The signal transducer and activator of transcription (STAT) proteins relay signals from cytokine receptors and receptor tyrosine kinases on the cell surface to the nucleus, where they affect the transcription of genes involved in normal cell functions, including growth, apoptosis and differentiation. STAT3 has been found to be constitutively active in head and neck squamous cell carcinoma (HNSCC) as well as in other epithelial malignancies. In HNSCC, STAT3 alters the cell cycle, prevents apoptosis, and mediates the proliferation and survival of tumour cells. Several therapeutic approaches are being developed to target STAT3, including molecules that block either dimerisation or DNA binding by STAT3, strategies to decrease STAT3 expression and drugs that inhibit STAT3 function. Strategies that block STAT3 may prove efficacious for cancer treatment.

We previously developed a transcription factor decoy targeting signal transducer and activator of transcription 3 (STAT3) and reported antitumor activity in both in vitro and in vivo models of squamous cell carcinoma of the head and neck (SCCHN). Based on the known existence of STAT1-STAT3 heterodimers, the high sequence homology between STAT1 and STAT3, as well as expression of both STAT1 and STAT3 in SCCHN, we examined whether the STAT3 decoy interferes with STAT1 signaling. SCCHN cell lines with different STAT1 expression levels (but similar STAT3 levels) were used. Both cell lines were sensitive to the growth-inhibitory effects of the STAT3 decoy compared with a mutant control decoy. Intact STAT1 signaling was demonstrated by interferon-␥ (IFN-␥)-mediated induction of STAT1 phosphorylation (Tyr701) and interferon-regulatory factor-1 (IRF-1) expression. Treatment with the STAT3 decoy (but not a mutant control decoy) resulted in inhibition of IRF-1 protein expression in both cell lines, indicating specific inhibition of STAT1 signaling by the STAT3 decoy. Because STAT1 is a potential tumor suppressor, we also investigated whether STAT1 signaling mitigated the therapeutic efficacy of the STAT3 decoy. In both PCI-15B and UM-22B cells, STAT1 siRNA treatment resulted in decreased STAT1 expression, without altering the antitumor activity of the STAT3 decoy. Likewise, the antitumor effects of the STAT3 decoy were not altered by STAT1 activation upon IFN-␥ treatment. These results suggest that the therapeutic mechanisms of STAT3 blockade using a transcription factor decoy are independent of STAT1 activation.Signal transducer and activator of transcription 3 (STAT3) has emerged as a potential molecular target for cancer therapy. STAT3 is constitutively activated and overexpressed in a variety of human malignancies, including breast, lung, prostate, brain, leukemia, multiple myeloma, and squamous cell carcinoma of the head and neck (SCCHN) (Grandis et al., 1998;Turkson and Jove, 2000). The expression levels of activated or tyrosine-phosphorylated STAT3 have been reported to correlate with decreased survival in several cancers, including oral tongue carcinoma (Masuda et al., 2002). Molecular targeting of STAT3 using a variety of strategies in preclinical models of human cancer has been shown to inhibit tumor growth (Turkson and Jove, 2000). We previously developed a transcription factor decoy based on the STAT3 DNA binding element and demonstrated that this decoy interferes with STAT3 signaling and decreases SCCHN tumor growth in vitro and in vivo (Leong et al., 2003;Xi et al., 2005).Transcription factor decoys are double-stranded DNA oligonucleotides that resemble the transcription factor-binding site in the promoters of target genes. Transcription factor decoys presumably bind transcription factors and sequester the targeted transcription factor, rendering it unavailable for transcription of downstream target genes. The sequence of the STAT3 decoy was derived from the serum-inducible element of the human c-fos...

BackgroundBone marrow stromal cells produce cytokines required for the normal growth and development of all eight hematopoietic cell lineages. Aberrant cytokine production by stromal cells contributes to blood cell dyscrasias. Consequently, factors that alter stromal cell cytokine production may significantly compromise the development of normal blood cells. We have shown that environmental chemicals, such as aromatic hydrocarbon receptor (AhR) agonists, suppress B lymphopoiesis by modulating bone marrow stromal cell function. Here, we extend these studies to evaluate the potential for two prototypic AhR agonists, 7,12-dimethylbenz [a]anthracene (DMBA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to alter stromal cell cytokine responses.MethodsBone marrow stromal cells were treated with AhR agonists and bacterial lipopolysaccharide (LPS) to mimic innate inflammatory cytokine responses and to study the effects of AhR ligands on those responses. Steady state cytokine RNA levels were screened by RNAse protection assays (RPA) and quantified by real-time PCR. Cytokine (IL-6) protein production was measured by ELISA. NF-κB EMSAs were used to study IL-6 transcriptional regulation.ResultsRPAs indicated that AhR+ bone marrow stromal cells consistently up-regulated genes encoding IL-6 and LIF in response to LPS, presumably through activation of Toll-like receptor 4. Pre-treatment with low doses of DMBA or TCDD selectively abrogated IL-6 gene induction but had no effect on LIF mRNA. Real-time-PCR indicated a significant inhibition of IL-6 mRNA by AhR ligands within 1 hour of LPS challenge which was reflected in a profound down-regulation of IL-6 protein induction, with DMBA and TCDD suppressing IL-6 levels as much as 65% and 88%, respectively. This potent inhibitory effect persisted for at least 72 hours. EMSAs measuring NF-κB binding to IL-6 promoter sequences, an event known to induce IL-6 transcription, indicated a significant decrease in the LPS-mediated induction of DNA-binding RelA/p50 and c-Rel/p50 heterodimers in the presence of DMBA.ConclusionsCommon environmental AhR agonists can suppress the response to bacterial lipopolysaccharide, a model for innate inflammatory responses, through down-regulation of IL-6, a cytokine critical to the growth of several hematopoietic cell subsets, including early B cells. This suppression occurs at least at the level of IL-6 gene transcription and may be regulated by NF-κB.

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