Activation of CCAAT/enhancer-binding protein beta by 2'-amino-3'-methoxyflavone (PD98059) leads to the induction of glutathione S-transferase A2

The induction of glutathione S-transferases by flavonoids is associated with cancer chemopreventive effects. We reported that 2'-amino-3'-methoxyflavone (PD98059), an MKK1 inhibitor, induces glutathione S-transferase A2 (rGSTA2). This report comparatively examines the role of CCAAT/enhancer-binding protein (C/EBP) and Nrf2 in the induction of rGSTA2 by PD98059. We first assessed whether the MKK1/ERK1/2 pathway regulated rGSTA2 induction. Northern and western blot analyses showed that PD98059 at the concentrations effective for the inhibition of MKK1 increased the rGSTA2 protein and mRNA levels in H4IIE cells. PD98059 also induced rGSTA2 in cells stably transfected with dominant-negative mutant of MKK1(-), which provided evidence that the inhibition of MKK1/ERK1/2 by PD98059 was not responsible for rGSTA2 induction. Gel shift assay and immunoblot analysis of subcellular fractions revealed that PD98059 caused nuclear translocation of C/EBP beta and increased C/EBP DNA binding, which was super-shifted with anti-C/EBP beta antibody. Nrf2 was not activated by PD98059. PD98059 increased the luciferase reporter gene activity in cells transfected with the C/EBP-containing -1.65 kb flanking region of the rGSTA2 gene. Deletion of the C/EBP-binding site or over-expression of dominant-negative mutant of C/EBP abolished the reporter gene activity. Flavone, a backbone structure of PD98059, also induced nuclear translocation of C/EBP beta and C/EBP-mediated rGSTA2 gene induction. Inhibition of phosphatidylinositol 3-kinase abolished C/EBP beta-mediated rGSTA2 induction by PD98059. These results provide evidence that PD98059 and flavone induce nuclear translocation of C/EBP beta and activate the C/EBP-binding site in the rGSTA2 gene, which constitutes the distinct pathway for the enzyme induction irrespective of the inhibition of MKK1/ERK activity.     

Complex regulation of the fibroblast growth factor-binding protein in MDA- MB-468 breast cancer cells by CCAAT/enhancer-binding protein beta

The fibroblast growth factor-binding protein (FGF-BP) binds and activates fibroblast growth factors in the extracellular matrix, and can have a rate-limiting role in tumor angiogenesis. Here we demonstrate high levels of FGF-BP expression in invasive human breast cancer, relative to normal breast and in situ carcinoma, and in MDA-MB-468 human breast cancer cells. In these cells, FGF-BP was up-regulated by treatment with epidermal growth factor (EGF), dependent on protein kinase C and p38 mitogen-activated protein kinase signaling. Mutational analysis revealed that the activator protein 1 and CCAAT/enhancer binding protein (C/EBP) sites on the FGF-BP gene promoter were required for the EGF effect, whereas deletion of the C/EBP site resulted in a significant increase in promoter basal activity indicating a basal repressive control mechanism. These data suggest that the C/EBP site is a central regulatory element for the regulation of FGF-BP promoter activity in MDA-MB-468 cells. We found that MDA-MB-468 cells express high endogenous levels of both the activating (LAP) and repressive (LIP) isoforms of C/EBPbeta. Overexpression of C/EBPbeta-LAP in MDA-MB-468 cells resulted in a large 80-fold increase in FGF-BP promoter basal activity, which was reversed by coexpression of LIP. Gel-shift analysis revealed that four LIP- and LAP-containing complexes (a-d) bind to the C/EBP site. DNA binding of the LIP and LAP-containing c complex and the b complex in the presence of EGF was modulated by inhibition of p38 mitogen-activated protein kinase, suggesting a role for these complexes in the EGF induction of the FGF-BP promoter. This study suggests that along with its well-defined role in mammary gland development, C/EBPbeta may well play a role in the pathology of breast cancer, in particular in the control of angiogenesis in the invasive phenotype.     

Post-transcriptional modulation of C/EBPα prompts monocytic differentiation and apoptosis in acute myelomonocytic leukaemia cells

CCAAT/enhancer binding protein alpha (C/EBPα) induction induces monocytic differentiation even in acute myeloid leukaemia (AML). In this study, the induction/activation of C/EBPα in myelomonocytic AML was investigated using a combination of all-trans retinoic acid (ATRA) and RAD001 (Everolimus), a mammalian target of rapamycin complex 1 (mTORC1) inhibitor. Combining these agents increased PU.1, C/EBPε and C/EBPα expression, increased the p42/p30 C/EBPα ratio, and decreased C/EBPα phosphorylation at serine 21, and was accompanied by growth inhibition, induction of CD11b expression and apoptosis in AML cell lines. Thus, agents that induce sufficient levels of C/EBPα expression might be useful in treating AML.     

Stress-induced NQO1 controls stability of C/EBPα against 20S proteasomal degradation to regulate p63 expression with implications in protection against chemical-induced skin cancer

Previously, we have shown a role of cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) in the stabilization of p63 against 20S proteasomal degradation resulting in thinning of the epithelium and chemical-induced skin cancer (Oncogene (2011) 30, 1098-1107). Current studies have demonstrated that NQO1 control of CCAAT-enhancer binding protein (C/EBPα) against 20S proteasomal degradation also contributes to the upregulation of p63 expression and protection. Western and immunohistochemistry analysis revealed that disruption of the NQO1 gene in mice and mouse keratinocytes led to degradation of C/EBPα and loss of p63 gene expression. p63 promoter mutagenesis, transfection and chromatin immunoprecipitation assays identified a C/EBPα-binding site between nucleotide position -185 and -174 that bound to C/EBPα and upregulated p63 gene expression. Co-immunoprecipitation and immunoblot analysis demonstrated that 20S proteasomes directly interacted and degraded C/EBPα. NQO1 direct interaction with C/EBPα led to stabilization of C/EBPα against 20S proteasomal degradation. NQO1 protection of C/EBPα required binding of NADH with NQO1. Exposure of skin and keratinocytes to the chemical stress agent benzo(a)pyrene led to induction of NQO1 and stabilization of C/EBPα protein, resulting in an increase in p63 RNA and protein in wild-type but not in NQO1-/- mice. Collectively, the current data combined with previous data suggest that stress induction of NQO1 through both stabilization of C/EBPα and increase in p63 and direct stabilization of p63 controls keratinocyte differentiation, leading to protection against chemical-induced skin carcinogenesis. The studies are significant as 2-4% human individuals are homozygous and 23% are heterozygous for the NQO1P187S mutation and might be susceptible to stress-induced skin diseases.