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Browsing by Author "Goulet, Robert J."
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Item The macrophage inhibitory cytokine integrates AKT/PKB and MAP kinase signaling pathways in breast cancer cells(Oxford University Press, 2005-05-01) Wollmann, Wyatt; Goodman, Mike L.; Bhat-Nakshatri, Poornima; Kishimoto, Hiromitsu; Goulet, Robert J.; Mehrotra, Sanjana; Morimiya, Akira; Badve, Sunil; Nakshatri, HarikrishnaMacrophage inhibitory cytokine 1 (MIC-1), a divergent member of the transforming growth factor beta superfamily, plays a role in the progression of a number of cancers, including breast, gastric, prostate and colorectal carcinomas. Serum MIC-1 levels are elevated in patients with metastatic prostate, breast and colorectal carcinomas. In vitro studies have revealed a cell type-specific role for MIC-1 in senescence and apoptosis. MIC-1 activates the survival kinase AKT/PKB in neuronal cells. Depending on the cell type, it activates or represses the MAP kinases ERK1/2. Mechanisms responsible for an increased MIC-1 expression in cancers and the consequences of MIC-1 overexpression, however, are not known. In this study, we show that AKT/PKB directly regulates the expression of MIC-1 in breast cancer cells. Sequences within −88 to +30 of the MIC-1 promoter are required for the AKT-mediated induction of MIC-1. This region of the promoter contains two SP-1 binding sites (SP-1B and SP-1C), which bind to the SP-1 and SP-3 proteins. Mutation of SP-1C but not SP-1B reduced the AKT-mediated activation of MIC-1. MIC-1 increased the basal ERK1 phosphorylation and prolonged the estrogen-stimulated ERK1 phosphorylation in MCF-7 breast cancer cells without altering the phosphorylation status of AKT/PKB. Immunohistochemistry with MIC-1 antibody revealed an MIC-1 expression within the cancer cells of primary breast cancer and in the MCF-7 xenografts. Furthermore, a limited analysis of RNA from primary breast cancers revealed an overexpression of MIC-1 in tumors, compared with normal tissues. These results suggest that AKT/PKB through MIC-1 could regulate the ERK1 activity and the MIC-1 expression levels may serve as a surrogate marker for the AKT activation in tumors.Item Negative Regulation of Transactivation Function but Not DNA Binding of NF-κB and AP-1 by IκBβ1 in Breast Cancer Cells(1999-06-25) Newton, Thomas R.; Patel, Nikhil M.; Bhat-Nakshatri, Poornima; Stauss, Carmen R.; Goulet, Robert J.; Nakshatri, HarikrishnaThe transcription factor NF-κB regulates the expression of genes involved in cancer cell invasion, metastasis, angiogenesis, and resistance to chemotherapy. In normal cells NF-κB is maintained in the cytoplasm by protein-protein interaction with inhibitor IκBs. In contrast, in cancer cells a substantial amount of NF-κB is in the nucleus and constitutively activates target genes. To understand the mechanisms of constitutive NF-κB activation, we have analyzed the function of IκBα and IκBβ in breast cancer cells. In most cases, constitutive NF-κB DNA binding correlated with reduced levels of either IκBα or IκBβ isoforms. Overexpression of IκBα but not IκBβ1 resulted in reduced constitutive DNA binding of NF-κB in MDA-MB-231 cells. Unexpectedly, IκBβ1 overexpression moderately increased 12-O-tetradecanoylphorbol-13-acetate- and interleukin-1-inducible NF-κB DNA binding. 12-O-Tetradecanoylphorbol-13-acetate- and interleukin-1-induced transactivation by NF-κB, however, was lower in IκBβ1-overexpressing cells. Mutants of IκBβ1 lacking the C-terminal casein kinase II phosphorylation sites, which form a stable complex with DNA bound NF-κB without inhibiting its transactivation in other cell types, repressed the transactivation by NF-κB in MDA-MB-231 cells. Consistent with the results of transient transfections, the expression of urokinase plasminogen activator, an NF-κB target gene, was reduced in IκBβ1-overexpressing cells. These results suggest that depending on the cell type, IκBβ1 represses the expression of NF-κB-regulated genes by inhibiting either DNA binding or transactivation function of NF-κB.