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 ErbB-2 overexpressing C4HD cells, from a murine progestin-dependent mammary tumor. ErbB-2 activity is an absolute requirement in the mechanisms of HRG stimulation of Stat3 activity. Recent findings have demonstrated ErbB-2 nuclear migration and its function as a transcription factor. In this work, we studied whether HRG induces ErbB-2 nuclear migration and its interaction with Stat3 in breast cancer cells. We demonstrated that HRG induces ErbB-2 nuclear migration and colocalization with Stat3 in C4HD and human T47D breast cancer cells. Cyclin D1 is a cancer-related gene that contains Stat3 binding sites (GAS sites) but lacks ErbB-2 response elements (HAS sites). By chromatin immunoprecipitation assays (ChIP) and sequential ChIP, we demonstrated that HRG induces in vivo binding of Stat3 and ErbB-2 to the GAS sites of the cyclin D1 promoter. This finding prompted us to evaluate the ability of HRG to regulate cyclin D1 expression. By inhibiting ErbB-2 and Stat3 activation or silencing their expression, we demonstrated that ErbB-2 and Stat3 participate in the mechanism of HRG-induced cyclin D1 protein expression. Next, we explored whether HRG induces cyclin D1 promoter directly via Stat3 binding to its response elements. C4HD and T47D cells transiently transfected with a cyclin D1 promoter luciferase construct showed an enhanced transcriptional activity with HRG treatment. Overexpression of increasing amounts of ErbB-2 wt resulted in a dose-dependent ErbB-2 capacity to enhance Stat3 transcriptional activity induced by HRG. On the other hand, transfection with increasing amounts of an ErbB-2 mutant that is defective in nuclear entry but retains its cell-surface location and functions (ErbB-2ΔNLS) resulted in abrogation of HRG-induced Stat3 activation of cyclin D1 promoter. Finally, we addressed the effect of targeting ErbB-2 in in vivo HRG-dependent growth of C4HD breast tumors. Transfection of C4HD cells with the ErbB-2ΔNLS expression vector significantly inhibited these cells’ ability to form tumors in syngeneic mice. Taken together, these results suggest a new role of ErbB-2 as a coactivator in the mechanism of HRG-induced transcriptional activation of Stat3. We also found nuclear ErbB-2 to be a requisite for HRG stimulation of in vitro and in vivo breast cancer growth.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1718.