Tumor‐Necrosis Factor‐α Modulates Mitogen‐Activated Protein Kinase Activity of Epidermal‐Growth‐Factor‐Stimulated MCF‐7 Breast Cancer Cells

Abstract

Tumor-necrosis factor(TNF)-a inhibited in a dose-dependent fashion the proliferation of epidermalgrowth-factor(EGF)-stimulated MCF-7 breast cancer cells with an IC,, of 0.25 nM. A comparable TNFa-mediated inhibition of ~42144 mitogen-activated protein (MAP) kinase activity was observed in 10 nM EGF-stimulated cells. The MAP kinase activity dropped 50% within 3 min of TNF-a (1 nM) addition to EGF-stimulated MCF-7 cells. EGF and TNF-a, when added independently, led to a transient stimulation of MAP kinase activity with maximal activations within 6-8 min and 1-2 min, respectively. These observations suggest that MAP kinase activity in EGF-stimulated MCF-7 cells is modulated by the growth-inhibitory receptor pathways of TNF-a. Phosphorylation measurements on western blots determined the involvement of several individual MAP kinases, namely ~42144 MAP kinases, p38 MAP kinase and c-Jun N,-terminal kinase 1 (JNKI), in EGF and TNF-a-induced signalling. Phosphorylation of p42 and p38 MAP kinases only was observed after treatment with either TNF-a or EGF. A combination of both ligands inhibited p42 and p38 MAP kinase phosphorylation in MCF-7 cells. In contrast, no JNKI phosphorylation was detected in these cells. Simultaneous addition of okadaic acid, a potent inhibitor of phosphatases 1 and 2A, blocked the decay of EGF-stimulated MAP kinase activity over 40 min. TNF-a added to EGF-stimulated and okadaic-acid-treated cells increased the MAP kinase activity twofold within 1 rnin. Similarly, okadaic acid treatment partly reverted the TNF-a-inhibited growth of MCF-7 cells. These experiments suggest that phosphatases are involved in the rapid shut-down by TNF-a of p42 MAP kinase activity.Keywords: mitogen-activated protein kinase ; tumor-necrosis factor-a; breast cancer; signal transduction ; cross-talk Tumor-necrosis factor(TNF)-a is a pluripotent cytokine, which can modulate the growth of both normal and tumor cells in vitro [ l , 21. Several tumor cell lines have been shown to be sensitive to the cytotoxic or cytostatic effects of TNF-a, among them the hormone-dependent human breast cancer MCF-7 cells [3-51. MCF-7 cells respond to both epidermal-growth factor (EGF) and TNF-a, with EGF inducing accelerated growth in vitro and TNF-a inhibiting it. The antimitogenic effect of TNFa is dose dependent in EGF-stimulated MCF-7 cells [5]. However, the site and nature of the interaction between the stimulatory EGF-receptor signalling pathway and the inhibitory TNFreceptor pathway has not been elucidated. TNF-a has been shown to induce serinelthreonine or tyrosine phosphorylation of various cellular proteins including mitogen-activated protein (MAP) kinases [6, 71.MAP kinases are a class of serinelthreonine protein kinases believed to occupy a pivotal position in the phosphorylation cascades triggered by extracellular stimuli such as EGF [8-111.Correspondence to H. Mueller, Department of Research, University Women's Clinic, Schanzenstrasse 46, CH-4031 Basel, SwitzerlandAhbreviations. EGF, epidermal-growth factor; IMEM-20, improved mi...

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