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Cancer Research 67, 1254-1261, February 1, 2007. doi: 10.1158/0008-5472.CAN-06-2439
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

A Small-Molecule Enhancer of Signal Transducer and Activator of Transcription 1 Transcriptional Activity Accentuates the Antiproliferative Effects of IFN-{gamma} in Human Cancer Cells

Rebecca A. Lynch, Julia Etchin, Traci E. Battle and David A. Frank

Department of Medical Oncology, Dana-Farber Cancer Institute, Departments of Medicine, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts

Requests for reprints: David A. Frank, Department of Medical Oncology, Mayer 522B, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. Phone: 617-632-4714; Fax: 1-617-632-6356; E-mail: david_frank{at}dfci.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The transcription factor signal transducer and activator of transcription (STAT) 1 can mediate antiproliferative and proapoptotic effects in cancer cells, and a number of mechanisms have been found whereby STAT1 signaling is attenuated in tumors thereby increasing their malignant behavior. Thus, enhancing gene transcription mediated by STAT1 may be an effective approach to cancer therapy. A high-throughput screen was developed to identify molecules that could enhance STAT1-dependent gene expression. Through this approach, it was found that 2-(1,8-naphthyridin-2-yl)phenol (2-NP) caused a 2-fold increase in STAT1-dependent reporter gene expression compared with that seen with maximally effective concentrations of IFN-{gamma} alone. This effect was specific to STAT1 because 2-NP had no effect on unrelated transcription factors such as nuclear factor (NF) {kappa}B or the highly homologous transcription factor STAT3. STAT1-dependent gene activation was enhanced by this compound in a variety of human and murine cell lines and was independent of the stimulus used. Furthermore, 2-NP enhanced the expression of the bona fide endogenous STAT1 target gene interferon regulatory factor 1. 2-NP increased the duration of STAT1 tyrosine phosphorylation in response to IFN-{gamma}, and this may underlie its enhancement of STAT1-dependent transcription. Reflecting the fact that STAT1 can exert tumor-suppressive effects, 2-NP enhanced the ability of IFN-{gamma} to inhibit the proliferation of human breast cancer and fibrosarcoma cells. Tumor cells lacking STAT1 were unaffected by either IFN-{gamma} or 2-NP. These findings indicate that enhancement of STAT1 transcriptional activity may have utility in anticancer therapies, and that cell-based screens for modulators of transcription factor function can be a useful approach for drug discovery. [Cancer Res 2007;67(3):1254–61]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The signal transducers and activators of transcription (STAT) family of transcription factors transduce signals generated by a wide variety of extracellular stimuli. As such, they play a critical role in the regulation of cell survival, proliferation, and differentiation in nearly all tissue types (1, 2). Reflecting the central position they play in these processes, inappropriate STAT function has been found in a wide array of human tumors (3, 4). Constitutive activation of STATs, most often STAT3 or STAT5, is a particularly common finding. However, increasing evidence has implicated the loss of function of another STAT family member, STAT1, in tumorigenesis. Whereas many STAT3 target genes promote cell cycle progression (5, 6), STAT1 mediates the expression of inhibitors of cyclin-dependent kinases (7) and represses genes necessary for cell cycle entry (8). Similarly, STAT3 and STAT5 enhance expression of prosurvival genes, whereas STAT1 can repress these genes (9) and trigger apoptosis (10, 11). Furthermore, STAT1 may be important in mediating the therapeutic effect of biological therapies for leukemia (12) and the induced differentiation of leukemia cells (13).

STAT1 may be particularly important in mediating antitumor effects of IFN-{gamma}. In addition to inhibiting proliferation and survival, IFN-{gamma} enhances the immunogenicity of tumor cells in part through enhancing STAT1-dependent expression of MHC proteins (14). This is likely of physiologic importance in that a loss of IFN-{gamma} responsiveness eliminates the immune clearance of tumors in mice (15, 16). A critical role for STAT1 has also been found in the response of human cancer cells to IFN-ß, in which the induction of the proapoptotic mediator tumor necrosis factor (TNF)-{alpha}–related apoptosis-inducing ligand requires STAT1 (17). In addition, STAT1 seems to be a key negative regulator of angiogenesis (18), and loss of STAT1 enhances metastasis and angiogenesis in murine systems (19). Reflecting the important role that this pathway plays in suppressing tumorigenesis, the loss of the ability to activate STAT1 via the IFN receptor has been found to occur in lung cancer (15) and prostate cancer (20). Conversely, increased phosphorylation and DNA binding activity of STAT1 is associated with decreased relapse and increased survival in patients with newly diagnosed breast cancer (21). The function of this transcription factor may be particularly important in patients receiving chemotherapy because STAT1 activation seems to be an important element in the killing of tumor cells in response to cytotoxic agents (22).

Given this central role that STAT1 plays in suppressing neoplastic behavior, an attractive therapeutic strategy is to enhance the gene activation events mediated by STAT1. To do this, a cell-based high-throughput screening system was developed to identify small organic molecules that enhance STAT1-dependent gene activation. Using this system, we identified a compound, 2-(1,8-naphthyridin-2-yl)phenol (2-NP), that enhances gene activation mediated by STAT1 over that seen with maximally efficacious concentration of IFN-{gamma} and other cytokines. Furthermore, 2-NP accentuates the antiproliferative effects of IFN-{gamma} on cancer cells in vitro, suggesting that modulation of the transcription factor function may be a useful new strategy in the treatment of cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfections. U3A and 2fTGH cells (kindly provided by Dr. George Stark, Department of Molecular Genetics, Cleveland Clinic, Cleveland, OH; ref. 23), MCF-7 cells, and NIH3T3 cells were grown in DMEM supplemented with 10% FCS at 37°C in 95% air and 5% CO2. Cell viability assays were done in duplicate using CellTiter-Glo (Promega, Madison, WI) to measure the luminescent output from the ATP present in viable cells.

To generate a cell-based system by which STAT1-dependent gene activation could be quantitated, a plasmid was constructed in which a firefly luciferase gene was placed under the control of a STAT-dependent promoter sequence. A neomycin phosphotransferase cassette was also introduced, and this plasmid was then transfected into NIH3T3 cells using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA). Cells were selected in G418 (500 µg/mL), and stable colonies of these STAT-luc/3T3 cells were picked and expanded. Clones were then assessed for low basal luciferase activity and prominent induction following treatment with IFN-{gamma}. Several such clones were expanded and used for subsequent experiments. As controls, parallel cell lines were generated in which luciferase was under the control of a NF{kappa}B-responsive promoter (NF{kappa}B-luc/3T3 cells) or constitutively expressed by a cytomegalovirus promoter (CMV-luc/3T3).

For transient transfections, a STAT-responsive firefly luciferase reporter was introduced along with a Renilla luciferase expression plasmid under the control of a thymidine kinase promoter (Promega) using LipofectAMINE 2000 (24). Sixteen hours after transfection, the cells were untreated or treated with 2-NP for 1 h, then treated with the indicated cytokine. Five hours later, cells were lysed, and relative luminescence was measured on a Luminoskan Ascent luminometer (ThermoLab Systems, Helsinki, Finland) using a dual-luciferase system (Promega). STAT-dependent luciferase production was normalized to the values from the control Renilla luciferase expression plasmid.

Screening for STAT1 modulators. For high-throughput screening, 4,000 STAT-luc/3T3 cells were plated in 30 µL per well in a 384-well plate. After being allowed to adhere overnight, 0.1 µL of compounds from the Peakdale Library (Institute of Chemistry and Cell Biology, Harvard Medical School, Boston, MA) were added to the plates using pin transfer. The compounds were stored in 100% DMSO, and thus, the final concentration of DMSO was 0.3%, a concentration we found had no effect on basal or induced luciferase expression in these cells. After incubation for 1 h, cells were then treated with IFN-{gamma} (R&D Systems, Minneapolis, MN) to a final concentration of 10 ng/mL. Five hours later, luciferase was quantitated using the Bright Glo Luciferase Assay Kit (Promega). For the NF{kappa}B-responsive cells, following incubation with compounds, NF{kappa}B-luc/3T3 cells were treated with TNF-{alpha} (R&D Systems; 10 ng/mL).

RT-PCR. RNA was isolated from cells using the RNeasy kit (Qiagen, Valencia, CA). RNA of 2 µg was reverse transcribed with a poly-dT primer using the SuperScript First-Strand synthesis kit (Invitrogen). Real-time PCR was then done with a SYBR Green master mix (Stratagene, La Jolla, CA) on an ABI Prism 7500 instrument. The primer pairs used were murine interferon regulatory factor (IRF)-1, 5'-CGTTGTGCCATGAACTCCCTGC-3' and 5'-GCTCTTAGTGTCTCGGCTGG-3'; and murine glyceraldehyde-3-phosphate dehydrogenase, 5'-CAAGAAGGTGGTGAAGCAGG-3' and 5'-CTCTTGCTCAGTGTCCTTGC-3'.

Western blotting. Cells were lysed in radioimmunoprecipitation assay buffer containing 50 mmol/L Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL pepstatin, and 1 mmol/L sodium orthovanadate. For Western blot analysis, 40 µg of protein were resolved on 7% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Blots were incubated with antibodies that recognize the tyrosine phosphorylated form of STAT1 (1:10,000; ref. 25), the serine phosphorylated form of STAT1 (1:10,000; ref. 26), total STAT1 (1:20,000; Santa Cruz Biotechnology, Santa Cruz, CA, SC-346), the tyrosine phosphorylated form of STAT3 (1:10,000; Cell Signaling Technology, Danvers, MA), and total STAT3 (1:10,000; Santa Cruz Biotechnology). Blots were incubated with goat anti-rabbit horseradish peroxidase–conjugated secondary antibodies (Calbiochem, La Jolla, CA) and detection was done using the Renaissance chemiluminescent ECL kit (NEN Dupont, Boston, MA). Western blots were then scanned and analyzed with NIH Image 1.61/ppc. Band densities from phospho-STAT1 Western blots were normalized to the band intensity from the total STAT1 Western blots.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of reporter cell lines. We first validated the specificity of the reporter cell lines by treating them with potent and specific activators of either STAT1 (with IFN-{gamma}) or NF{kappa}B (with TNF-{alpha}). STAT-luc/3T3 cells and NF{kappa}B-luc/3T3 cells were left untreated or treated with IFN-{gamma} or TNF-{alpha}, and luciferase activity was determined. Treatment of STAT-luc/3T3 cells with IFN-{gamma} led to a 5-fold induction of luciferase activity (Fig. 1 ). By contrast, treatment of these cells with TNF-{alpha} caused no significant change in luciferase activity. NF{kappa}B-luc/3T3 cells displayed the opposite response, showing no significant change in luciferase activity in response to IFN-{gamma}, but displaying a 2- to 3-fold induction in response to TNF-{alpha}. Both assays were highly reproducible and showed little interwell variability. Reflecting this, the Z' factor (27) for the STAT-luc/3T3 system was 0.92, and for the NF{kappa}B-luc/3T3 system, it was 0.81. The characteristics of these cells remained unchanged over 6 months in culture. Thus, these cell lines display high sensitivity and specificity as reporters for activity of each of these pathways.


Figure 1
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  		Figure 1. STAT-luciferase reporter cells are sensitive and specific indicators of STAT1 activation. STAT-luc or NF{kappa}B-luc NIH3T3 cells were untreated or treated with IFN-{gamma} (10 ng/mL) or TNF-{alpha} (10 ng/mL) for 5 h, after which luciferase activity was quantitated. Columns, mean of three experiments; bars, SE.

 
Screening for enhancers of STAT1 transcriptional activity. Given the importance of STAT1 as a potential tumor suppressor, the isolation of small organic molecules that enhance the activity of this protein would represent a novel approach to cancer therapy that may complement standard therapies. To identify such molecules, STAT-luc/3T3 cells were seeded in 384-well plates and then treated with 5,120 compounds from the Peakdale libraries at a concentration of 16 µg/mL in a final concentration of 0.3% DMSO. The Peakdale compounds are characterized by their structural diversity. They display appropriate molecular weight and lipophilicity for cell-permeable drugs, and they lack functional groups likely to cause toxicity or instability. After 1 h, cells were stimulated with IFN-{gamma}, and 5 h later, luciferase activity was quantitated relative to cells that had been treated with IFN-{gamma} alone. Although the STAT-responsive promoter used in the STAT-luciferase vector can also respond to STAT3, when NIH3T3 cells are treated with IFN-{gamma}, STAT1 is the only STAT family member that is activated (data not shown). From this screen, we identified three compounds that increased STAT1-dependent luciferase activity by more than 1.5-fold. Among these, 2-NP (Fig. 2A ) was the most efficacious enhancer of STAT1-dependent luciferase activity.


Figure 2
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  		Figure 2. A, chemical structure of 2-NP. B–D, 2-NP specifically enhances STAT1-dependent gene expression. B, STAT-luc/NIH3T3 cells were left untreated or pretreated with 2-NP for 1 h at the indicated concentration, then incubated in the absence (open columns) or presence of IFN-{gamma} (10 ng/mL; filled columns). After 5 h, luciferase activity was quantitated. *, P < 0.05 by two-sided t test when compared with untreated cells. C, NF{kappa}B-luc/NIH3T3 cells were left untreated or pretreated with 2-NP (45 µmol/L) for 1 h and then incubated in the absence or presence of TNF-{alpha} (10 ng/mL). After 5 h, luciferase activity was quantitated. D, CMV-luc/NIH3T3 cells were left untreated or treated with 2-NP (45 µmol/L) for 5 h, after which luciferase activity was quantitated. Columns, means of three experiments; bars, SE.

 
2-NP specifically enhances STAT1-dependent gene expression. To determine the effects of 2-NP on STAT1-dependent gene activation, STAT-luc/3T3 cells were left untreated or pretreated with increasing concentrations of 2-NP for 1 h, then stimulated with IFN-{gamma}. Luminescence was then quantitated 5 h later. In the absence of 2-NP, a dose of 10 ng/mL of IFN-{gamma} led to a maximal induction of luciferase activity, 8-fold above baseline. At this concentration of IFN-{gamma}, pretreatment with 2-NP led to a dose-dependent enhancement in STAT1-driven luciferase activity (Fig. 2B). At a dose of 45 µmol/L, 2-NP showed maximal effects, enhancing STAT1-dependent luciferase activity by 2.5-fold. 2-NP alone had minimal effects on luciferase expression in STAT-luc/3T3 cells. The DMSO vehicle had no effect on STAT1-dependent gene expression (data not shown). Thus, the major effect of 2-NP is to enhance the STAT1-dependent transcriptional activity induced by IFN-{gamma}.

To exclude the possibility that 2-NP was enhancing luciferase activity in these cells by a nonspecific mechanism, we tested the effect of this compound on gene activation mediated by an independent transcription factor, NF{kappa}B. In NF{kappa}B-luc/3T3 cells, in which luciferase is under the control of a NF{kappa}B-responsive promoter, TNF-{alpha} treatment led to a 2-fold induction of luciferase activity. In the presence or absence of TNF-{alpha}, 2-NP had no significant effect on luciferase activity in these cells (Fig. 2C). Furthermore, in cells in which luciferase was constitutively expressed, driven by a cytomegalovirus promoter, 2-NP also had no effect on luciferase activity (Fig. 2D). Thus, 2-NP does not act by nonspecifically increasing transcription or translation or directly enhancing luciferase enzymatic activity.

To determine whether 2-NP enhanced STAT1-dependent gene activation in other cell types, we transiently transfected the STAT-luc reporter plasmid into SK-N-MC neuroblastoma cells, along with a plasmid-encoding Renilla luciferase under the control of a thymidine kinase promoter to serve as a control of transfection efficiency. In these cells, IFN-{gamma} induced a 2.5-fold induction in normalized luciferase activity (Fig. 3A ). Whereas the vehicle DMSO had no effect on this induction, pretreating the cells for 1 h before IFN-{gamma} treatment led to further 2-fold induction in activity, similar in magnitude to the enhancement seen in NIH3T3 cells. Similar effects were seen in MCF-7 cells, indicating that this effect of 2-NP is not cell type or species restricted (data not shown).


Figure 3
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  		Figure 3. 2-NP enhances STAT1-dependent but not STAT3-dependent gene expression. A, SK-N-MC human neuroblastoma cells were transiently transfected with a STAT-luciferase reporter plasmid as well as an expression plasmid for Renilla luciferase under the control of a thymidine kinase promoter. Cells were then left untreated or pretreated with 2-NP (45 µmol/L) for 1 h and then incubated in the absence or presence of IFN-{gamma} (10 ng/mL). After 5 h, luciferase activity was quantitated. B, U3A human fibrosarcoma cells, which lack STAT1, were transiently transfected with a STAT-luciferase reporter plasmid as well as an expression plasmid for Renilla luciferase under the control of a thymidine kinase promoter. Cells were then left untreated or pretreated with 2-NP (45 µmol/L) for 1 h and then incubated in the absence or presence of IL-6 (10 ng/mL) to induce STAT3 activation (10 ng/mL). After 5 h, luciferase activity was quantitated. STAT-dependent luciferase was normalized to the control Renilla luciferase. Columns, means of three experiments; bars, SE. *, P < 0.05 by two-sided t test.

 
It is known that the STAT reporter construct used can also confer STAT3 transcriptional control on a reporter gene. We thus considered the possibility that 2-NP might enhance the activity of this highly homologous STAT family member. U3A cells, which lack STAT1, were stably transfected with the STAT-luc construct. Stimulation of these cells with the cytokine IL-6 (10 ng/mL), which induces activation solely of STAT3, led to a 5-fold induction in luciferase activity (Fig. 3B). 2-NP, either alone or in the presence of IL-6, had no effect on luciferase activity. Thus, 2-NP enhances STAT1-dependent gene induction, without affecting the function of STAT3.

2-NP enhances the induction of an endogenous STAT1 target gene. To facilitate high-throughput screening of STAT1 activators, we used a heterologous luciferase reporter construct. However, it was necessary to determine whether 2-NP could enhance the expression of a bona fide STAT1 target gene in its genomic context. To address this question, NIH3T3 cells were treated with vehicle alone or with 2-NP for 1 h, after which they were stimulated with IFN-{gamma} for 30 min. RNA was harvested, and the expression of IRF-1, a well-validated STAT1 target gene, was determined by quantitative reverse transcription-PCR. (Fig. 4 ). A maximally effective concentration of IFN-{gamma} alone led to a 13-fold induction in IRF-1 mRNA. Preincubation with 2-NP increased the magnitude of induction of IRF-1 to 25-fold over basal levels. Thus, in addition to enhancing the expression of model STAT1-responsive genes, 2-NP enhances the expression of endogenous STAT1 target genes.


Figure 4
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  		Figure 4. 2-NP enhances the expression of the endogenous STAT1 target gene IRF-1. NIH3T3 cells were left untreated or were treated with 2-NP (45 µmol/L) for 1 h. Cell were then incubated in the absence or presence of IFN-{gamma} (10 ng/mL) for 2 h, after which RNA was harvested. IRF-1 mRNA was quantitated by real-time reverse transcription-PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase expression. Columns, mean fold changes of three experiments done in triplicate; bars, SE. *, P < 0.05 by two-sided t test.

 
2-NP leads to the prolongation of STAT1 tyrosine phosphorylation. Given the prominent and specific ability of 2-NP to enhance STAT1-dependent gene activation, we considered the possibility that 2-NP was enhancing STAT1 tyrosine phosphorylation. NIH3T3 cells were untreated or incubated with 2-NP and then either left untreated or stimulated with IFN-{gamma} at varying doses or for differing intervals. Cell extracts were prepared, and the phosphorylation of STAT1 on tyrosine 701 was determined by Western blotting and normalized to total STAT1 levels. At an early time point, 30 min, 2-NP had little effect on STAT1 tyrosine phosphorylation induced by IFN-{gamma} over a range of concentrations (Fig. 5A ). IFN-{gamma}–induced STAT1 tyrosine phosphorylation is usually rapid and transient, returning toward baseline after 2 to 4 h. However, when cells were pretreated with 2-NP, STAT1 phosphorylation remained elevated for up to 4 h (Fig. 5B). No changes in STAT1 serine phosphorylation were noted (data not shown). To examine whether 2-NP also prolonged the tyrosine phosphorylation of STAT3, U3A cells were incubated with 2-NP or the DMSO vehicle, and then stimulated with IL-6 (10 ng/mL). Cell extracts were prepared over a 4-h time span, and Western blots were done for STAT3 phosphorylated on tyrosine 705 and for total STAT3 (Fig. 5C). No difference was seen in the magnitude or kinetics of STAT3 phosphorylation in the presence of 2-NP. Thus, the ability of 2-NP to specifically enhance STAT1-dependent gene activation may occur by specifically prolonging the tyrosine phosphorylation of STAT1.


Figure 5
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  		Figure 5. 2-NP prolongs the tyrosine phosphorylation (P-tyr) of STAT1, but not STAT3. NIH3T3 cells were untreated or treated with 2-NP (45 µmol/L) for 1 h, as indicated. They were then treated with IFN-{gamma} for 30 min at the indicated concentration (A), or with 10 ng/mL IFN-{gamma} for the indicated times (B), after which cell extracts were prepared, and Western blots were done to tyrosine-phosphorylated STAT1 (top) or total STAT1 (bottom). The band density for the tyrosine-phosphorylated STAT1 was quantitated and normalized to total STAT1. C, U3A cells were untreated or treated with 2-NP (45 µmol/L) for 1 h, as indicated. They were then treated with IL-6 (10 ng/mL) for the indicated times, after which cell extracts were prepared, and Western blots were done to tyrosine-phosphorylated STAT3 (top) or total STAT3 (bottom).

 
2-NP accentuates the antiproliferative effects of IFN-{gamma} on cancer cells. The rationale for developing STAT1 enhancers was based on evidence that STAT1 plays an important role in suppressing tumorigenesis. To determine whether 2-NP could enhance an antitumor effect mediated by STAT1, we examined the effect of 2-NP on the cytostatic effect of IFN-{gamma} on the human breast cancer cell line MCF-7. IFN-{gamma} alone leads to a modest but reproducible dose-dependent inhibition of MCF-7 cell proliferation with a maximal effect of 12% inhibition at a concentration of 5 µg/mL (Fig. 6A ). 2-NP alone had minimal effects on MCF-7 proliferation, indicating that it does not have nonspecific toxic or cytostatic effects. However, the addition of 2-NP to IFN-{gamma}led to a 4-fold enhancement in the inhibition of proliferation. Thus, the STAT1 enhancer 2-NP can accentuate the growth-inhibitory effects induced by IFN-{gamma}.


Figure 6
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  		Figure 6. 2-NP enhances the antiproliferative effects of IFN-{gamma} on human cancer cells. (A) MCF-7, (B) 2fTGH, or (C) U3A cells lacking STAT1 were preincubated with 2-NP (45 µmol/L) for 1 h, as indicated. They were then untreated or treated with IFN-{gamma} (10 ng/mL) for 48 h, after which, viable cells were quantitated by ATP-based luminescence. Columns, means of three experiments; bars, SE. *, P < 0.05 by two-sided t test.

 
To confirm that this growth-inhibitory effect of 2-NP was STAT1 dependent, we used the human fibrosarcoma cell line 2fTGH. A variant of this cell line, U3A, was derived that lacks STAT1 (23). Similar to MCF-7 cells, IFN-{gamma} causes a modest reduction in proliferation, which is accentuated by 2-NP (Fig. 6B). Interestingly, 2-NP alone also causes some reduction in proliferation of these cells, perhaps through the enhancement of low-level basal STAT1 activation (28). However, neither IFN-{gamma}nor 2-NP showed any effect on the proliferation of the isogenic U3A cells lacking STAT1 (Fig. 6C). Thus, 2-NP does not cause nonspecific toxicity and exhibits growth-inhibitory activity only in the presence of functional STAT1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Most therapeutic approaches to advanced cancers rely on cytotoxic agents that induce cell death in a nonspecific manner. As a result, these agents induce significant toxicity and exhibit a low therapeutic index. It would be beneficial if the next generation of anticancer therapies made use of specific molecular pathways that can be used to inhibit the malignant behavior of tumor cells. One such target is STAT1, which exhibits properties of a tumor suppressor. This transcription factor can mediate cell cycle arrest, enhanced apoptosis, and therapeutic differentiation and, thus, may suppress tumorigenicity by a number of means. Furthermore, STAT1 can be activated by IFNs, cytokines that have been used therapeutically for many years for a variety of malignant, infectious, and autoimmune diseases with minimal toxicity.

In considering how to enhance STAT1-dependent gene expression, several strategies can be envisioned. One approach is to focus on pathways that down-regulate STAT1 function and to develop small molecules that inhibit their function using structure-based drug design. Such targets might include phosphatases that mediate STAT1 dephosphorylation (29, 30), pathways leading to STAT1 degradation (31), or members of the suppressors of cytokine signaling family that dampen the activation of STAT1 (32). We decided to take an open-ended approach based on a cell-based screening assay in which STAT1-dependent gene activation could be detected in a high-throughput fashion by measuring the activity of a reporter gene. This strategy has the advantage that active compounds must be cell permeable or otherwise able to reach their cellular targets, and that molecules displaying nonspecific toxicity are rapidly excluded. Furthermore, it allows the identification of active compounds independent of a predetermined mechanism of action. This provides the ability to find compounds that may function by a variety of mechanisms and may reveal levels of functional regulation that might not have been previously appreciated.

Using this approach to identify molecules that could enhance STAT1-dependent gene activation over that seen with maximally active concentrations of IFN-{gamma}, we screened 5,120 compounds. The compounds were tested at a single concentration of 16 µg/mL, ~30 to 50 µmol/L for most of these molecules. This concentration was known to be nontoxic for nearly all of the compounds, and at this relatively high concentration, it was thought to be less likely that an active agent would be missed. Thus, this single concentration was used to minimize the costs of the screen. However, screening at a single concentration does increase the chance of false negative results, for example, of a compound displaying an "inverted U" dose-response curve.

From this screen, 2-NP was found to induce the greatest increase in STAT1-dependent luciferase expression. 2-NP not only enhanced the expression of a STAT1-responsive reporter gene, but it also increased the expression of an endogenous STAT1 target gene, and it accentuated the STAT1-mediated inhibition of proliferation of cancer cells. However, 2-NP showed specificity in this effect in that it had no effect on gene activation mediated by the highly homologous protein STAT3, or by the unrelated transcription factor NF{kappa}B. Because constitutively active cysteine-containing STAT1 (and STAT3) mutants seem to work by prolonging STAT tyrosine phosphorylation (28), we considered the possibility that 2-NP might be mediating a similar effect. At early time points, 2-NP has a negligible effect at enhancing STAT1 tyrosine phosphorylation. However, 2-NP seems to prolong STAT1 phosphorylation, and its ability to enhance STAT1-dependent gene expression may relate to this effect. This action may be mediated by inhibiting a STAT1 phosphatase, blocking STAT1 degradation (31) or prolonging the interaction of STAT1 with its cognate DNA sequence (28, 33). Because 2-NP does not exert a similar effect on STAT3, elucidating the mechanism for this effect might uncover distinct aspects of the regulation of these two homologous transcription factors.

The enhancement of transcription mediated by 2-NP, both in reporter gene systems and of the endogenous target IRF-1, is ~2-fold over that seen with maximally effective concentrations of cytokines. Although this seems to be a small level of enhancement, evidence from gene expression microarrays and quantitative analysis of gene expression indicates that changes in expression of as little as 1.5-fold may be important in mediating significant changes in cellular physiology (6). Furthermore, this level of activity allows a significant enhancement of the growth suppression mediated by IFN-{gamma} in cancer cells. Thus, compounds that can enhance STAT1-dependent gene activation to this magnitude can clearly mediate biologically important effects in cellular function.

Maximal activity of 2-NP is seen at concentrations in the low micromolar range (Fig. 2B). It remains to be determined whether this potency will be adequate for use in animals with acceptable toxicity. However, the minimal toxicity exhibited by 2-NP in vitro, especially in the U3A cells, which lack STAT1, suggests that any "off-target" effects of 2-NP will be limited. Once a specific cellular target for the STAT1-enhancing effects of 2-NP is identified, the goal will be to enhance the function of this molecule through structure-activity considerations or to develop other compounds with similar activity, maximizing potency and minimizing the likelihood of toxicity or nonspecific effects.

There are several potential medical uses for 2-NP for malignant and nonmalignant diseases. IFNs are already used in the treatment of a number of forms of hematopoietic cancers, such as chronic myelogenous leukemia (34) and hairy cell leukemia (35), as well as nonhematopoietic cancers, such as melanoma (36) and renal cell carcinoma (37). Thus, 2-NP might show particular efficacy in enhancing the activity of IFNs for these diseases. However, STAT1 may be important in mediating endogenous antitumor signals whether emanating from cytokines released around tumor cells, such as IFNs, or from cell-cell interactions (38). Thus, enhancing STAT1-dependent gene activation may be useful even in the absence of cytokines or other agents that can induce the tyrosine phosphorylation of this transcription factor. Furthermore, there is evidence that STAT1 activation is important in mediating cell cycle arrest and apoptosis induced by cytotoxic agents (22). Thus, a STAT1 enhancer such as 2-NP may help increase the efficacy of conventional anticancer treatments. Finally, STAT1 activation in endothelial cells may be an important negative regulator of angiogenesis (18). Thus, 2-NP alone, or in combination with other agents targeting tumor vasculature, may be important in optimizing antiangiogenesis treatments.

In addition to the anticancer effects mediated by STAT1, activation of this transcription factor may play an important role in a range of non-neoplastic diseases. For example, IFNs, with their natural roles likely centering on their antiviral effects, are a central component of the treatment of infectious diseases such as hepatitis B (39) and hepatitis C (40). In addition, STAT1 activation in immune cells likely underlies the efficacy of IFNs in autoimmune diseases such as multiple sclerosis (41). Thus, the ability to specifically accentuate the biological effects of IFNs may have widespread utility.

In conclusion, using a cell-based screen to isolate compounds that can modulate STAT1-dependent gene activation, we have identified 2-NP as a selective and efficacious enhancer of STAT1 activity. Through mechanistic analysis and further preclinical studies, it will be possible to define the potential clinical uses of compounds with this activity in both neoplastic and non-neoplastic diseases.


   Acknowledgments
 
Grant support: G&P Foundation for Cancer Research and the Brent Leahey Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the National Cancer Institute and the Initiative for Chemical Genetics, who provided support for this publication, and the Chemical Biology Platform of the Broad Institute of Harvard and the Massachusetts Institute of Technology for their assistance in this work.

Received 7/ 3/06. Revised 10/16/06. Accepted 11/20/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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