Canonical and non-canonical membrane progesterone receptors (PR) in murine mammary carcinomas. Agonistic effects of progestins and antiprogestins mediating rapid non-genomic effects

BOTTINO MARÍA CECILIA; MONDILLO CAROLINA; ROJAS PAOLA; PIGNATARO OMAR; LÜTHY ISABEL; AMORNPHIMOLTHAM PANOMWAL; GUTKIND SILVIO; LANARI CLAUDIA

Los Angeles, California, USA

Congreso; AACR Annual Meeting; 2007

We have reported the presence of two progesterone (Pg) binding sites in mammary carcinomas of the medroxyprogesterone acetate (MPA) breast cancer model (Helguero et al, J.Steorid Molec Biol, 84(1):9-14, 2003): a high affinity (Kd= 43 ± 9 pM) and low capacity (Q= 9 ± 3 fmol/mg protein) and the canonical lower affinity (Kd = 9.2 ± 4.2 nM) with higher capacity PR (Q = 376 ± 64 fmol/mg of protein). Pg-binding at both sites was displaced by the antagonist RU 486. We have also demonstrated that MPA stimulates cell growth in the same mammary carcinomas with EC50 at the same two Kd. RU 486 inhibited cell proliferation at concentrations higher than 0.1 nM. By confocal imaging using Pg coupled to BSA-FITC we were able to demonstrate Pg binding sites at the cell membrane. Non canonical PR (mPR alpha, beta and gamma) belonging to the family of G coupled seven transmembrane domain receptors, have been recently described (Zhu et al, PNAS, 100: 2237–2242, 2003). These receptors show a Kd of 28–39nM and fail to bind classical antiprogestins. Their physiological role is still uncertain. Pg, upon binding to these receptors, induces a decrease in cAMP levels (Zhu et al, PNAS, 100: 2231–2236, 2003).The aim of this study was a) to evaluate the presence of canonical PR at the cell membrane, b) to evaluate the expression of non-canonical mPR in these tumor cells and c) to study the effects of MPA and RU 486 on MAPK activation and cAMP regulation. We used the C4-HD and C4-HI tumors and two C4-HD-derived cell lines MC4-L2 and MC4-L5. Starting with 15 grams of tumors or with 7.5 107cells we isolated 100 mg of protein of purified membrane fractions in which, by western blots, PR A and PR B were detected. By inmunofluorescence and confocal imaging PR signal colocalized with membrane caveolin-1. Expression of the three mPR (alpha, beta and gamma) was detected by RT-PCR and qPCR in the C4-HD tumor and in the cell lines. Thus, these results suggest that canonical and non-canonical mPR are present at the cell membrane. The effects of MPA and RU486 (0.1 and 10 nM) on MAPK phosphorylation and cAMP levels (1, 3 or 10 min) were studied. MPA and RU 486 induced a similar increase in both cAMP (RIA, p<0.05) and in MAPK phosporylation (as judged by western blots and immunofluorescence analysis). Since a) MPA stimulates while RU 486 inhibits cell proliferation b) similar rapid effects were obtained with both agents and c) non-canonical mPR do not bind RU486, we propose that canonical PR located at the cell membrane are responsible for these non-genomic effects. The activation of these pathways could sensitize the cells to other signals that will finally drive to proliferation or inhibition of cell growth.