Persistent Phenotypic Responses of Human Mammary Epithelial Cells Induced by Ionizing Radiation

Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. We have shown that irradiating human mammary epithelial cells (HMEC) cultured with that transforming growth factor β1 (TGFβ) can generate a persistent phenotype in daughter cells characterized by spindle cell morphology, increased mesenchymal markers, decreased epithelial markers and increased cellular motility and invasion, which are hallmarks of epithelial to mesenchymal transition (EMT). Neither radiation nor TGFβ alone elicited EMT, although IR increased chronic TGFβ signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFβ-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFβ, and inhibition of Erk activation, blocked the EMT phenotype. Preliminary studies suggest that eqi-toxic doses of sparsely and densely ionizing radiation resulted in comparable EMT when cells were cultivated in the presence of TGFβ, Furthermore radiation dose response studies show that this effect has a very low threshold in that a single exposure of 3-200 cGy radiation elicits the ‘same’ phenotypic switch, which is consistent with non-targeted effects. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to radiation carcinogenesis

New rationales for using TGFbeta inhibitors in radiotherapy.

PURPOSE: The first reports that ionizing radiation (IR) induces rapid and persistent activation of transforming growth factor beta1 (TGFbeta) were nearly two decades ago. Subsequent studies have shown that TGFbeta is a major mediator of cellular and tissue responses to IR and have revealed novel facets of its complex biology. RESULTS: We and others have recently shown that inhibition of production or signaling of TGFbeta in epithelial cells modulates radiosensitivity and impedes activation of the DNA damage response program. The primary transducer of cellular response to DNA damage caused by ionizing radiation is the nuclear protein kinase ataxia telangiectasia mutated, whose activity is severely compromised when TGFbeta is inhibited. Thus, in conjunction, with its well-recognized contribution to normal tissue fibrosis, the role of TGFbeta in the genotoxic stress program provides a previously unsuspected avenue to modulate radiotherapy. CONCLUSIONS: We hypothesize that identification of the circumstances and tumors in which TGFbeta manipulation enhances tumor cell radiosensitivity, while protecting normal tissues, could significantly increase therapeutic index

Ionizing radiation predisposes non-malignant human mammary epithelial cells to undergo TGF beta-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). Non-malignant HMEC (MCF10A, HMT3522 S1 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 morphological shift from cuboidal to spindle-shaped. This phenotype was accompanied by 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{trademark}. Neither radiation nor TGF{beta} alone elicited EMT, even though 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/MAPK signaling. We hypothesized that IR-induced MAPK activation primes non-malignant HMEC to undergo TGF{beta}-mediated EMT. Consistent with this, Erk phosphorylation were transiently induced by irradiation, persisted in irradiated cells treated with TGF{beta}, and treatment with U0126, a Mek inhibitor, blocked the EMT phenotype. Together, these data demonstrate that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression