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    Research Grants Awarded

    Role of CRIF1 in NRF2 signaling pathway

    Study Section:
    RISK and Prevention, Epidemiology

    Scientific Abstract:
    Background . Oxidative stress and the resulting highly reactive free radicals can contribute to many chronic diseases, including breast cancer. Mammalian cells reduce the potential harmful consequences of oxidative stressors by activating phase 2 cytoprotective genes, which encode detoxification and antioxidant enzymes. Many phase 2 genes are regulated by upstream antioxidant response elements (ARE), targets of NRF2, a leucine zipper transcription factor. Nevertheless, the regulation of NRF2 activity is still not completely understood and may occur at many different levels. Our studies found that NRF2 physically interacts with CRIF1, a cell cycle regulatory protein. We also found that overexpressed CRIF1 inhibits NRF2’s activity and causes cultured cells to have increased sensitivity to oxidative stress. Objective/Hypothesis . CRIF1 confers sensitivity to oxidative stress by physically associating with NRF2 and by inhibiting NRF2-dependent phase 2 gene expression. Specific Aims . The major goals of this project are to investigate: 1) the CRIF1-NRF2 physical interaction; 2) whether CRIF1 affects NRF2-dependent oxidative stress-induced gene expression; 3) the molecular mechanism(s) underlying CRIF1 induced sensitivity to oxidative stress. Study Design. We will validate the CRIF1-NRF2 interaction using immunoprecipitation-Western blotting and GST-capture assays. We will also determine if CRIF1 affects the total amounts of NRF2 (or NRF2 partners, such as Keap1) mRNA by using real-time RT-PCR and Western blotting analysis. Using promoter activity assays the effects of CRIF1 on NRF2-mediated transcription of phase 2 genes will be investigated. CRIF1 deletion and point mutation DNA constructs will be used to identify essential regions/domains, and siRNA knockdown studies will be used to validate the specificity of the anticipated findings. Chromatin immunoprecipitation assays will be used to determine if CRIF1 binds (AREs) of phase 2 genes along with NRF2 or inhibits NRF2 binding to these promoters. Finally, we will investigate CRIF1-dependent and oxidative stress-driven cell sensitization mechanisms by determining whether CRIF1 regulates redox balance by altering total intracellular levels of phase 2 proteins. Potential Outcomes and Benefits of the Research . These studies will significantly increase of understanding of CRIF1 and NRF2 function(s) in regulating gene expression and oxidative stress defense mechanisms in human breast cancer cells.

    Lay Abstract:
    Background . Oxidative stress, a condition in which harmful chemicals become “activated”, can seriously damage important molecules in human cells. Although cells routinely respond to this stress, an inadequate response can contribute to cancer, aging and neurodegenerative diseases. Researchers discovered a molecule, NRF2, which becomes activated by oxidative stress and responds to it by increasing the number of gene products able to detoxify harmful chemicals. The mechanism of controlling NRF2 activity is still not completely understood, but it may occur at many different stages. As is often the case with important control proteins such as NRF2, cells employ multiple ways to influence their activity. We are interested in NRF2, a positive control protein, because we have discovered a protein that appears to influence NRF2’s activity in a new way. This protein, CRIF1 previously identified as having other effects, acts as a negative control protein for NRF2 because it inactivates the positive effects of NRF2 against oxidative stress. Objective/Hypothesis. We propose to test two hypotheses; 1) CRIF1 makes cells more sensitive to the harmful effects of oxidative stress by blocking the ability of NRF2 to protect cells against these harmful effects; 2) this sensitizing effect is caused by the binding of CRIF1 to NRF2, blocking the ability of NRF2 to act as a positive-acting transcription factor. Specific Aims . We propose to determine; 1) if CRIF1 binds to NRF2; 2) if CRIF1 inhibits NRF2 transcription promoting activity; 3) how CRIF1 makes cells more sensitive to oxidative stress. Study Design . We will use state-of–the-art methods to; 1) identify interacting of CRIF1 and NRF2 molecules and identify the part of NRF2 that binds CRIF1; 2) determine which stage in NRF2 activation is affected by CRIF1; 3) test whether CRIF1 confers cell sensitivity to oxidative stress by the mechanism hypothesized. Potential Outcomes and Benefits of the Research. These studies will increase our understanding of breast cancer biology and should impact cancer control efforts in several ways. If our hypothesis is correct, dysregulated expression of CRIF1 in breast cancer cells may alter the amounts of NRF2-induced protective gene products made after cells detect harmful chemicals. Thus, we predict that CRIF1 likely regulates cells’ sensitivity to chemicals that produce oxidative stress. The proposed research should identify new targets for cancer prevention and treatment.