Radiation-induced release of transforming growth factor alpha activates the epidermal growth factor receptor and mitogen-activated protein kinase pathway in carcinoma cells, leading to increased proliferation and protection from radiation-induced cell death

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizin g radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of t he mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (J NK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immedi ate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathway s, which were surprisingly followed by later prolonged secondary activation s (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activat ion of the MAPK pathway was abolished by molecular inhibition of either EGF R or Ras function. In contrast, molecular inhibition of EGFR function aboli shed the secondary but not the primary activation of the JNK pathway. Inhib ition of tumor necrosis factor alpha receptor function by use of neutralizi ng monoclonal antibodies blunted primary activation of the JNK pathway. Add ition of a neutralizing monoclonal antibody versus transforming growth fact or alpha (TGF alpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secon dary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro- TGF alpha cleavage 120-180 min after exposure. In agreement with radiation- induced release of a soluble factor, activation of the EGFR and the MAPK an d JNK pathways could be induced in nonirradiated cells by the transfer of m edia from irradiated cells 120 min after irradiation. The ability of the tr ansferred media to cause MAPK and JNK activation was blocked when media wer e incubated with a neutralizing antibody to TGF alpha. Thus radiation cause s primary and secondary activation of the EGFR and the MAPK and JNK pathway s in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage an d autocrine action of TGF alpha. Neutralization of TGF alpha function by an anti-TGF alpha antibody or inhibition of MAPK function by MEK1/2 inhibitor s (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irrad iation in apoptosis, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium b romide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGF alpha-EGFR-MAPK signaling module represents a strategy to decre ase carcinoma cell growth and survival after irradiation.