Ionizing radiation predisposes nonmalignant human mammary epithelial cells to undergo transforming growth factor β-induced epithelial to mesenchymal transition

Transforming growth factor beta 1 (TGF beta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGF beta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGF beta-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 SI, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGF beta (0.4 ng/mL) or double treated. All double-treated (IR + TGF beta) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, beta-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGF beta alone elicited EMT, although IR increased chronic TGF beta signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogenactivated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGF beta-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGF beta, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.