IBYME   02675
INSTITUTO DE BIOLOGIA Y MEDICINA EXPERIMENTAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
. Signal transducer and activator of transcription 3 (Stat3) enhances Progesterone Receptor (PR) activation of transcription in breast cancer cells.
Autor/es:
CECILIA J. PROIETTI, WENDY BEGUELIN, MARÍA CELESTE DÍAZ FLAQUÉ, MARTÍN RIVAS, MERCEDES TKACH, EDUARDO CHARREAU, ROXANA SCHILLACI AND PATRICIA V. ELIZALDE
Lugar:
Carefree, Arizona, USA
Reunión:
Congreso; Extra-Nuclear Steroid Receptors: Integration with Multiple Signaling Pathways; 2008
Resumen:
Dear Applicant,
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We have previously demonstrated that the
synthetic progestin medroxyprogesterone acetate (MPA) is able to induce Stat3
transcriptional activation, which is in turn an absolute requirement for
progestin stimulation of in vitro and in vivo breast cancer growth.
We have performed our study both in C4HD cells from an experimental model of
hormonal carcinogenesis in which MPA induced mammary adenocarcinomas in Balb/c
mice, and in the human breast cancer cell line T47D (Mol Cell Biol, 25: 4826,
2005).Therefore, we here assessed whether the requirement of Stat3 activation
in progestin-stimulated breast cancer cell proliferation could be due to
bi-directional cross-talks between progestins and Stat3, where Stat3 in turn
regulates PR transcriptional activation. We studied whether Stat3, acting as a
coactivator, could modulate PR function, both when PR binds to specific
progesterone response elements (PRE) in the promoter regions of target genes,
such as bcl-X gene, and when PR regulates the transcription of p21 gene, which lacks canonical PREs in its
promoter region. Our present findings evidenced that MPA treatment of C4HD
cells induced an increase in bcl-XL mRNA levels. This effect
was completely abolished by transfection with a dominant negative Stat3
expression vector, Stat3Y705-F, and in the presence of the progestin antagonist
RU486. In addition, we also found that MPA treatment of both C4HD and T47D
cells, induced an increase in the levels of p21 protein expression, which was
further enhanced in a dose-dependent manner in the presence of a plasmid
expressing a constitutively activated Stat3 mutant, Stat3-C. To study the
effect of Stat3 on PR-mediated transcriptional activity, C4HD and T47D cells
were transiently transfected with a luciferase reporter plasmid under the
control of the bcl-XP4 promoter, together with Stat3-C. MPA treatment resulted in an increase in
luciferase activity, in accordance with previous results describing the
presence of two hormone responsive
elements present in the fourth promoter of bcl-X gene. We found
that Stat3-C enhanced progestin-induced PR transcriptional activity in a
dose-dependent manner and that this effect was abolished by RU486 and by
cotransfection with Stat3Y705-F. Similar
results were obtained with the
well-characterized progesterone-responsive luciferase reporter plasmid
MMTV-luc. We also
studied the effect of Stat3 on PR capacity
to regulate p21 transcription by tethering to DNA-bound trans-acting factor Sp1.
C4HD and T47D cells were transiently transfected with a p21 promoter
reporter construct containing two Sp1 binding sites, together with increasing
amounts of Stat3-C. We found that Stat3-C enhanced progestin-induced PR
transcriptional activity in a dose-dependent manner. This effect was completely
abolished when Stat3 expression was silenced using an siRNA strategy. Finally,
we explored the specific association of Stat3 and PR to the PRE region of the bcl-X
gene in the context of living cells, by performing Chromatin
Immunoprecipitation (ChIP) and Sequential ChIP Assays. Our findings unraveled that
MPA treatment of primary cultures of C4HD cells induced PR and Stat3
recruitment to the bcl-X promoter. We also evaluated the specific
association of Stat3 and PR to the Sp-1 binding site in the p21 promoter and found
that MPA treatment of T47D cells induced
Sp-1, PR and Stat3 recruitment to the p21 promoter.
These results provide the
first evidence that Stat3 acts as a coactivator of PR when PR binds directly to
target genes containing PREs in the promoter region, or when PR regulates
transcription by tethering to Sp1.