(STAT2) Protein: Stable Expression Blocks Interferon Action

Vol. 2, 453–459, May 2003 Molecular Cancer Therapeutics 453

Dominant Negative Signal Transducer and Activator of Transcription 2 (STAT2) Protein: Stable Expression Blocks Interferon ␣ Action in Skin Squamous Cell Carcinoma Cells1

John L. Clifford,2 Xiulan Yang, Eugene Walch, provide a novel in vitro model for the study of type I Michael Wang, and Scott M. Lippman IFN action in human skin cells. Department of Clinical Cancer Prevention, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 Introduction Non-melanoma skin cancer is the most common cancer in Abstract the United States, with over one million new cases of the two 3 We have demonstrated previously that suppression of most common forms, SCC and basal cell carcinoma, antic- some or all of the IFN-stimulated gene factor 3 (ISGF-3) ipated in 2002 (1). The more clinically aggressive form is SCC proteins in skin squamous cell carcinomas is an early of the skin (2), which has been increasing in incidence since event in squamous skin carcinogenesis. This finding the 1960s at annual rates from 4% to as much as 10% in led to the hypothesis that suppressed expression recent years. Unlike early-stage SCC, advanced SCC is ag- of ISGF-3 proteins may lead to reduced IFN gressive, often resistant to local therapy, requires repeated responsiveness, which in turn may contribute to skin surgical resections and courses of radiotherapy, and ac- malignancy by conferring a growth and/or survival counts for approximately 2000 deaths each year in the advantage. To test this hypothesis, we have developed United States. Better control of advanced skin SCC is clearly a skin cell-based model for inhibiting the IFN-␣ necessary, presenting a great challenge to clinical oncolo- signaling pathway through the forced expression of gists. a dominant negative-acting signal transducer and IFNs regulate proliferation, differentiation, and immune activator of transcription 2 (dnSTAT2) protein. function (3). IFN-␣ has been shown to be an active chemo- Expression of dnSTAT2 suppressed cell growth preventive agent in the treatment of IEN of the skin (4, 5). inhibition with a pharmacologically achievable IFN-␣ as well as the other type 1 IFN, IFN-␤, binds to cell concentration (100 IU/ml) of IFN-␣ in the IFN-␣- surface receptors composed of two distinct subunits, sensitive skin squamous cell carcinoma cell line IFNAR1 and IFNAR2, causing their oligomerization (6). This SRB12-p9. dnSTAT2 also suppressed the IFN-␣- results in the activation (autophosphorylation and/or induced phosphorylation of signal transducer and transphosphorylation) of receptor-associated kinases JAK1 activator of transcription (STAT) 1 and STAT2, which and tyk2, members of the JAK family of receptor-associated are early events following IFN-␣ treatment, but did not tyrosine kinases. JAK1 and tyk2 phosphorylate the receptor suppress the IFN-␥-induced phosphorylation of STAT1. subunits along with several different STATs, members of a Finally, the dnSTAT2 protein suppressed the up- family of latent cytoplasmic transcription factors, resulting in regulation of several IFN-␣-inducible genes that were STAT dimerization and translocation to the nucleus, where identified in this system by cDNA microarray screening. they modulate gene transcription. STAT1 and STAT2 are We conclude that the cell growth-inhibitory effect of likely to be the most important STATs mediating IFN-␣ ef- IFN-␣ in skin cells requires an intact STAT2 protein and fects and are phosphorylated on tyrosine 701 and tyrosine is therefore mediated by the ISGF-3 complex. These 690, respectively (3). Upon phosphorylation, STAT1 and results support STAT2 as an important molecular STAT2 proteins complex with a third protein, IRF9 (also target for skin cancer chemoprevention. Furthermore, called p48), to form the ISGF-3 transcription factor (7). After we propose that these dnSTAT2-expressing cells translocation to the nucleus, the IRF9 component of ISGF-3 binds to specific DNA elements found in the promoters of most type 1 IFN-responsive genes (7). The activation of both STAT1 and STAT2 by IFN-␣ can be abolished by mutating tyrosine 701 or tyrosine 690, respec- Received 12/16/02; revised 3/4/03; accepted 3/6/03. The costs of publication of this article were defrayed in part by the tively (8, 9). The mutant form of STAT1 (STAT1-Y701F) can payment of page charges. This article must therefore be hereby marked act in a dominant negative fashion to block ISGF-3 formation advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by National Cancer Institute Grant 1 P01 CA68233, National Institute of Environmental Health Sciences Grant 5 P30 ES07784, and an Institutional Research Grant to J. L. C. 3 The abbreviations used are: SCC, squamous cell carcinoma; IEN, intra- 2 To whom requests for reprints should be addressed, at The University of epithelial neoplasia; ISGF-3, IFN-stimulated gene factor 3; STAT, signal Texas M. D. Anderson Cancer Center, Box 236, 1515 Holcombe Boule- transducer and activator of transcription; dnSTAT, dominant negative vard, Houston, TX 77030. Phone: (713) 792-0627; Fax: (713) 792-0628; STAT; RT-PCR, reverse transcription-PCR; IFNAR, IFN-␣ receptor; JAK, E-mail: [email protected]. Janus-activated kinase.

(8). The corresponding dnSTAT2 (STAT2-Y690F) prevented 3Ј) into the newly generated XhoI site. The resulting vector, the phosphorylation of STAT1 upon IFN-␣ treatment, thereby pSG5-dnSTAT2-FLAG, was linearized with AatII and electro- presumably blocking IFN-␣ responses mediated by ISGF-3 porated along with the bacterial neomycin phosphotrans- (9). Current evidence suggests that STAT2 is specific for the ferase gene expression vector pKJ1, and stably expressing IFN-␣ pathway, unlike other STAT family members, which cell clones were isolated essentially as described previously can mediate signaling by multiple inducers (6). It is therefore (17). Cells (5 ϫ 106) suspended in 800 ␮l of PBS were expected that expression of dnSTAT2 protein will only block electroporated with 5 ␮g of linearized, purified pSG5- the IFN-␣ pathway and not others, clearly linking any phe- dnSTAT2 and 0.5 ␮g of the bacterial neomycin phospho- notypic change to the IFN-␣ pathway. It should also be noted transferase gene (neoR) expression vector pHR56 (kindly that IFN-␣ can activate other STATs besides STAT1 and provided by P. Chambon) with a Bio-Rad Gene Pulser set at STAT2 (3). For example, IFN-␣ can induce the phosphoryl- 200 V and 960 ␮F. Cells were then plated at a density of ation and DNA binding of STAT3 and STAT4 proteins, approximately 1 ϫ 106 cells/10-cm culture plate and, after thereby regulating additional sets of genes not common to 24 h, subjected to neomycin selection (300 ␮g/ml G418 the ISGF-3 pathway (10, 11). sulfate, Life Technologies Inc., Rockville, MD) for up to 14 ␣ In an effort to better understand the role of IFN- in skin days. Individual colonies were isolated, propagated, and di- carcinogenesis, we have previously determined the expres- vided into two aliquots, one for freezing and the other for ␣ ␤ sion pattern of the ISGF-3 proteins (STAT1 / , STAT2, and expansion and Western blotting. IRF9) in normal skin, skin SCC, and actinic keratoses from Western Blotting. Whole cell extracts of stably trans- patient biopsies (12, 13). Our results indicated a suppressed fected cells were purified as described previously (17). Pro- expression of one or more of these proteins in the majority of teins were quantitated by the Bradford assay (Pierce, Rock- patients tested, as determined by manual scoring and quan- ford, IL), and 50–100 ␮g protein/lane were electrophoresed titative densitometry. We have observed a similar decrease in by SDS-PAGE and electrophoretically transferred to nitro- expression of the same set of IFN signaling proteins in actinic cellulose membranes. After transfer, blots were blocked with keratoses (skin IEN; Ref. 14), indicating that the suppression 3% milk powder for1hatroom temperature, followed by may be an early event in skin carcinogenesis (13). These data incubation for1hatroom temperature with rabbit polyclonal have led to the hypothesis that the suppressed expression of antibodies to the FLAG epitope (Sigma-Aldrich, St. Louis, these proteins may result in reduced responsiveness to type MO), STAT2 (Santa Cruz Biotechnology, Santa Cruz, CA), 1 IFNs, conferring a growth or survival advantage to those STAT1 (Santa Cruz Biotechnology), phospho-STAT2 (Up- cells. To test this hypothesis, we have attempted to perma- state Biotechnology, Lake Placid, NY), phospho-STAT1 (Cell nently block IFN-␣ signaling in a skin cell-based system ␤ through the forced expression of the dnSTAT2 protein. We Signaling Technology, Beverly, MA), and -actin (Sigma- demonstrate that expression of the dnSTAT2 protein can Aldrich). Blots were then washed three times (15 min each specifically suppress IFN-␣ responses in the IFN-␣-sensitive time) with PBS and 0.05% Tween and incubated with horse- SRB12-p9 human skin SCC cell line. radish peroxidase-conjugated donkey antirabbit secondary antibody for1hatroom temperature, followed by an additional three washes with PBS and 0.05% Tween. The blot Materials and Methods was then incubated for 10 s to 1 min in chemiluminescence Cell Culture and Generation of Stable dnSTAT2-express- detection solution (Amersham Life Science Inc., Piscataway, ing Cell Lines. The human skin SCC cell line SRB12-p9 was NJ) and autoradiographed. derived by single cell cloning from SRB-12 cells (a gift from Cell Growth Inhibition Assays and IFN Treatments. Cell Dr. Janet Price; Department of Cancer Biology, The Univer- sity of Texas M. D. Anderson Cancer Center). Cells were growth inhibition assays were carried out essentially as de- cultured in a humidified atmosphere at 5% CO2, in a 1:1 scribed previously (18). Cells were plated in 24-well plates at mixture of DMEM and Ham’s F-12 medium, plus 10% fetal identical densities in normal culture media 1 day before ␣ bovine serum (15). The pSG5-dnSTAT2 expression vector treatment with 100 IU/ml human IFN- (PBL Biomedical Lab- was constructed by ligating the EcoRI insert fragment of oratories, New Brunswick, NY). Media were changed after 2 ␣ pBSK-STAT2 (kindly provided by J. E. Darnell; Rockefeller days, and the cells received fresh IFN- for an additional 2 University) into the EcoRI site of the pSG5 expression vector days. The percentage of cell growth inhibition was deter- (16), followed by sequential PCR-based site-specific mu- mined by cell counting using a Coulter cell counter (Coulter tagenesis using the oligonucleotides 5Ј-ATGCCTTCTGACT- Electronics Inc., Hialeah, FL). The percentage of growth in- TCAGATCTAGGAACCACATTTC-3Ј and 5Ј-GAAATGTGGT- hibition was calculated using the equation: (1 Ϫ R/C) ϫ 100, TCCTAGATCTGAAGTCAGAAGGCAT-3Ј to introduce a BglII where R and C represent the number of cells in IFN-␣-treated site immediately downstream of codon 851 and the oli- and control culture, respectively. For all other treatments gonucleotides 5Ј-GGAACGGAGGAAATTCCTGAAACACAG- with IFN-␣, cells were grown in normal culture media and GC- TC-3Ј and 5Ј-GAGCCTGTGTTTCAGGAATTTCCTCC- switched to 0.5% FBS for 48 h followed by a 2-h incubation GTTCC-3Ј to convert tyrosine 690 into phenylalanine. The in serum free media, immediately before treatment with hu- FLAG epitope extension was created by inserting a DNA man IFN-␣. For phospho-STAT Western blot experiments, fragment consisting of a complementary oligonucleotide pair cells were treated with 100 IU/ml IFN-␣ for 30 min. For RNA (5Ј-GATCGGACAAAGACGATGACGATAAATAGTAGATC-3Ј- isolation for cDNA microarray screening, cells were treated and 5Ј-GATCGATCTACTATTTATCGTCATCGTCTTTGTCC- with 100 IU/ml IFN-␣ for either 1 or 12 h, and for RT-PCR

assays, cells were treated with 100 or 500 IU/ml IFN-␣ for 12 and 24 h. cDNA Microarray Screening. Total RNA was isolated by homogenization of fresh or snap-frozen epidermal cells using TriReagent (Molecular Research Center Inc., Cincinnati, OH), followed by standard organic extraction and precipitation. Purity and yield were determined by UV absorbance spectra over the range of 220–320 nm. Samples of total RNA (10–30 ␮g) were fractionated on 1% agarose gels containing 0.66 M formaldehyde to determine integrity, and 100 mg total RNA/ sample was used for fluorescent probe synthesis. Probe synthesis and array hybridization were performed within The University of Texas M. D. Anderson Cancer Center Cancer Genomics Core Laboratory (Dr. W. Zhang, director), using established methods. Briefly, RNA was reverse transcribed and labeled by an indirect labeling method according to the manufacturer’s instructions (Clontech, Palo Alto, CA). The probe from untreated cells was labeled with Cy5, a red fluorescent dye, and the probe from IFN-␣-treated cells was labeled with Cy3, a green dye. Slide hybridization was also Fig. 1. Stable expression of the dnSTAT2 protein. A, structure of the conducted in the core facility according to the manufactur- dnSTAT2 insert used in the pSG5-dnSTAT2 expression construct. TAD, er’s instructions (Clontech). Fluorometric detection of probed transcriptional activation domain; F, FLAG epitope extension; SH2 and SH3, Src homology domains 2 and 3, respectively. B, Western blot of slides was carried out for Cy5 (A650 nm) and Cy3 (A550 nm) with whole cell extracts from the human SCC cell line, SRB12-p9, stably gray scale images. A colorized, merged image showed a red transfected with the combination of pSG5 vector and pKJ1 vectors (neoR, Lane 1) or pSG5-dnSTAT2 and pKJ1 (Lanes 2 and 3). c1 and c3 indicate signal for spots hybridizing primarily with the control skin dnSTAT2-expressing clones 1 and 3. kD, molecular mass in kilodaltons. probe, a green signal for spots hybridizing primarily with the treated skin probe, and a yellow signal for spots hybridizing equally with both probes. Signal:noise ratio adjustment and Results normalization of signals were conducted within The Univer- Generation of dnSTAT2-expressing SCC Cells. Initially, sity of Texas M. D. Anderson Cancer Center Cancer Genom- SRB12-p9 cells, which were derived from the SRB-12 cell line ics Core Laboratory using Arrayvision quantification software (15), were stably transfected with an expression vector encod- (Imaging Research, St. Catherines, Ontario, Canada). ing a dnSTAT2 protein linked to the FLAG octapeptide at the Semiquantitative RT-PCR. Semiquantitative RT-PCR COOH terminus, controlled by the SV40 promoter (Fig. 1A). The was performed essentially as described previously (19). RNA expression of dnSTAT2-transfected cell clones was determined was purified as described above and quantitated by absorb- by Western blotting with an anti-FLAG antibody (Fig. 1B, top R ance. RNA (1 ␮g/reaction) and the appropriate 20-mer oli- panel). Note that the faint bands observed in the neo lane are gonucleotides (50 pmol/reaction) were combined with a 10ϫ nonspecific. The same blots were stripped and reprobed with PCR mix [final concentrations, 50 mM KCl, 10 mM Tris (pH an anti-STAT2 antibody that recognizes both endogenous and ␮ ␮ dnSTAT2, revealing a Ͼ10-fold increase in total STAT2 (endog- 8.3), 1.5 M Mg2Cl, and 200 M each of dATP, dCTP, dGTP, and dTTP] to a final volume of 100 ml and subjected to the enous STAT2 plus dnSTAT2) immunoreactivity in the dnSTAT2- following PCR parameters: (94°, 3 min; 94° to 50° slope, 10 expressing cell clones (Fig. 1B, middle panel; data not shown). The increase in total STAT2 is likely due to high expression min; 50°C, 22 min) ϫ 1 cycle; followed by (94°, 1 min; 55°, levels of dnSTAT2, not to an increase in endogenous STAT2 30 s; 72°, 1 min) ϫ 15–40 cycles. A mix of 5 ml of a Taq expression (see explanation below). polymerase (2.5 units/tube) and avian myeloblastosis virus dnSTAT2 Blocks Cell Growth Inhibition by IFN-␣ reverse transcriptase (4 units/tube) was added to each tube through the ISGF-3 Pathway. The parental SRB12-p9 cells immediately after the 94° to 50° slope. Aliquots of each (Fig. 2, Par) are typically growth inhibited 45–75% after 4 reaction were collected over a broad range of cycle numbers days of treatment with 100 IU/ml IFN-␣, a pharmacologically and electrophoresed in a 2% agarose gel containing achievable concentration. For the independent dnSTAT2 cell ethidium bromide. RT-PCR products that were just below the clones shown in Fig. 1B (c1 and c3), there was approximately visual limit of detection were blotted onto nylon membranes 15% growth inhibition compared with 47.5% and 43.9% for by capillary transfer in high-salt buffer. Blots were probed the parental SRB12-p9 and neoR cells, respectively, aver- 32 with [␥- P]ATP]-end-labeled oligonucleotide probes com- aged from 3 independent experiments (Fig. 2). We have plementary to sequences contained between the oligonu- previously demonstrated that the cell growth inhibitory ac- cleotides used for the RT-PCR. The expression of the glyc- tion of IFN-␣ in the SRB12-p9 parental cells is primarily due eraldehyde phosphate dehydrogenase gene, which is to apoptosis induction and not to inhibition of proliferation (5). ubiquitously expressed, was determined for each RNA sam- We next sought to determine whether the observed differ- ple to control for variations in RNA quantity. ences in IFN-␣-induced growth inhibition are due to a spe-

Fig. 2. Expression of dnSTAT2 suppresses the cell growth-inhibitory effect of IFN-␣. Bars indicate percentage of cell growth inhibition from counting assays for cells treated for 4 days with 100 IU/ml IFN-␣ (see “Materials and Methods”). Par, parental SRB12-p9 cells; c1 and c3, dnSTAT2-expressing clones 1 and 3. Error bars, the mean Ϯ SEM, where n ϭ 3.

cific suppression in IFN-␣ signaling through the IFNAR/ ISGF-3 pathway. We compared the IFN-␣-induced phosphorylation of STAT2 and STAT1, which occurs rapidly in response to IFNAR activation, between parental and dnSTAT2-expressing cells using antibodies specific for the tyrosine-phosphorylated forms of STAT2 and STAT1. Tyro- sine phosphorylation of both STAT2 and STAT1, induced by a 30-min treatment with 100 IU/ml IFN-␣, was almost com- pletely blocked in the dnSTAT2-expressing cells [Fig. 3A, phospho STAT2 and phospho STAT1, compare Lanes ␣ (parental) and neoR (controls) with corresponding lanes for clones 1 and 3 (Lanes c1 and c3)]. The level of total STAT1 Fig. 3. Expression of dnSTAT2 suppresses the phosphorylation of and STAT2 proteins was unchanged by the IFN-␣ treatment, STAT2 and STAT1. A, Western blotting of whole cell extracts from and STAT1 levels were unaffected by dnSTAT2 expression SRB12-p9 cells and the same SRB12-p9 stably transfected clones shown ␣ (Fig. 3A, STAT2 and STAT1). Total STAT2 immunoreactivity in Figs. 1 and 2, treated for 30 min with 100 IU/ml IFN- . Top panel, blot was probed with a STAT2 phospho-tyrosine 690-specific antibody, was markedly higher in the dnSTAT2-expressing cells and stripped, and reprobed with an anti-STAT2 antibody (second panel). Third was also unchanged by IFN-␣ treatment (Fig. 3A, second panel, blot was probed with a STAT1 phospho-tyrosine 701-specific antibody, stripped, and reprobed with an antibody against both the ␣ and panel). We believe that these higher total STAT2 levels were ␤ isoforms of human STAT1 (fourth panel) and then stripped and reprobed not due to an increase in endogenous STAT2 expression with an antibody to ␤-actin (bottom panel). B, cells were cultured as because the phospho-STAT2 antibody (Fig. 3A, top panel) described in A before treatment with 100 IU/ml human IFN-␥ for 30 min. The blot was sequentially probed with the indicated antibodies as de- will only recognize the endogenous STAT2 (because scribed in A. kD, molecular mass in kilodaltons. dnSTAT2 cannot be phosphorylated at tyrosine 690). The markedly lower signal for phospho-STAT2 in Lanes ␣ for c1 and c3 is consistent with a reduction in the proportion of Cancer Center Cancer Genomics Core Laboratory, we have endogenous STAT2 protein in those cells, compared with the identified several early IFN-␣-inducible genes in SRB12-p9 parental (Par) and neoR lines. These findings parallel the growth cells. Three separate cDNA array screens were conducted. inhibition results and strongly suggest that the ISGF-3 complex The first compared the expression of genes between control mediates this effect. The ability of IFN-␥ to induce phosphoryl- cells and cells treated with 100 IU/ml IFN-␣ for 1 h, using an ation of STAT1 was unaffected in the c3 cells (Fig. 3B), indicat- array containing 2304 duplicate spotted unique cDNA frag- ing that the presence of dnSTAT2 did not interfere with the ments. The second and third screens compared control cells activation of STAT1 by the IFN-␥ receptor. with cells treated with 100 IU/ml IFN-␣ for 12 h using two dnSTAT2 Suppresses Induction Several IFN-␣-respon- arrays, one containing 1127 duplicate spotted cDNA frag- sive Genes. The recent development of cDNA microarray ments corresponding to genes previously shown to be active technology has made it possible to compare gene expres- in several signal transduction pathways, and the other array sion profiles between two different cell or tissue samples. In containing 5700 spotted expressed sequence tag fragments. collaboration with The University of Texas M. D. Anderson All screens identified a small number of gene expression

Fig. 4. Expression of dnSTAT2 suppresses the induction of several IFN-␣- inducible genes. A, semiquantitative RT-PCR is shown for four genes identified as IFN-␣ inducible by cDNA microarray screening. Glyceraldehyde phosphate dehydrogenase (GAPDH) expression was determined for each RNA sample to control for variations in RNA quantity. Vector-transfected control cells (neoR) and dnSTAT2 clone 3 cells (c3) were treated as described above with 100 IU/ml IFN-␣ (lower end of triangle) or 500 IU/ml IFN-␣ (upper end of triangle) for 24 h. B, densitomet- ric analysis of RT-PCR blots of the five indicated IFN-␣-inducible genes were carried out for cells treated as described in A for 12 or 24 h. White bars indicate untreated controls, black bars and striped bars indicate 100 and 500 IU/ml IFN-␣, respectively.

changes (where 0.5 Ն Cy5/Cy3 Ն 2.0), as was expected with otherwise highly IFN-␣-sensitive SRB12-p9 cells. Of the two the short treatment time and relatively low dose of IFN-␣ dnSTAT2-expressing cell lines analyzed, c3 cells, which ex- used. A total of 16 genes were shown to be up-regulated by press the higher amount of dnSTAT2 protein, show a stron- IFN-␣ in the screens, and 5 genes were suppressed by IFN-␣ ger overall suppression of IFN-␣-induced gene expression. in the screens. To date, individual gene expression changes The c3 cells also show a slightly greater suppression of were confirmed for five of the up-regulated genes by RT- IFN-␣-induced phosphorylation of STAT1, which was ob- PCR analysis of SRB12-p9 cells treated with 100 or 500 served in several independent experiments (Fig. 3A; data not IU/ml IFN-␣ for 1, 12, and 24 h (Fig. 4, A and B; data not shown). These results provide further evidence for the spec- shown). All five of the confirmed genes, myxovirus resistance ificity of the dnSTAT2 effect. 1(MxA1), IFN-␣-inducible protein 6-16 (IFI-6-16), IFN- The specific mechanism by which the dnSTAT2 (Y690F) induced protein 56 (IFI-56), IFN-regulatory factor 7 (IRF-7), phosphorylation site mutation blocks IFN-␣ signaling re- and the IFN-inducible protein 9-27 (9-27), have previously mains to be determined. Wild-type STAT2 normally interacts been shown to be IFN-␣ inducible in other systems (3). constitutively with the IFNAR2 chain, in both its tyrosine- We next determined whether the IFN-␣ induction of any of phosphorylated and unphosphorylated forms (20). Upon these five genes was suppressed in the c1 and c3 cells. All binding of IFN-␣ to the receptor complex, STAT2 is recruited five genes were still induced by a 12-h treatment with 100 or by way of its Src homology domain 2 to the phospho-tyro- 500 IU/ml IFN-␣ in the c1 cells and by a 12-h treatment with sine 466 site of the IFNAR1 chain (21). This event is followed 500 IU/ml IFN-␣ in the c3 cells (Fig. 4B). Three of five genes by the phosphorylation of STAT2 on tyrosine 690 and re- ␣ were less induced at both IFN- concentrations for c1 cells, cruitment of STAT1 to the complex, whereupon STAT1 is and all five genes were less induced for c3 cells. After a 24-h tyrosine phosphorylated (8). Our observation that IFN-␣- ␣ treatment with either 100 or 500 IU/ml IFN- , all five genes induced phosphorylation of STAT1 is suppressed in the are less induced in both dnSTAT2 cell lines, with the excep- dnSTAT2-expressing cells suggests that the dnSTAT2 pro- ␣ tion of IRF-7, for c1 cells treated with 500 IU/ml IFN- (Fig. tein acts proximal to the STAT1 tyrosine phosphorylation ␣ 4B). There is an overall greater suppression of IFN- -induced step. We observe a partial block of IFN-␣ responses, con- gene expression in the c3 cells compared with c1 cells (Fig. 1B). sistent with the findings of other investigators who have observed a partial suppression of IFN-␣-induced transcrip- Discussion tion by dnSTAT2 in transient transcription assays (22). Those In this study we demonstrate by several criteria that stable investigators speculated that the dnSTAT2 protein exerts its expression of dnSTAT2 can reduce IFN-␣ responsiveness of effect by competing with endogenous STAT2 for binding to

the IFNAR1 chain, thereby only partially blocking recruitment transcription and that this modification was inhibited in cer- of STAT1 (22). Nevertheless, we cannot rule out the possi- tain transformed cell lines (31). This indicates an inverse bility that alternate pathways besides ISGF-3 activation are correlation between STAT1 and STAT2 expression and ma- available for induction of the growth inhibitory response or lignancy. These results, along with the finding that some DNA the induction of ISGs by IFN-␣ in the c1 and c3 cells. tumor viruses inhibit IFN action, are in keeping with the The inability of the dnSTAT2 protein to block IFN-␥- assertion that IFNs have a role as tumor suppressors (32). induced phosphorylation of STAT1 rules out other possible IFN-␣ also could potentially play a role in tumor surveillance effects of dnSTAT2 expression on STAT1 function and allows in normal or premalignant skin. In a mechanism similar to type us to conclude that STAT1 function remains intact. Further 1 IFN signaling, type 2 IFN (IFN-␥) activates a distinct set of study will be necessary to determine the exact point of action genes through the formation of STAT1 homodimers (3, 6). of the dnSTAT2 protein. It should be noted that STAT3 phos- Kaplan et al. (33) have previously demonstrated that mice lack- phorylation, which can also be induced by IFN-␣ (10), is ing either IFN-␥ receptors or STAT1 protein develop spontane- constitutive in SRB12-p9 cells and is not affected by expres- ous and chemically induced tumors more frequently than wild- sion of dnSTAT2 (data not shown). type mice. They also demonstrated defects in IFN-␥ signaling in Importantly, the cell growth inhibitory effect of 100 IU/ml several lung adenocarcinoma cell lines, but they did not find IFN-␣, a pharmacologically relevant concentration, on the similar defects in IFN-␣ signaling, leaving a question as to the dnSTAT2-expressing cells is markedly reduced compared potential role of IFN-␣ in tumor surveillance. Because STAT1 is with the parental cells. We have recently shown that IFN-␣- shared between the type 1 and type 2 IFN pathways, our induced growth inhibition of the parental SRB12-p9 cells previous finding that its expression is reduced in human tumors does not correlate with changes in cell cycle distribution but (18, 19) could reflect a defect in both pathways. Whether im- rather with an induction of apoptosis (5). A recent study by paired IFN-␣ signaling results in diminished tumor surveillance Guo et al. (23) provides a possible explanation for this effect. capacity in normal skin remains to be investigated. Those investigators showed that IFI-56, one of the IFN- We have demonstrated the utility of dnSTAT2 expression inducible genes that we find suppressed in the dnSTAT2 for permanently suppressing IFN-␣ signaling in a skin cell- cells (Fig. 4, A and B), plays a role in inhibiting protein based model system. Current and future studies using the translation by binding to the P48 subunit [not to be confused dnSTAT2 protein expressed in HaCaT cells, an immortalized with the p48 (IRF9) subunit of ISGF-3] of the translation nontumorigenic skin cell line that serves as a model for skin initiation factor eIF3 (23). It is possible that IFI-56 could act in premalignancy (34), and transgenic mice expressing a manner similar to another IFN-inducible gene, double- dnSTAT2 under the control of the skin-selective cytokeratin stranded RNA-dependent protein kinase (PKR), which has 5 promoter, along with STAT2 knockout mice, are aimed at also been shown to suppress protein translation by binding directly testing our hypothesis that reduced IFN responsive- to eIF2␣ (24). In this latter study, PKR action was blocked ness can lead to skin malignancy. In addition, other investi- through expression of a dominant negative variant, resulting gators have generated a fusion protein between IRF9 and the in resistance to apoptosis induction by treatment with double- COOH-terminal transcription activation domain of STAT2, stranded RNA for 3T3 L1 cells (24). The exact mechanism of which can constitutively activate ISGF-3 target genes and action of PKR in mediating apoptosis induction is not com- produce a type I IFN response in human fibrosarcoma cells pletely understood, but it appears to involve the up-regulation (35). We plan to further test our hypothesis by expressing this of several proapoptotic proteins. We speculate that IFI-56 may fusion protein in tumorigenic skin SCC cells and in mouse act in a similar manner. Additional studies are planned to de- skin to determine whether constitutive activation of type I IFN termine the mechanisms of IFN-␣-induced apoptosis and the signaling has a tumor-suppressive or -preventive effect. This possible involvement of IFI-56 in this system. mechanistic study, together with our earlier report in skin IEN A role for IFN signaling in skin SCC development and (13), supports STAT2 as an important molecular target for progression is not clear at present. Previous studies have skin cancer chemoprevention drug development. shown that IFN-␣ can suppress the proliferation of primary cultured keratinocytes (25) and induce apoptosis in skin SCC Acknowledgments cells (5, 12). A requirement for STAT1 activation for the We thank Reuben Lotan, John DiGiovanni, David Menter, and members of antiproliferative effects of IFN-␣, as well as the apoptotic the laboratory for helpful discussions and advice. We also thank James E. effect of tumor necrosis factor ␣, has been demonstrated Darnell for the STAT2 cDNA, Pierre Chambon for the pSG5 and pHR56 (26, 27). Further support for the possibility that reduced expression vectors, and Dr. Janet Price for the SRB12 cell line. The IFN-␣ responsiveness could be related to malignancy comes University of Texas M. D. Anderson Cancer Center Cancer Genomics Core Laboratory is supported by the Tobacco Settlement Fund as appro- from studies aimed at determining the mechanism of IFN priated by the Texas Legislature, a generous donation from the Kadoorie resistance in various cancer cell types. Other investigators Foundation, and the Cancer Center Core Grant from the National Cancer have shown that cultured cancer cells have deficiencies in Institute. We thank Kendall Morse for critical reading of the manuscript. ISGF-3 activity, and in one case, a series of melanoma cell lines exhibited reduced IFN response relative to primary References melanocytes (28, 29). It was also shown that IRF9 and STAT1 1. Jemal, A., Thomas, A., Murray, T., and Thun, M. Cancer statistics, levels and DNA binding activity increased during monocytic 2002. CA Cancer J. Clin., 52: 23–47, 2002. differentiation of U937 cells (30). Recently, it was found that 2. Alam, M., and Ratner, D. Cutaneous squamous-cell carcinoma. arginine methylation of STAT1 is required for IFN-␣-induced N. Engl. J. Med., 344: 975–983, 2001.

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Downloaded from mct.aacrjournals.org on September 30, 2021. © 2003 American Association for Cancer Research. Dominant Negative Signal Transducer and Activator of Transcription 2 (STAT2) Protein: Stable Expression Blocks Interferon α Action in Skin Squamous Cell Carcinoma Cells1

John L. Clifford, Xiulan Yang, Eugene Walch, et al.

Mol Cancer Ther 2003;2:453-459.

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