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 recent findings indicate that pyrrolidine dithiocarbamate (PDTC), a small metal chelator compound, impinges on the ability of pro-inflammatory cytokines to promote expression of acute phase proteins in cultured hepatocytes (He et al., 2006). More recently, we performed a microarray analysis (Agilent human 1A 22 K Oligo microarray) coupled with quantitative PCR to demonstrate alteration in the expression of a subset of genes involved in the regulation of NF-kappaB signaling in human HepG2 hepatoma cells post-PDTC treatment. Additional experiments are underway to provide mechanistic insights into the protective role of PDTC against inflammation-associated pathologies not only in cultured cells, but also in more complex biological systems.? ? In the second leg of the study, we worked on projects that relate to the regulation and control of cancer cell signaling by the actin-binding protein, filamin A (FLNa). The work was initially carried out using human M2 melanoma cancer cells that lack the gene encoding FLNa, and M2 cells where FLNa has been stably reintroduced (clone M2A7). (He et al., 2003). The lack of FLNa was associated with enhanced stimulation of insulin-dependent Ras/MAPK pathway, but repression of tumor necrosis factor-mediated pro-inflammatory responses induced by NF-kappaB in M2 cells. Such pattern provides a molecular rationale for the antagonism between insulin and pro-inflammatory cytokines, and highlights the need to further investigate the role of FLNa as regulator of cytokine and insulin signaling in models of liver cancer cells.? ? Matrix metalloproteinases (MMPs) are enzymes capable of regulating extracellular matrix remodeling that occurs with normal developmental growth and tumor progression of malignant cells. Among its members, the gelatinases A (MMP-2) and B (MMP-9) are thought to be key enzymes for degrading type IV collagen, a major component of the basement membrane. Filamin A (FLNa) is an actin-binding protein that has been implicated in proliferation, cell migration, spreading, and intracellular signaling by providing a scaffold for the recruitment of accessory molecules that influence cell spreading and migration functions. We hypothesized that the expression of MMP-2 and/or MMP-9 will be under the control of the cytoskeletal protein FLNa. The results showed constitutive and phorbol ester-induced expression and secretion of active MMP-9 in M2 cells to levels that were significantly higher than in FLNa-expressing M2A7 cells. This contrasted sharply with the weak expression of MMP-2 in both cell lines. A number of biochemical, genetic and pharmacological approaches were used to demonstrate that FLNa-mediated suppression of the Ras/MAPK signaling cascade played a significant role in the transcriptional down-regulation of MMP-9 gene expression. Ubiquitination-dependent degradation of the guanine-nucleotide exchange factor Ras-GRF1 was found to be partly responsible for this reduced Ras/MAPK activation in FLNa-expressing cells. These data suggest that Ras-GRF1 is a novel modulator of MMP-9 expression, at least in human melanoma cells. Normal tissue remodeling and tumor cell invasion are associated with an increase in MMP-9 activity, and reduced expression or localized mutations in the gene encoding FLNa may be responsible for the increased risk of certain cancer types. On-going work involving the characterization of FLNa-mediated downregulation of Ras-GRF1 in cytokine and insulin signaling in HepG2 hepatoma cells is currently underway. The role of FLNa in Ras-GRF1 stability and subsequent control in MMP-9 expression may offer a potential target for therapeutic interventions.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Intramural Research (Z01)
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National Institute on Aging
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Fiori, Jennifer L; Zhu, Tie-Nian; O'Connell, Michael P et al. (2009) Filamin A modulates kinase activation and intracellular trafficking of epidermal growth factor receptors in human melanoma cells. Endocrinology 150:2551-60
He, Hua-Jun; Zong, Yaping; Bernier, Michel et al. (2009) Sensing the insulin signaling pathway with an antibody array. Proteomics Clin Appl 3:1440-50
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