Nuclear interaction of ErbB2 and Stat3 in the cyclin D1 promoter: role of ErbB2 as coactivator in breast cancer cells

BEGUELIN W, PROIETTI C, DÍAZ FLAQUÉ MC, ROSEMBLIT C, RIVAS M, SUNDBLAD V, TKACH M, CHARREAU EH, SCHILLACI R, ELIZALDE PV.

San Diego, California, USA.

Congreso; Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications, AACR Special Conference; 2009

American Association for Cancer Research

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.