Endogenous Coactivator ARA70 Interacts with Estrogen Receptor α (ERα) and Modulates the Functional ERα/Androgen Receptor Interplay in MCF-7 Cells

Marilena Lanzino,Francesca De Amicis,Michael J Mcphaul,Stefania Marsico,Maria Luisa Panno,Sebastiano Andò

doi:10.1074/jbc.m413576200

Marilena Lanzino, Francesca De Amicis + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m413576200

Copy DOI

Abstract

Overexpression of androgen receptor (AR) decreases estrogen receptor alpha (ERalpha) transactivation, which plays a basic role in hormone-dependent breast cancer. This transcriptional interference can be due to shared coactivators. Here we demonstrated that in MCF-7 cells ARA70, an AR-specific coactivator, interacted with endogenous ERalpha, increasing its transcriptional activity, and it was recruited to the pS2 gene promoter. Moreover, a dominant negative ARA70 down-regulated ERalpha transcriptional activity as well as pS2 mRNA. ARA70 overexpression reversed the AR down-regulatory effect on ERalpha signaling. However, in the presence of a progressive increase of transfected AR, ARA70 switched into enhancing the inhibitory effect of AR on ERalpha signaling. These opposite effects of ARA70 were further evidenced by coimmunoprecipitation assay in MCF-7wt, MCF-7-overexpressing AR, and HeLa cells, exogenously expressing an excess of ERalpha with respect to AR or an excess of AR with respect to ERalpha. Thus, ARA70 is a coactivator for ERalpha and may represent a functional link between ERalpha/AR modulating their cross-talk in models of estrogen signaling in MCF-7 and HeLa cells.

Highlights

Overexpression of androgen receptor (AR) decreases estrogen receptor ␣ (ER␣) transactivation, which plays a basic role in hormone-dependent breast cancer
ER␣ Transcriptional Activity Is Inhibited by Overexpressed AR—We investigated whether the down-regulatory effect of AR on estrogen signaling may be related to the induced reduction of the ER␣ content and to a direct interference on ER␣ transcriptional machinery
ER␣-ARA70 Complex Binds the Promoter Region of the Estrogen Target Gene pS2 in MCF-7 Cells—After showing that ARA70 is able to modulate ER␣ transcriptional activity, to explore further whether the ER␣-ARA70 interaction may be important in vivo, we investigated the recruitment of ER␣-ARA70 complex to the promoter of the well characterized estrogen-responsive target gene pS2, either in presence or absence of E2

Summary

EXPERIMENTAL PROCEDURES

Materials—Dulbecco’s modified Eagle’s medium (DMEM/F-12), Lglutamine, Eagle’s non-essential amino acids, penicillin, streptomycin, calf serum, bovine serum albumin, and phosphate-buffered saline were purchased from Eurobio (Les Ullis Cedex, France). For chromatin immunoprecipitation (ChIP) assay samples, cells were grown in 150-mm plates and transfected, using the FuGENE 6 reagent, with an appropriate amount of the various plasmids as indicated in the figure legends. Supernatants were collected and diluted in 1.3 ml of IP buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl, pH 8.1, 16.7 mM NaCl) followed by immunoclearing with 80 ␮l of sonicated salmon sperm DNA/protein A-agarose for 1 h at 4 °C. In Re-ChIP experiments, complexes were eluted by incubation for 30 min in Re-IP buffer (0.5 mM dithiothreitol, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.1, 150 mM NaCl) and subjected again to the ChIP procedure, using anti-ER␣ antibody (F10, Santa Cruz Biotechnology) and normal mouse antibody (Santa Cruz Biotechnology) for the negative control. Data were analyzed by analysis of variance test using the STATPAC computer program

RESULTS

DISCUSSION

Luisa Panno and Sebastiano Andò