Targeting of Nrf2 induces DNA damage signaling and protects colonic epithelial cells from ionizing radiation.

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator for antioxidant and anti-inflammation enzymes that binds to its endogenous inhibitor protein, Kelch-like ECH (erythroid cell-derived protein with CNC homology)-associated protein 1, in the cytoplasm under normal conditions. Various endogenous or environmental oxidative stresses, such as ionizing radiation (IR), can disrupt the Nrf2-Kelch-like ECH-associated protein 1 complex. This allows Nrf2 to translocate from the cytoplasm into the nucleus to induce transcription of heme oxygenase-1 and other cytoprotective enzymes through binding to antioxidant responsive elements. However, how Nrf2 protects cells from IR-induced damage remains unclear. Here, we report that Nrf2 activation by the synthetic triterpenoids, bardoxolone methyl (BARD) and 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid-ethyl amide, protects colonic epithelial cells against IR-induced damage, in part, by enhancing signaling of the DNA damage response. Pretreatment with BARD reduced the frequency of both G1 and S/G2 chromosome aberrations and enhanced the disappearance of repairosomes (C-terminal binding protein interacting protein, Rad51, and p53 binding protein-1 foci) after IR. BARD protected cells from IR toxicity in a Nrf2-dependent manner. The p53 binding protein-1 promoter contains three antioxidant responsive elements in which Nrf2 directly binds following BARD treatment. In addition, 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid-ethyl amide provided before exposure to a lethal dose of whole-body irradiation protected WT mice from DNA damage and acute gastrointestinal toxicity, which resulted in improved overall survival. These results demonstrate that Nrf2 activation by synthetic triterpenoids is a promising candidate target to protect the gastrointestinal tract against acute IR in vitro and in vivo.

Branching morphogenesis of immortalized human bronchial epithelial cells in three-dimensional culture.

While mouse models have contributed in our understanding of lung development, repair and regeneration, inherent differences between the murine and human airways requires the development of new models using human airway epithelial cells. In this study, we describe a three-dimensional model system using human bronchial epithelial cells (HBECs) cultured on reconstituted basement membrane. HBECs form complex budding and branching structures on reconstituted basement membrane when co-cultured with human lung fetal fibroblasts. These structures are reminiscent of the branching epithelia during lung development. The HBECs also retain markers indicative of epithelial cell types from both the central and distal airways suggesting their multipotent potential. In addition, we illustrate how the model can be utilized to understand respiratory diseases such as lung cancer. The 3D novel cell culture system recapitulates stromal-epithelial interactions in vitro that can be utilized to understand important aspects of lung development and diseases.

Proton radiation-induced cancer progression.

There are considerable health risks related to ionizing and proton radiation exposure. While there is a long history of health risks associated with ionizing (photon) radiation exposure, there is a limited understanding of the long-term health risks associated with proton radiation exposure. Since proton radiation is becoming more common in cancer therapy, the long-term biological effects of proton radiation remain less well characterized in terms of radiotherapy and well as for astronauts during deep space explorations. In this study, we compared the long-term side effects of proton radiation to equivalent doses of X-rays in the initiation and progression of premalignant lesions in a lung cancer susceptible mouse model (K-rasLA1). We show proton irradiation causes more complex DNA damage that is not completely repaired resulting in increased oxidative stress in the lungs both acutely and persistently. We further observed K-rasLA1 mice irradiated with protons had an increased number and size of initiated and premalignant lesions and adenomas that were often infiltrated with inflammatory cells. Proton irradiated mice had a lower median survival and increased carcinoma incidence as compared to unirradiated controls and X-rays exposed mice. Our conclusion is that exposure to proton irradiation enhances the progression of premalignant lesions to invasive carcinomas through persistent DNA damage, chronic oxidative stress, and immunosuppression.

Radiation-enhanced lung cancer progression in a transgenic mouse model of lung cancer is predictive of outcomes in human lung and breast cancer.

PURPOSE: Carcinogenesis is an adaptive process between nascent tumor cells and their microenvironment, including the modification of inflammatory responses from antitumorigenic to protumorigenic. Radiation exposure can stimulate inflammatory responses that inhibit or promote carcinogenesis. The purpose of this study is to determine the impact of radiation exposure on lung cancer progression in vivo and assess the relevance of this knowledge to human carcinogenesis. EXPERIMENTAL DESIGN: K-ras(LA1) mice were irradiated with various doses and dose regimens and then monitored until death. Microarray analyses were performed using Illumina BeadChips on whole lung tissue 70 days after irradiation with a fractionated or acute dose of radiation and compared with age-matched unirradiated controls. Unique group classifiers were derived by comparative genomic analysis of three experimental cohorts. Survival analyses were performed using principal component analysis and k-means clustering on three lung adenocarcinoma, three breast adenocarcinoma, and two lung squamous carcinoma annotated microarray datasets. RESULTS: Radiation exposure accelerates lung cancer progression in the K-ras(LA1) lung cancer mouse model with dose fractionation being more permissive for cancer progression. A nonrandom inflammatory signature associated with this progression was elicited from whole lung tissue containing only benign lesions and predicts human lung and breast cancer patient survival across multiple datasets. Immunohistochemical analyses suggest that tumor cells drive predictive signature. CONCLUSIONS: These results demonstrate that radiation exposure can cooperate with benign lesions in a transgenic model of cancer by affecting inflammatory pathways, and that clinically relevant similarities exist between human lung and breast carcinogenesis.

Mitigation of radiation-induced damage by targeting EGFR in noncancerous human epithelial cells.

Methyl-2-cyano-3,12 dioxoolean-1,9 diene-28-oate (CDDO-Me) is an antioxidative, anti-inflammatory modulator, which activates the nuclear factor-erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. While CDDO-Me has radioprotective activity through Nrf2 activation in vitro and in vivo, its ability to mitigate radiation-induced damage when provided after irradiation has not been studied. Here we investigated whether CDDO-Me mitigates ionizing radiation (IR)-induced DNA damage in immortalized normal human colonic epithelial cells (HCECs) and bronchial epithelial cells (HBECs). DNA damage and clonogenic survival were assessed after treatment with CDDO-Me postirradiation. We observed that treatment with CDDO-Me within 30 min after irradiation improved both DNA damage repair and clonogenic survival independently of Nrf2. CDDO-Me activates the epidermal growth factor receptor (EGFR) related DNA repair responses. In the presence of CDDO-Me, EGFR is phosphorylated and translocates into the nucleus where it interacts with DNA-PKcs. CDDO-Me-mediated mitigation activity can be abrogated through depletion of EGFR, ectopic overexpression of mutant EGFR or inhibition of DNA-PKcs. While post-treatment of CDDO-Me protected noncancerous HCECs and HBECs against IR, cancer cells (HCT116 and MCF7) were not protected by CDDO-Me. These results suggest that targeting EGFR using CDDO-Me is a promising radiation mitigator with potential utility for first responders to nuclear accidents.

Multipotent capacity of immortalized human bronchial epithelial cells.

While the adult murine lung utilizes multiple compartmentally restricted progenitor cells during homeostasis and repair, much less is known about the progenitor cells from the human lung. Translating the murine stem cell model to humans is hindered by anatomical differences between species. Here we show that human bronchial epithelial cells (HBECs) display characteristics of multipotent stem cells of the lung. These HBECs express markers indicative of several epithelial types of the adult lung when experimentally tested in cell culture. When cultured in three different three-dimensional (3D) systems, subtle changes in the microenvironment result in unique responses including the ability of HBECs to differentiate into multiple central and peripheral lung cell types. These new findings indicate that the adult human lung contains a multipotent progenitor cell whose differentiation potential is primarily dictated by the microenvironment. The HBEC system is not only important in understanding mechanisms for specific cell lineage differentiation, but also for examining changes that correlate with human lung diseases including lung cancer.