Etanercept as a new tool for treatment of Herceptin-resistant breast cancer induced by tumor necrosis factor alpha.

RIVAS M, TKACH M, BéGUELIN W, PROIETTI C, DíAZ FLAQUé M, MARONNA E, FRAHM I, CHARREAU E, ELIZALDE P, SCHILLACI R

Washington DC, USA

Congreso; 101st Annual Meeting American Association for Cancer Research; 2010

American Association for Cancer Research

Breast cancer overexpressing ErbB2 is associated with high aggressiveness and elevated metastatic potential. Several therapies have been designed targeting receptor tyrosine kinase ErbB2, such as the low molecular weight inhibitor Lapatinib and the monoclonal antibody Herceptin. We have already demonstrated that tumor necrosis factor alpha (TNF) induces proliferation of BT-474 and SKBR-3 human and C4HD murine breast cancer cells which overexpress ErbB2, through the transactivation of ErbB2 and subsequent activation of transcription factor NF-κB, and through increase of Cyclin D1 protein expression. In the present study we evaluated the effectiveness of clinical drugs which block ErbB2, in order to inhibit NF-κB transcriptional activity and TNF-induced proliferation. We transfected BT-474 and SKBR-3 breast cancer cells with a luciferase reporter gene under control of a NF-κB response element and observed that the pharmacological ErbB2 inhibitor AG825 (100 μM)and the Lapatinib analog GW2974 (1 μM) blocked NF-κB activity induced by TNF. However, when we used 10 μg/ml Herceptin, TNF still activated NF-κB. We also monitored NF-κB activation through Western blot of phosphorylated IκBα, the protein which inhibits NF-κB traslocation to the nucleus, with same results. By reporter gene assays, we then explored activation of Cyclin D1 promoter, a target gene of NF-κB. While both AG825 and GW2974 blocked TNF-induced Cyclin D1 promoter activation, Herceptin did not. The endogenous protein Cyclin D1 mirrored the profile obtained with reporter gene assays. BT-474 proliferation increased in the presence of TNF (75 ± 12% vs control cells) measured by 3H-thimidine incorporation, cell count and flow cytometry of cells stained with propidium iodide. AG825 and GW2974 completely blocked TNF-induced proliferation, but Herceptin failed to do so. Bearing in mind that TNF is frequently expressed in invasive breast tumors, our results suggest that TNF may be one of the factors which confer Herceptin resistance.We therefore hypothesized that blocking TNF through the TNFR2-FcIgG fusion protein Etanercept may overcome Herceptin resistance. We observed that Etanercept (5 mg/kg, i.p. twice wk) inhibited TNF-induced in vitro proliferation of C4HD cells, a murine breast adenocarcinoma that produces TNF. When this tumor was growth in nude mice and treated with Etanercept the tumor size was reduced by 37.6 ± 1.1 % (P<0.05). Interestingly, histological examination of said tumors revealed that there were fewer mitotic figures and a lower percentage of necrotic and fibrotic areas in the tumors from mice treated with Etanercept, as compared to the group treated with an irrelevant IgG. Our results propose Etanercept as a new agent to overcome clinically observed Herceptin resistance.