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STAT1 plays a pivotal role in signal transduction and transcriptional activation in response to type I and II IFNs. Regulation of STAT1 expression has significant consequences in human cancer cells, where STAT1 deficiencies have been associated with cellular resistance to type I IFN. Distinct promoter, enhancer, and repressor regions have previously been described in the regulatory part of the human STAT1 gene extending as far as the second intron. A putative IFN-stimulated response element sequence in the STAT1 promoter is inducible by type I IFN and binds the IFN-α/β–induced complex, ISGF3. Together with the previously characterized IRF-E/GAS/IRF-E (IGI) motif, these positive regulatory elements provide a means for intracellular amplification of STAT1 expression, which is necessary for increasing cell responsiveness to the IFNs. In contrast, the transcriptional repressor REST binds to an RE-1 element in the STAT1 repressor region and in doing so represses transcription from the STAT1 gene regulatory region in melanoma cells lines. Repression significantly decreased in a REST-null cell line. Altering REST function from a transcriptional repressor into an activator as REST-VP16 increased expression from RE-1–targeted reporters. RNA expression of 65 melanoma cell lines by microarray and selected lines with known IFN responsiveness showed significant inverse correlations between STAT1/REST that were related to cellular responses to IFN. Thus REST, through the intronic RE-1 element, provides a means for downregulating STAT1 expression, affecting melanoma responsiveness to IFN. Intracellular levels of REST may be a useful marker to test for IFN resistance and as a novel therapeutic target in IFN-resistant melanomas. Mol Cancer Ther; 12(7); 1288–98. ©2013 AACR.

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PDF file - 675K, Supplementary Figure 1. Characterization of an ISRE in the human STAT1 gene promoter. Supplementary Figure 2. ChIP assay demonstrates binding of REST to the STAT1 RE-1 element in melanoma cell lines. Supplementary Figure 3. Full-length, uncropped gel image from Fig. 5A of REST mRNA and 18S rRNA levels analysed in MM96 and INS-1 cells by RT-PCR. Supplementary Figure 4. Full-length, uncropped blot images from Fig. 5B of A. REST, and B. α-tubulin protein levels determined in MM96 and INS-1 cells by western blotting. Supplementary Figure 5. Full-length, uncropped gel image from Fig. 7A of REST mRNA and 18S rRNA levels determined by RT-PCR in SK-Mel-28 and MM96 cells. Supplementary Figure 6. Full-length, uncropped images of western blot from Fig. 7B showing REST and STAT1 protein levels detected in SK-Mel-28 and MM96 cells Supplementary Figure 7. The same blot as in Suppl. Fig. 6, stripped and reprobed with α-tubulin antibody. Non-specific background blotch is visible in lane 7. Relevant bands and molecular weights are indicated by arrows in the figure.

Dynamic remodelling of the extracellular matrix (ECM) is a key feature of cancer progression. Enzymes that modify the ECM, such as matrix metalloproteinases (MMPs), have long been recognised as important targets of anticancer therapy. Inflammatory cytokines are known to play a key role in regulating protease expression in cancer. Here we describe the identification of gamma-activated site (GA S)-like, signal transducer and activator of transcription (STAT) binding elements (SBEs) within the proximal promoters of the MMP-1 and MMP-3 genes, which in association with AP-1 components (c-Fos or Jun), bind STAT-1 in a homodimer like complex (HDLC). We further demonstrate that MMP expression and binding of this complex to SBEs can either be enhanced by interleukin (IL)-6, or reduced by interferon gamma (IFN-γ), and that IL-6 regulation of MMPs is not STAT-3 dependent. Collectively, this data adds to existing understanding of the mechanism underlying cytokine regulation of MMP expression via STAT-1, and increases our understanding of the links between inflammation and malignancy in colon cancer.

STAT1 plays a pivotal role in signal transduction and transcriptional activation in response to type I and II IFNs. Regulation of STAT1 expression has significant consequences in human cancer cells, where STAT1 deficiencies have been associated with cellular resistance to type I IFN. Distinct promoter, enhancer, and repressor regions have previously been described in the regulatory part of the human STAT1 gene extending as far as the second intron. A putative IFN-stimulated response element sequence in the STAT1 promoter is inducible by type I IFN and binds the IFN-α/β–induced complex, ISGF3. Together with the previously characterized IRF-E/GAS/IRF-E (IGI) motif, these positive regulatory elements provide a means for intracellular amplification of STAT1 expression, which is necessary for increasing cell responsiveness to the IFNs. In contrast, the transcriptional repressor REST binds to an RE-1 element in the STAT1 repressor region and in doing so represses transcription from the STAT1 gene regulatory region in melanoma cells lines. Repression significantly decreased in a REST-null cell line. Altering REST function from a transcriptional repressor into an activator as REST-VP16 increased expression from RE-1–targeted reporters. RNA expression of 65 melanoma cell lines by microarray and selected lines with known IFN responsiveness showed significant inverse correlations between STAT1/REST that were related to cellular responses to IFN. Thus REST, through the intronic RE-1 element, provides a means for downregulating STAT1 expression, affecting melanoma responsiveness to IFN. Intracellular levels of REST may be a useful marker to test for IFN resistance and as a novel therapeutic target in IFN-resistant melanomas. Mol Cancer Ther; 12(7); 1288–98. ©2013 AACR.

Luciferase reporter constructs are widely used for analysis of gene regulation when characterizing promoter and enhancer elements. We report that the recently developed codon-modified Renilla luciferase construct included as an internal standard for cotransfection must be used with great caution with respect to the amount of DNA transfected. Also, the dual-luciferase reporter vectors encoding Photinus pyralis firefly or Renilla reniformis luciferase showed a linear increase in dose-response with increasing amounts of transfected DNA, but at higher levels of transfected DNA, a reduction in expressed levels of luciferase activity resulted. In addition, treatment with type I interferon (IFN) was found to significantly reduce levels of P. pyralis firefly and Renilla luciferase activity. In contrast, cells transfected with a green fluorescent protein (GFP) reporter construct showed no significant IFN-associated change. The reduction in luciferase activity resulting from IFN treatment was not due to IFN-mediated cytotoxicity, as no change in cellular propidium iodide (PI) staining was observed by flow cytometry. IFN treatment did not alter the levels of firefly luciferase activity in cell culture supernatants or the luciferase mRNA levels determined by quantitative real-time RT-PCR analysis. Based on these results, it is probable that the IFN-induced reduction in levels of luciferase activity detected in reporter assays occurs via a posttranscriptional mechanism. Thus, it is important to be aware of these complications when using luciferase reporter systems in general or for analyzing cytokine-mediated responsive regulation of target genes, particularly by the type I IFNs.

Recently, a role for IFN γ in the genesis of melanoma was reported. Here, we define the mechanism whereby IFN γ becomes a growth stimulator of melanoma cells. In this study, IFN γ stimulated STAT5 to activate the expression of c- myc , an oncogene linked with reduced melanoma patient survival. In a preliminary retrospective study of 27 stage III melanoma patients who received IFN α 2 therapy, STAT5A and c- myc expression levels in metastatic lymph node biopsies were highly correlated (Pearson’s r = 0.914; P myc or STAT5 relative to STAT1 were prognostic markers indicating reduced patient survival. In vitro studies showed that STAT5 activated the c- myc proximal promoter via a functional STAT5A binding site, conferring significant inducibility by IFN γ or EGF (but not IFN α ) in luciferase reporter assays of melanoma cells overexpressing STAT5A. IFN γ caused markedly different responses in wild-type A375 cells compared with A375-STAT5A cells, supporting a key role for STAT5A in IFN γ -mediated regulation of c- myc expression in melanoma. ChIP assays confirmed direct STAT5A binding during IFN γ -mediated activation of the c- myc proximal SBE regulatory region. 5′ RACE and Q-PCR showed that increased levels of c- myc in A375-STAT5A cells induced by IFN γ were associated with a shift in the initiation of transcription near to or at the proximal STAT5 binding element. These results indicate that overexpression of STAT5 in cancer cells is responsible for growth-stimulation by IFN γ .

Interleukin-6 (IL-6) is a major regulator of matrix metalloproteinase (MMP) expression in cancers. A patient matched control study of human colorectal cancers revealed that levels of IL-6 expression correlated strongly with those of MMP-1 and MMP-3 . In human colon cancer cell lines, MMP-3 expression was increased by treatment with IL-6, and suppressed by interferon-gamma (IFN- γ ). Analysis of the MMP-1 and MMP-3 proximal promoters by gel shift and ChIP assays revealed noncanonical STAT binding elements (SBEs) which bound STAT-1 in a complex with AP-1/Fos constitutively and in an IL-6/PMA-inducible manner. STAT-3 was not observed to bind to these novel proximal promoter SBEs. Using northern blot analysis with a STAT-3 knockout colon cancer cell line confirmed that STAT-3 was not required for the IL-6 inducibility of MMP-1 and -3 expression. However, STAT-1 specific siRNA knockdown significantly inhibited induction, validating the requirement for STAT-1 in transcriptional activation. These results strengthen a role for STAT-1 in cytokine induction of malignancy-associated factors such as MMPs in colorectal cancer.