Heregulin (HRG) induces transcriptional activation of signal transducer and activator of transcription 3 (Stat3) via ErbB-2 and Src-dependent mechanism in breast cancer cells

CECILIA J. PROIETTI, CINTHIA ROSEMBLIT, MARIANA SALATINO, WENDY BÉGUELIN, ROXANA SCHILLACI, ROMINA CARNEVALE, MARTÍN RIVAS, EDUARDO H. CHARREAU, PATRICIA V. ELIZALDE

Washington DC, USA

Congreso; Annual Meeting of the American Association for Cancer Research; 2006

We have already exhaustively characterized mechanisms of progestin-induced growth in an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice. In this model system, proliferation is driven by a complex cross talk between progestins and growth factors pathways. We have previously demonstrated that MPA induced Stat3 transcriptional activation is a requisite for MPA stimulation of C4HD cell growth (Mol Cell Biol, 25: 4826, 2005). To gain further insight in the interactions among progestins, HRG/ErbBs, and Stats in breast tumor growth, we explored the capacity of HRG to modulate Stat3 transcriptional activation. We found that HRG treatment of C4HD cells for 48 h up-regulated Stat3 protein expression. Furthermore, HRG was able to induce Stat3 tyrosine phosphorylation after 5-10 min stimulation in C4HD cells. This effect was abolished when ErbB-2 expression was blocked using antisense oligodeoxynucleotides (ASODNs) to its mRNA and when HRG binding to ErbB-3 was inhibited using anti-ErbB-3 monoclonal antibody. Blockage of Jak2 activity, by the use of Dominant Negative (DN) Jak2 expression vector, and of Src activity with the selective Src family kinase inhibitor, PP2, also inhibited HRG-induced Stat3 tyrosine phosphorylation. Electrophoretic mobility shift assay (EMSA) showed that HRG treatment of C4HD cells for 15 min induced Stat3 binding to DNA, using as labeled probe the sis-inducible element (SIE) of the human c-fos promoter. Presence of specific Stat3-DNA complexes after HRG treatment was demonstrated by competition with excess unlabeled oligonucleotides, and by lack of competition with mutated oligodeoxinucleotide probes. The inclusion of a rabbit polyclonal anti-Stat3 antibody in the EMSA reaction supershifted the protein-DNA complex. Transient transfections of C4HD cells with a luciferase reporter gene containing four copies of the m67 high-affinity Stat 3 binding site gene, demonstrated that HRG promoted a significant increase in luciferase activity. HRG-induced Stat3 transcriptional activation was abolished by blockade of ErbB-2, Jak2 and Src activities. Furthermore, inhibition of Stat3 activation by transfection of C4HD cells with the DN Stat3 expression vector, DN Stat3Y705-F, inhibited HRG capacity to induce C4HD cells proliferation. Our findings have, for the first time, demonstrated that Stat3 activation is a requisite for HRG-induced breast cancer cell growth.