Obesity and related inflammatory processes have been associated with increased incidence of hepatocellular carcinoma, where initiation of intracellular signaling through cytokine-mediated NF-kappaB and Jak/STAT3 pathways leads to decreased apoptosis and increased proliferation and invasion via expression of target genes. Our overall goal is to understand the contribution of chronic low-grade inflammation as an important underlying condition for tumorigenesis. To achieve this goal, the project has been divided into three distinct parts.? ? Part I: It was previously reported that the compound pyrrolidine dithiocarbamate (PDTC) is able to dampen pro-inflammatory cytokine signaling in a number of cultured cell lines and in in vivo models of inflammation. There is great interest in delineating the mechanisms whereby hepatic inflammation is regulated. During the last year, we performed a more detailed investigation of the possible effects on gene expression arising from the interaction between PDTC and human HepG2 hepatoma cells by a combination of microarray display and quantitative real-time polymerase chain reaction assays. Evidences are presented to indicate that genes for cholesterol, fatty acid, and phospholipids synthesis were suppressed, while genes participating in cell arrest were induced. Of significance, the upregulation of some stress-related genes and genes involved in a diverse network of cellular functions was associated with trimerization of heat shock factor-1 (HSF-1) and its translocation from the cytosol to the nucleus of PDTC-treated cells. Chromatin immunoprecipitation assays and siRNA-mediated silencing of HSF-1 have strengthened these findings. It was also found that PDTC represses the promoters of genes that mediate acute inflammation and mitogenesis though HSF-1 interaction with heat shock-responsive elements present in the promoter of target genes. This work forms the basis of a new study with HepG2 tumor-bearing nude mice. These studies provide mechanistic insights into some of the biological actions of PDTC in vitro and in in vivo models of oxidative stress and inflammation. They also identify HSF-1 as a key player in the complex scenario of cellular homeostasis and tumor biology, which deserve further and more extensive studies as a potential new therapeutic target.? ? Part II: S-glutathionylation is a physiological, reversible protein modification of cysteine residues with glutathione in response to mild oxidative stress. Since the key cell growth regulator STAT3 is particularly susceptible to redox regulation, we hypothesized that oxidative modification of cysteine residues of STAT3 by S-glutathionylation may occur. During the last year, we demonstrated that the cysteine residues of STAT3 are modified by a thiol alkylating agent and are the targets of S-glutathionylation. The experimental approach involved the treatment of human HepG2 hepatoma cells with the metal chelator pyrrolidine dithiocarbamate (PDTC), glutathione disulfide or diamide. Under these experimental conditions, STAT3 protein thiol reactivity was reversibly attenuated with concomitant increase in the S-glutathionylation of STAT3. There was a marked reduction in interleukin (IL)-6-dependent STAT3 signaling, including decreased STAT3 tyrosine phosphorylation and lower association with the molecular chaperone Hsp90, loss in nuclear translocation of STAT3 and impaired expression of target genes, such as fibrinogen-γ and glucose-6-phosphatase. In a cell-free system, diamide induced glutathionylation of STAT3, which was decreased upon addition of glutaredoxin (GRX)-1, a deglutathionylation enzyme, or the reducing agent, dithiothreitol. Glutationylated STAT3 was a poor JAK2 substrate in vitro, and it exhibited low DNA-binding activity. Cellular GRX-1 activity was inhibited by diamide and PDTC treatment; however, ectopic expression of GRX-1 was accompanied by a modest increase in phosphorylation, nuclear translocation and DNA-binding ability of STAT3 in response to IL-6. These results are the first to show S-glutathionylation of STAT3, a modification that may exert regulatory function in STAT3 signaling. Studies like these may ultimately lead to new therapies for IL-6-mediated proinflammatory disorders and cancer.? ? Part III: Filamin A is an actin-binding protein that has been implicated in proliferation, cell migration, invasion and carcinogenesis. We have recently reported the existence of a link between filamin A and the signaling pathways that control the expression of matrix metalloproteinase 9 (MMP-9) in human melanoma cells (Zhu et al., 2007). MMP-9 is involved in the breakdown of type IV collagen, a major component of the basement membrane. The ability of filamin A to affect the intracellular trafficking of various classes of receptors led us to evaluate the contribution of filamin A on the activation and propagation of EGF receptor (EGFR) signals, which ultimately results in cancer cell invasiveness. In the past year, we obtained a number of human melanoma cell lines with varying metastatic potential and showed a positive correlation between the expression of filamin A and EGFR with metastasis. We found that pharmacological inhibition of EGFR kinase sharply reduces migration of metastatic melanoma cells as measured by wound-healing assays. Similarly, RNA interference-mediated silencing of filamin A led to reduction in EGF-mediated cell migration. To further support the hypothesis that filamin A plays a key role in EGFR-dependent malignancy, various techniques will be used to characterize ligand-mediated activation of EGFR, its internalization and intracellular sorting, as well as lysosomal degradation in metastatic melanoma cells expressing various levels of filamin A. Similar approach will be used in other human cancer cell types, focusing on the contribution of filamin A in EGFR activation and function. The existence of a link between filamin A and the early events associated with carcinogenesis could have potential therapeutic applications.
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|Zhu, Tie-Nian; He, Hua-Jun; Kole, Sutapa et al. (2007) Filamin A-mediated down-regulation of the exchange factor Ras-GRF1 correlates with decreased matrix metalloproteinase-9 expression in human melanoma cells. J Biol Chem 282:14816-26|
|He, Hua-Jun; Zhu, Tie-Nian; Xie, Yi et al. (2006) Pyrrolidine dithiocarbamate inhibits interleukin-6 signaling through impaired STAT3 activation and association with transcriptional coactivators in hepatocytes. J Biol Chem 281:31369-79|