INVESTIGADORES
ROSEMBLIT Cinthia
congresos y reuniones científicas
Título:
Nuclear interaction of ErbB2 and Stat3 in the cyclin D1 promoter: Role of ErbB2 as coactivator in breast cancer cells
Autor/es:
BEGUELIN W; PROIETTI CJ ; DIAZ FALQUE MC; ROSEMBLIT C ; RIVAS MA ; SUNDBLAD V ; TKACH M ; CHARREAU EH ; SCHILLACI R ; ELIZALDE PV
Lugar:
San Diego, California
Reunión:
Congreso; Advances in Breast Cancer Research. AACR Special Meeting; 2009
Institución organizadora:
American Association for Cancer Research (AACR)
Resumen:
Nuclear interaction of ErbB2 and Stat3 in the cyclin D1 promoter: role
of ErbB2 as coactivator in breast cancer cells
Wendy Béguelin, Cecilia J. Proietti, María Celeste Díaz Flaqué, Cinthia
Rosemblit, Martín Rivas, Victoria Sundblad, Mercedes Tkach, Eduardo H.
Charreau, Roxana Schillaci, Patricia V. Elizalde.
We have previously demonstrated that heregulin (HRG), a ligand for ErbB
receptors, activates signal transducer and activator of transcription 3
(Stat3) in primary cultures of ErbB2 overexpressing C4HD cells, from a
murine progestin-dependent mammary tumor. ErbB2 activity is an absolute
requirement in the mechanisms of HRG stimulation of Stat3 activity.
Recent findings have demonstrated ErbB2 nuclear migration and its
function as a transcription factor. In this work, we studied whether HRG
induces ErbB2 nuclear migration and its interaction with Stat3. By
immunofluoresence staining and confocal microscopy we demonstrated that
HRG treatment for 30 to 120 min induces ErbB2 nuclear migration and
colocalization with Stat3 in C4HD and human breast cancer T47D cells.
Physical association of both proteins in the nucleus was evidenced by
coimmunoprecipitation studies. Cyclin D1 is a cancer-related gene that
contains Stat3 binding sites (GAS sites) but lacks ErbB2 response
elements (HAS sites). By chromatin immunoprecipitation assays, we
demonstrated that HRG induces in vivo binding of Stat3 and ErbB2 to the
GAS sites of the cyclin D1 promoter. Simultaneous binding of Stat3 and
ErbB2 to the GAS sites was shown by sequential ChIP studies. This
finding prompted us to evaluate the ability of HRG to regulate cyclin D1
protein expression. Western Blot assays indicated that HRG treatment
for 6 to 48 hs induces cyclin D1 expression in C4HD and T47D cells.
Inhibition of ErbB2 activity by AG825 or knockdown of ErbB2 expression
with ErbB2 siRNAs and abolishment of HRG-induced Stat3 activation with
Jak inhibitor I or silencing Stat3 expression with Stat3 siRNAs
significantly inhibited HRG capacity to induce cyclin D1 expression.
Next, we explored whether HRG induces the cyclin D1 promoter directly
via Stat3 binding to its response elements. C4HD and T47D cells
transiently transfected with cyclin D1 promoter luciferase construct
showed an enhanced transcriptional activity with HRG treatment.
HRG-induced luciferase activity was indeed increased by cotransfection
with a constitutively active form of Stat3 and was inhibited with a
dominant negative Stat3 expression vector. Overexpression of increasing
amounts of ErbB2 wt resulted in a dose-dependent ErbB2 capacity to
enhance HRG-induced Stat3 transcriptional activity. On the other hand,
transfection with increasing amounts of an ErbB2 mutant that is
defective in nuclear entry but retains its cell-surface location and
functions (ErbB2DNLS) resulted in abrogation of HRG-induced Stat3
activation of the cyclin D1 promoter. Finally, we addressed the effect
of targeting ErbB2 in in vivo HRG-dependent growth of C4HD breast
tumors. Transfection of C4HD cells with the ErbB2DNLS expression vector
significantly inhibited these cells ability to form tumors in syngeneic
mice. Taken together, these results suggest a new role of ErbB2 as a
coactivator in the mechanism of HRG-induced transcriptional activation
of Stat3. We also found nuclear ErbB2 to be a requisite for HRG
stimulation of in vitro and in vivo breast cancer growth.