Heregulin (HRG) induces signal transducer and activator of transcription 3 (Stat3) phosphorylation through the assembly of a multimeric protein complex among ErbB-2, Src and progesterone receptor (PR) in breast cancer cells

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

Los Ángeles, USA

Congreso; AACR Annual Meeting; 2007

American Associatiom of Cancer Research (AACR)

We have already demonstrated that HRG modulates Stat3 tyrosine phosphorylation via ErbB-2, Src and Janus kinases in an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice and in the human breast cancer line T47D. (Proc Amer Assoc Cancer Res 2006;47:3645). In the present work, we explored whether a multimeric protein complex was assembled in  the mechanism of HRG induction of Stat3 tyrosine phosphorylation. Physical association among proteins was explored by performing coimmnuprecipitation experiments. First, whole cell protein extracts from C4HD or T47D cells were immunoprecipitated with an anti-ErbB-2 antibody and immunoblotted with an anti-Stat3 antibody. HRG was able to induce strong association between ErbB-2 and Stat3, which was abolished by inhibition of ErbB-2 phosphorylation with the selective ErbB-2 inhibitor AG485. In addition, we found that HRG caused ErbB-2 association with phospho-Stat3. As expected, since inhibition of ErbB-2 activity resulted in abolishment of Stat3 phosphorylation on tyrosine 705, no association between phospho-Stat3 and ErbB-2 was observed in presence of AG825. In the same protein complex we found that HRG increased ErbB-2 and Src physical association, which was inhibited in presence of the selective Src family kinase inhibitor PP2 and AG825. We obtained similar results when we reversed the coimmunoprecipitation-immunoblotting procedure using Stat3 as the immunoprecipitating antibody. Since our previous work showed the capacity of HRG to activate PR (Mol Cell Biol, 23.3: 1095, 2003), we also explored the participation of PR in HRG-induced Stat3 tyrosine phosphorylation. The progestin antagonist RU486 inhibited the assembly of the complex as determinated by, blocking the capacity of HRG to induce Stat3 tyrosine phosphorylation. Our findings showed that HRG induces the assembly of a multimeric protein complex among ErbB-2, Src and PR, which must be functional to recruit and phosphorylate Stat3 on tyrosine 705 in human and murine breast cancer cells