Tumor necrosis factor alpha (TNF)-induced breast cancer cell proliferation is mediated through TNF receptor type 1-activation of NF-êB via JNK and Akt.

MARTÍN A. RIVAS, ROMINA P. CARNEVALE, CINTHIA ROSEMBLIT, CECILIA J. PROIETTI, WENDY BEGUELIN, EDUARDO H. CHARREAU, PETER BROUCKAERT, PATRICIA V. ELIZALDE, ROXANA SCHILLACI.

Los Angeles, USA

Congreso; Annual Meeting of the American Association for Cancer Research; 2007

American Association for Cancer Research

TNFa enhances proliferation of chemically-induced mammary tumors and of T47D human cell line through not fully understood pathways. We have already shown that TNFa induces proliferation of murine breast cancer cells C4HD, from a mammary adenocarcinoma induced by the synthetic progestin medroxyprogesterone acetate in female Balb/c mice, and of human breast cancer cell line T47D through the activation of p42/p44 MAPK, JNK and PI-3K/Akt (Proc Am Assoc Cancer Res 2006;48:1647). In order to gain further insight into the role of TNFa receptor (TNFR) 1 and TNFR2 in breast cancer cell proliferation, here we used genetically engineered TNFa muteins that bind selectively either murine TNFR1 (R1-TNF) or TNFR2 (R2-TNF), or both receptors (R1R2-TNF). Proliferation assays, performed by [3H]-thymidine incorporation, showed that R1-TNF and R1R2-TNF induced a dose-dependent proliferation similar to the one observed in response to wild type (wt) TNFa, with the highest proliferation reached at 20 ng/ml. However, R2-TNF induced proliferation modestly at 200 ng/ml. By western blot, we observed that 20 ng/ml R1-TNF were able to induce p42/p44 MAPK, JNK and Akt phosphorylation to the same extent as R1R2-TNF and wtTNFa. On the other hand, 200 ng/ml R2-TNF were able to induce activation of p42/p44 MAPK but minimally of JNK and Akt. In addition, we determined the participation of each receptor in TNFa-induced NF-êB transcriptional activation. C4HD cells were transiently transfected with a êB-luc reporter construct and stimulated with muteins. We observed that TNFa, R1-TNF or R1R2-TNF induced a 3-fold activation of NF-êB while R2-TNF failed to stimulate luciferase activity. Furthermore, we used pharmacological inhibitors of p42/p44 MAPK (PD98059 and U0126), JNK (SP600125) and PI-3K/Akt (LY294002) pathways to determine their involvement in NF-êB activation and IêBa phosphorylation of C4HD and T47D cells. Blockage of JNK and PI-3K/Akt abolished TNFa-induced NF-êB transcripcional activity and IêBa phosphorylation. Blockage of p42/p44 MAPK had only a partial inhibitory effect on TNFa-induced NF-êB activation, and no decrease in IêBa phosphorylation was detected. Finally, we performed proliferation assays in the presence of Bay 11-7082, a pharmacological inhibitor that prevents IkBa phosphorylation, and we observed a complete inhibition of TNFa-induced C4HD or T47D cells proliferation. Our results strongly suggest that breast cancer cell proliferation induced by TNFa is mainly mediated through TNFR1 which, through the activation of JNK and Akt, stimulates NF-êB transcriptional activity. Despite the fact that p42/p44 MAPK is essential for TNFa’s proliferative effects and both receptors are able to induce its activation, the downstream transcription factor seems not to be NF-êB. Finally, we showed that NF-kB is a key transcription factor for TNFa-induced breast cancer cells proliferation. Tumor necrosis factor alpha (TNFa)-induced breast cancer cell proliferation is mediated through TNFa receptor type 1-activation of NF-êB via JNK and Akt.