Progestin nongenomic actions regulate cell proliferation and proteases activity in breast cancer cells

ROMINA CARNEVALE; ROXANA SCHILLACI; ALEJANDRO J. URTREGER; MARIANA SALATINO; CECILIA PROIETTI; MARTIN RIVAS; CINTHIA ROSEMBLIT; EDUARDO CHARREAU; V. BOONYARATANAKORNKIT; D. EDUARDS; ELISA D. BAL DE KIER JOFFÉ; PATRICIA ELIZALDE

Washington, EEUU.

Congreso; 97th Annual Meeting of the American Association for Cancer Research; 2006

American Association for Cancer Research (AACR)

Rapid, nongenomic steroids actions have been described in a wide variety of cell models. Recently, we demonstrated that progesterone regulates proliferation and proteases activity via rapid activation of MAPKs and PI-3K/Akt pathways in the progestin-dependent murine mammary tumor line C4HD (Proc. Am. Assoc. Cancer Res. 46:299, 2005). In this study, we investigated the involvement of progesterone receptor (PR) in medroxyprogesterone acetate (MPA)-induced rapid activation of MAPKs and PI-3K/Akt pathways using reconstitution experiments with either wild-type PR-B or C587A-PR, a transcriptionally impaired PR-B mutant. We also explored MPA capacity to regulate cell proliferation and proteases activity via nongenomic mechanisms. Here we used two PR null mammary tumor cells: the murine mammary metastatic tumor line LM3 and human T47D-Y cells. Both lines were transiently transfected with expression vectors containing the human PR-B (LM3-PRB; T47D-Y-PRB) or the C587A-PR mutant (LM3-C587A-PR; T47D-Y-C587A-PR). MPA (10nM) treatment for 5 to 10 min induced p42/p44MAPKs and PI-3K/Akt activation in both LM3 and T47D-Y cells transfected with either PR-B or C587A-PR. Preincubation of these cells with the progestin antagonist RU486 significantly inhibited MPA-induced p42/p44MAPKs and PI-3K/Akt activation. We next studied MPA effects in proliferation by incorporation of [3H] thymidine. MPA significantly stimulated LM3-PRB (46±7%), LM3-C587A-PR (43±4%), T47D-Y-PRB (43±8%) and T47D-Y-C587A-PR (40±5%) cell proliferation. Preincubation with RU486 significantly inhibited MPA-induced proliferation of both cell types transfected with either PR-B or C587A-PR. Abolishment of p42/p44MAPKs activity with U0126 (5ìM) or PI-3K/Akt activity with Ly294002 (2ìM) resulted in abrogation of MPA-induced growth in these cells. Finally, we studied the effect of MPA treatment on urokinase plasminogen activator (uPA) activity, an important enzyme involved in tumor cell invasion and metastasis, by casein and plasminogen zymography. Treatment of LM3-PRB and LM3-C587A-PR cells with MPA reduced uPA activity (37±7% and 35±5% respectively). This effect was blocked by pretreatment of these cells with RU486. Preincubation of LM3-PRB and LM3-C587A-PR with either U0126 or Ly294002 resulted in a significant reduction of MPA-inhibition of uPA activity. These results provide the first evidence of MPA-inhibition of uPA activity by a nongenomic mechanism mediated by p42/p44MAPKs and PI-3K/Akt pathways in breast cancer cells. Our findings demonstrated that PR mediates progestin regulation of cell proliferation and proteases activity in both human and murine mammary tumor cells by activation of cytoplasmic signaling pathways and independently of PR transcriptional activity.