Membrane classic progesterone receptors in murine mammary carcinomas: Agonist effects of the antiprogestin RU 486 mediating rapid non genomic effects.

BOTTINO MC; ROJAS P; LUTHY IA; LANARI C

San Diego, EEUU

Congreso; American Association of Cancer Research; 2008

Association of Cancer Research

Membrane classic progesterone receptors in murine mammary carcinomas: agonist effects of the antiprogestin RU 486 mediating rapid non genomic effects. MC Bottino, P. Rojas, I Luthy  C Lanari. Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires, Argentina. Murine mammary carcinomas of the medroxyprogesterone acetate (MPA) breast cancer model express high levels of estrogen and progesterone receptors (PR) and may regress completely with antiprogestin treatment. We have previously investigated the presence of classical PR at the cell membrane of these tumors and the presence of both, PRA and PRB, was detected by western blots using purified membrane extracts. In addition, a discrete co-localization of PR and caveolin 1 was detected at the cell membrane using confocal imaging. We have also demonstrated that both, progestins and antiprogestins, increased ERK phosphorylation and cAMP levels. Taking into account that under these experimental conditions RU 486 inhibits cell proliferation, we hypothesized that concentrations of RU 486 lower than those able to elicit genomic effects would increase cell proliferation. The aim of this study was: a) to evaluate the ability of MPA and RU 486 to modify the membrane localization of PR and b) to investigate whether low concentrations of RU 486 increased cell proliferation. Primary cultures of C4-HI tumor were set up. Epithelial cells were grown in chamber slides and incubated with DMEM-F12 with 1% ch FCS for 24 hs. Cells were then incubated for 20 min with MPA or RU 486 (10 nM) and PR and pSer190 PR were detected using Ab 7 and Ab 11 Neomarkers antibodies respectively. Both treatments increased membrane and nuclear pSer190 PR staining. The effect on cell proliferation was studied by 3H-thymidine uptake. The cells were starved for 24 hs and were treated for 48 hs with increasing concentrations of RU 486 (10-15 to 10-7M). A pulse of thymidine was added in the last 20 hs. A significant increase in cell proliferation was observed at concentrations ranging from 10-14 to 10-10 M (p<0.001), while an inhibitory effect was observed at concentrations higher than 1 nM (p<0.05). In addition, cells were incubated in the presence of 10 nM MPA with RU 486 (0.01 nM or 10 nM). As expected, RU486 induced a significant inhibition of MPA-induced cell proliferation at 10 nM while a stimulatory effect was observed at 0.01 nM. Since RU 486 and MPA induce ERK and PR phosphorylation independently to their ability to stimulate or inhibit cell proliferation, we propose that these non genomic effects are priming the cell for a further genomic effect. In the absence of this genomic effect due to the low concentrations of ligand unable to activate nuclear PR, the increase in ERK phosphorylation will result in an increase in cell proliferation. These results highlight the relevance of adequate antagonist concentrations in the management of breast cancer.