NUCLEAR ESTROGEN RECEPTORS ALPHA ARE INVOLVED IN PROGESTIN-DEPENDENT AND INDEPENDENT MAMMARY TUMOR GROWTH

SEBASTIAN GIULIANELLI; VIRGINIA NOVARO; CLAUDIA LANARI.

Denver, Colorado. USA.

Conferencia; 100th AACR Annual Meeting; 2009

AACR

Mouse mammary carcinomas induced by medroxyprogesterone acetate (MPA) are maintained by serial transplantations in BALB/c female mice. Hormone-dependent (HD) tumors need the exogenous administration of MPA while hormone-independent (HI) variants are able to grow in vivo without hormone supply. Both tumor types retain high levels of estrogen (ER) and progesterone receptors (PR). However, isolated HD and HI tumor cells in culture have the same hormone requirements: they are stimulated by progestins and growth factors, and they are inhibited by estrogens, antiprogestins and tamoxifen. Moreover, the pure antiestrogen ICI 182780 inhibited MPA-induced cell proliferation in both tumor types suggesting that ERa is a key protein involved in tumor growth in this model. We have already demonstrated that PR expression is only partially dependent on ERa and that both proteins co-localize in the nucleus, suggesting that nuclear interactions between both receptors might be necessary to induce cell proliferation. The main goal of this study was to determine whether MPA increases ERa phosphorylation and activation in HD tumors and to evaluate the role of ERa in HI tumor growth. High levels of pSer118 and pSer167-ERa were detected by immunohistochemistry in HI tumors and in HD tumors growing only with MPA, suggesting that in the HI tumors ERa is constitutively activated. In primary cultures of tumor cells, we detected by immunofluorescence (IF) and western blot (WB), higher levels of phospho-ERa in quiescent HI cells as compared with HD cells, indicating that activated ERa is not sufficient to induce cell proliferation. We also demonstrated that the PI3K/Akt pathway is involved in the activation of ERa since incubation of HI cells with LY294002 (10mM) reduced pSer167-ERa levels and cell proliferation. However, the MEK/Erk pathway seems not to be involved since PD98059 (1mM) did not decrease ERa phosphorylation. To investigate ERa activation by MPA, HD cells were incubated 30 min with 10nM MPA. An increase in pSer118 and pSer167-ERa levels was observed by WB which correlated with increased binding to ER consensus sequence in the DNA (ERE) as confirmed by electrophoretic mobility shift assays (EMSA). Nuclear co-localization between pSer294-PR and pSer118-ERa, sites involved in transcriptional activation, also increased in MPA-treated HD cells. Interestingly, HI cells showed a high basal ability of ERa to bind to the ERE sequence, which was not amplified by hormone treatment. As a whole, our results indicate that MPA induces ERa phosphorylation and activation in HD tumors. The nuclear association between PR and ERa  appears as a novel mechanism in cell proliferation mediated by progestins in this model. The fact that a higher basal activation and phosphorylation of ERa via PI3K/Akt pathway was observed in HI cells, suggests that constitutive activation of ERa may be participating in the HI phenotype.