RESUMO
Although nuclear factor E2-related factor-2 (Nrf2) protects from carcinogen-induced tumorigenesis, underlying the rationale for using Nrf2 inducers in chemoprevention, this antioxidative transcription factor may also act as a proto-oncogene. Thus, an enhanced Nrf2 activity promotes formation and chemoresistance of colon cancer. One mechanism causing persistent Nrf2 activation is the adaptation of epithelial cells to oxidative stress during chronic inflammation, e.g. colonocytes in inflammatory bowel diseases, and the multifunctional stress response gene immediate early response-3 (IER3) has a crucial role under these conditions. We now demonstrate that colonic tissue from Ier3(-/-) mice subject of dextran sodium sulfate colitis exhibit greater Nrf2 activity than Ier3(+/+) mice, manifesting as increased nuclear Nrf2 protein level and Nrf2 target gene expression. Likewise, human NCM460 colonocytes subjected to shRNA-mediated IER3 knockdown exhibit greater Nrf2 activity compared with control cells, whereas IER3 overexpression attenuated Nrf2 activation. IER3-deficient NCM460 cells exhibited reduced reactive oxygen species levels, indicating increased antioxidative protection, as well as lower sensitivity to TRAIL or anticancer drug-induced apoptosis and greater clonogenicity. Knockdown of Nrf2 expression reversed these IER3-dependent effects. Further, the enhancing effect of IER3 deficiency on Nrf2 activity relates to the control of the inhibitory tyrosine kinase Fyn by the PI3K/Akt pathway. Thus, the PI3K inhibitor LY294002 or knockdown of Akt or Fyn expression abrogated the impact of IER3 deficiency on Nrf2 activity. In conclusion, the interference of IER3 with the PI3K/Akt-Fyn pathway represents a novel mechanism of Nrf2 regulation that may get lost in tumors and by which IER3 exerts its stress-adaptive and tumor-suppressive activity.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Colite/metabolismo , Colo/metabolismo , Células Epiteliais/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Cromonas/farmacologia , Colite/induzido quimicamente , Colite/genética , Colo/patologia , Sulfato de Dextrana/toxicidade , Inibidores Enzimáticos/farmacologia , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Proteínas Imediatamente Precoces/genética , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Morfolinas/farmacologia , Fator 2 Relacionado a NF-E2/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) exhibits one of the worst survival rates of all cancers. While death rates show declining trends in the majority of cancers, PDAC registers rising rates. Based on the recently described crosstalk between TGF-ß1 and Nrf2 in the PDAC development, the involvement of ATF3 and its splice variant ΔZip2 in TGF-ß1- and Nrf2-driven pancreatic tumorigenesis was investigated. As demonstrated here, PDAC (Panc1, T3M4) cells or premalignant H6c7 pancreatic ductal epithelial cells differentially express ΔZip2- and ATF3, relating to stronger Nrf2 activity seen in Panc1 cells and TGF-ß1 activity in T3M4 or H6c7 cells, respectively. Treatment with the electrophile/oxidative stress inducer tBHQ or the cytostatic drug gemcitabine strongly elevated ΔZip2 expression in a Nrf2-dependent fashion. The differential expression of ATF3 and ΔZip2 in response to Nrf2 and TGF-ß1 relates to differential ATF3-gene promoter usage, giving rise of distinct splice variants. Nrf2-dependent ΔZip2 expression confers resistance against gemcitabine-induced apoptosis, only partially relating to interference with ATF3 and its proapoptotic activity, e.g., through CHOP-expression. In fact, ΔZip2 autonomously activates expression of cIAP anti-apoptotic proteins. Moreover, ΔZip2 favors and ATF3 suppresses growth and clonal expansion of PDAC cells, again partially independent of each other. Using a Panc1 tumor xenograft model in SCID-beige mice, the opposite activities of ATF3 and ΔZip2 on tumor-growth and chemoresistance were verified in vivo. Immunohistochemical analyses confirmed ΔZip2 and Nrf2 coexpression in cancerous and PanIN structures of human PDAC and chronic pancreatitis tissues, respectively, which to some extent was reciprocal to ATF3 expression. It is concluded that depending on selective ATF3-gene promoter usage by Nrf2, the ΔZip2 expression is induced in response to electrophile/oxidative (here through tBHQ) and xenobiotic (here through gemcitabine) stress, providing apoptosis protection and growth advantages to pancreatic ductal epithelial cells. This condition may substantially add to pancreatic carcinogenesis driven by chronic inflammation.