Tumor necrosis factor alpha (TNFα) induces proliferation of breast cancer cells by a mechanism that requires TNF receptor type 1 (TNFR1) and type 2 (TNFR2)

1647 TNFα is a pleiotropic cytokine that stimulates growth of normal mammary epithelial cells and of several breast cancer cell lines. We have previously demonstrated that TNFα was able to induce proliferation through activation of p42/p44 MAPK, JNK and Akt pathways in C4HD cells from an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate induced mammary adenocarcinomas in female Balb/c mice (Proc. Am. Assoc. Cancer Res. 45:103, 2004). Here, we explore the participation of TNFR1 and TNFR2 in TNFα-triggered signaling pathways and the molecular mechanisms underlying TNFα-induced C4HD cells proliferation. Western blot analysis showed that C4HD cells express both TNFR1 and TNFR2. Preincubation of C4HD cells with blocking TNFR1 or TNFR2 antibodies resulted in inhibition of TNFα-induced C4HD cells proliferation of 58.2 ± 4.5% and 76.1 ± 15.9% respectively and in a complete inhibition of TNFα-induced p42/p44 MAPK, JNK and Akt activation. Stimulating antibodies to TNFR1 or TNFR2 induced C4HD cell proliferation at the same level as TNFα alone (89.7 ± 5.9% and 113.1 ± 19.9% respectively) and were also able to activate p42/p44 MAPK, JNK and Akt signaling pathways. To explore whether TNFR1 and TNFR2 activation induce NF-κB transcriptional activity, C4HD cells were transiently transfected with a luciferase reporter gene driven by κB-response element. Treatment of C4HD cells with TNFα (20 ng/ml) or the TNFRs agonist antibodies induced transcriptional activity of NF-κB. Finally, we addressed the effect of TNFα on the expression of the anti-apoptotic protein Bcl-X L and the cell cycle promoter protein cyclin D1, both NF-κB-induced genes. Stimulation of C4HD cells with TNFα induced an increase in Bcl-X L and cyclin D1 expression levels. Similar results were observed in the human breast cancer cell line T47D, in which TNFα was also mitogenic, suggesting that Bcl-X L and cyclin D1 molecules were downstream targets of the proliferative effect of TNFα on breast cancer cells. These results demonstrated that while activation of either TNFR1 or TNFR2 was enough to induce activation of p42/p44 MAPK, JNK and Akt and proliferation of C4HD cells, blockage of only one of these receptors inhibited these effects. Our findings demonstrated that anti-TNFα agents could be a promising therapeutic approach against breast cancer. In the case that both TNFR1 and TNFR2 were present in tumor cells, blockage of only one would be sufficient to disrupt TNFα effects.