 7-hydroxystaurosporine (UCN-01) (Yokoyama et al,2000) and with 4-hydroxytamoxifen (Schoenlein et al,2007). Mifepristone also blocked the growth of MCF-7 sublines made resistant to 4-hydroxytamoxifen (Gaddy et al,2004) and was lethal to MDA-MB-231 cells that are devoid of estrogen and progesterone receptors (Liang et al,2003).…”