Tumor necrosis factor transactivates ErbB2 in breast cancer cells

MARTÍN A. RIVAS, MERCEDES TKACH, CECILIA J. PROIETTI, CINTHIA ROSEMBLIT, WENDY BEGUELIN, VICTORIA SUNDBLAD, M. CELESTE DÍAZ FLAQUÉ, EDUARDO H. CHARREAU, PATRICIAV. ELIZALDE, ROXANA SCHILLACI

San Antonio, TX, USA

Simposio; CTRC-AACRSan Antonio Breast Cancer Symposium; 2008

Dear Applicant, <!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> We have previously shown that TNFα induces proliferation of the murine mammary adenocarcinoma C4HD through activation of the PI-3K/Akt signaling pathway that converges on the transcriptional activation of NF-κB. Since C4HD tumor overexpresses ErbB-2 and given that this tyrosine kinase plays a critical role in C4HD cell proliferation, we wondered whether interactions between TNFα and ErbB-2 could be taking place. Our findings revealed that treatment of C4HD cells with the ErbB2 inhibitor AG825 blocked TNFα-induced proliferation. Similar results were obtained using the human ErbB2-overexpressing cell line SK-BR-3. Then, we studied the effect of TNFα on ErbB2 phosphorylation in C4HD and SK-BR-3 cells. We found that TNFα increased total ErbB2 tyrosine phosphorylation in C4HD and SK-BR-3 cells, as well as on the specific residues tyrosines 927 and 1172 in murine cells and on its human homologues 877 and 1222. These effects where not caused by the release of ErbBs ligands from the cell membrane by TNFα, since treatment with the metalloprotease inhibitor GM6001 did not affect TNFα-induced ErbB2 phosphorylation. We then studied if c-Src was involved in the above effect given that it is known to directly phosphorylate ErbB2 in the Tyr 877 residue. We found that addition of PP2, a Src family inhibitor, completely inhibited phosphorylation of Tyr 927/877 ErbB2, and that to a lesser degree it inhibited Tyr 1172/1222 ErbB2 in both cell types. We also performed an in vitro cold phosphorylation assay where we observed that c-Src immunoprecipitated from C4HD cells treated with TNFa was able to phosphorylate ErbB2 on Tyr 927 residue. Taken together, these results indicate that TNFa induces phosphorylation of ErbB-2 at Tyr877/927 residue and that c-Src is the tyrosine kinase involved in this effect. In addition, we observed that upon TNFα stimulation, ErbB2 associated with ErbB3 leading to PI-3K/Akt pathway activation. Treatment of cells with AG825 inhibited Akt and NF-κB activation by TNFα, as evidenced by western blots of phospho proteins and reporter gene studies, respectively. The above data for the first time identify TNFα as a cytokine able to transactivate ErbB2, disclosing c-Src involvement in such effect. Also we demonstrated that TNFa ability to activate Akt and NF-kB transcriptional activation is dependent on ErbB2 phosphorylation in breast cancer cells that overexpress ErbB2. Interestingly, TNFα appears as a new player in ErbB2-overexpressing breast tumors, and its eventual worth as a prognostic factor in anti ErbB2 therapy is yet to be determined.