Oncogenic Ki-Ras Inhibits the Expression of Interferon-responsive Genes through Inhibition of STAT1 and STAT2 Expression
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m304721200
ISSN1083-351X
AutoresLidija Klampfer, Jie Huang, Georgia Corner, John M. Mariadason, Diego Arango, Takehiko Sasazuki, Senji Shirasawa, Leonard H. Augenlicht,
Tópico(s)NF-κB Signaling Pathways
ResumoEndogenous interferon γ (IFNγ) promotes the host response to primary tumors, and IFNγ-insensitive tumors display increased tumorigenicity and can evade tumor surveillance mechanisms. Here we demonstrate that activating mutations of Ki-ras are sufficient to inhibit the expression of STAT1 and STAT2, transcription factors required for signaling by IFNs, providing a potential mechanism for the insensitivity of tumors to IFNs. We demonstrated that colon cancer cell lines with Ki-ras mutations display reduced expression of IFN-responsive genes compared with the cell lines that have retained wild type Ras and that inactivation of the mutant Ki-ras allele in the HCT116 colon cancer cell line is sufficient to restore the expression of STAT1, STAT2, and IRF-9. Accordingly, the expression of 27 interferon-inducible genes was reduced in HCT116 cells compared with the isogenic clones with targeted deletion of the mutant Ki-ras allele, Hkh2 and Hke-3. The expression of IFNγ receptors did not differ among the isogenic cell lines. IFNγ stimulated transcription of a STAT1-dependent reporter gene was impaired by RasV12, demonstrating a transmodulation of IFN/STAT signaling by activated Ras. Finally, we demonstrated that the expression of RasV12 in 293T cells is sufficient to inhibit the endogenous expression of STAT1 and STAT2, confirming the negative regulation of IFN signaling by oncogenic Ras. Our data demonstrate that the signaling initiated by activated Ki-ras interferes with the IFN/STAT signaling pathway and modulates the responsiveness of cancer cells to interferons. Furthermore, the data suggest that tumors harboring activating Ki-ras mutations may escape tumor surveillance mechanisms due to reduced responsiveness to IFNγ. Endogenous interferon γ (IFNγ) promotes the host response to primary tumors, and IFNγ-insensitive tumors display increased tumorigenicity and can evade tumor surveillance mechanisms. Here we demonstrate that activating mutations of Ki-ras are sufficient to inhibit the expression of STAT1 and STAT2, transcription factors required for signaling by IFNs, providing a potential mechanism for the insensitivity of tumors to IFNs. We demonstrated that colon cancer cell lines with Ki-ras mutations display reduced expression of IFN-responsive genes compared with the cell lines that have retained wild type Ras and that inactivation of the mutant Ki-ras allele in the HCT116 colon cancer cell line is sufficient to restore the expression of STAT1, STAT2, and IRF-9. Accordingly, the expression of 27 interferon-inducible genes was reduced in HCT116 cells compared with the isogenic clones with targeted deletion of the mutant Ki-ras allele, Hkh2 and Hke-3. The expression of IFNγ receptors did not differ among the isogenic cell lines. IFNγ stimulated transcription of a STAT1-dependent reporter gene was impaired by RasV12, demonstrating a transmodulation of IFN/STAT signaling by activated Ras. Finally, we demonstrated that the expression of RasV12 in 293T cells is sufficient to inhibit the endogenous expression of STAT1 and STAT2, confirming the negative regulation of IFN signaling by oncogenic Ras. Our data demonstrate that the signaling initiated by activated Ki-ras interferes with the IFN/STAT signaling pathway and modulates the responsiveness of cancer cells to interferons. Furthermore, the data suggest that tumors harboring activating Ki-ras mutations may escape tumor surveillance mechanisms due to reduced responsiveness to IFNγ. Point mutations that activate the Ki-ras protooncogene are present in up to 50% of sporadic colorectal tumors (1Kinzler K.W. Vogelstein B. Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4260) Google Scholar). The majority of the Ki-ras mutations are gain-of-function mutations at codon 12 and 13, and activated Ki-ras has been shown to synergize with both APC (2D'Abaco G.M. Whitehead R.H. Burgess A.W. Mol. Cell. Biol. 1996; 16: 884-891Crossref PubMed Scopus (102) Google Scholar) and p53 mutations (3Sevignani C. Wlodarski P. Kirillova J. Mercer W.E. Danielson K.G. Iozzo R.V. Calabretta B. J. Clin. Invest. 1998; 101: 1572-1580Crossref PubMed Scopus (32) Google Scholar) in the transformation of colonic epithelial cells. In addition, recent reports suggested that the acquisition of an activated Ki-ras mutation may be sufficient for transformation of epithelial cells (4Janssen K.P. el-Marjou F. Pinto D. Sastre X. Rouillard D. Fouquet C. Soussi T. Louvard D. Robine S. Gastroenterology. 2002; 123: 492-504Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 5Smith G. Carey F.A. Beattie J. Wilkie M.J. Lightfoot T.J. Coxhead J. Garner R.C. Steele R.J. Wolf C.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9433-9438Crossref PubMed Scopus (378) Google Scholar). Oncogenic forms of Ras are locked in their active state and thereby transduce signals for transformation, angiogenesis, invasion, and metastasis in the absence of extracellular stimuli (6Rebollo A. Martinez A.C. Blood. 1999; 94: 2971-2980Crossref PubMed Google Scholar, 7Shields J.M. Pruitt K. McFall A. Shaub A. Der C.J. Trends Cell Biol. 2000; 10: 147-154Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Signaling transmitted through Ras results in activation of several downstream effectors, with the Raf-ERK1/ERK2, PI3/AKT, and RalGDS pathways playing crucial roles in the modulation of target gene expression (8Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (918) Google Scholar). In addition, activated Ras has been shown to positively regulate signaling by several transcription factors, such as ELK-1, serum response factor (SRF), members of the AP-1 family, and NF-κB (9Malumbres M. Pellicer A. Front. Biosci. 1998; 3: d887-d912Crossref PubMed Scopus (11) Google Scholar). In contrast, Ras signaling has been shown to inhibit nuclear accumulation and transcriptional activity of Smad2 and Smad3 and thereby represses signaling by TGF-β, an important negative regulator of epithelial cell proliferation (10Kretzschmar M. Doody J. Timokhina I. Massague J. Genes Dev. 1999; 13: 804-816Crossref PubMed Scopus (849) Google Scholar). To identify additional effectors of Ras signaling, we performed a genome-wide survey of Ras target genes utilizing the system of isogenic cell lines, HCT116, Hkh2, and Hke-3, which differ only in the presence of the activated Ki-ras allele. In the HCT116 colon carcinoma cell line targeted deletion of the activated Ki-ras resulted in reduced proliferation, accompanied by reduced expression of c-myc, loss of the capacity for anchorage independent growth, and reduced tumorigenicity of cells in vivo (11Shirasawa S. Furuse M. Yokoyama N. Sasazuki T. Science. 1993; 260: 85-88Crossref PubMed Scopus (598) Google Scholar). In accord with published data (12Zuber J. Tchernitsa O.I. Hinzmann B. Schmitz A.C. Grips M. Hellriegel M. Sers C. Rosenthal A. Schafer R. Nat. Genet. 2000; 24: 144-152Crossref PubMed Scopus (242) Google Scholar), target genes of activated Ki-Ras included signaling molecules, transcription factors, and proteins modulating cell cycle progression and apoptosis. Among the genes whose expression was inhibited in cells that harbor Ki-ras mutation were several IFN target genes, including STAT1, STAT2, and p48 (interferon regulatory factor (IRF)-9), transcription factors which themselves mediate signaling by both type I (α/β) and type II (γ) IFNs. 1The abbreviations used are: IFNinterferonRTreverse transcriptaseβ2Mβ2-microglobulinWTwild typeMTmutantMHCmajor histocompatibility complexIRFinterferon regulatory factorPI3Kphosphatidylinostiol 3-kinaseCBPcAMP-responsive element-binding protein-binding protein.1The abbreviations used are: IFNinterferonRTreverse transcriptaseβ2Mβ2-microglobulinWTwild typeMTmutantMHCmajor histocompatibility complexIRFinterferon regulatory factorPI3Kphosphatidylinostiol 3-kinaseCBPcAMP-responsive element-binding protein-binding protein. interferon reverse transcriptase β2-microglobulin wild type mutant major histocompatibility complex interferon regulatory factor phosphatidylinostiol 3-kinase cAMP-responsive element-binding protein-binding protein. interferon reverse transcriptase β2-microglobulin wild type mutant major histocompatibility complex interferon regulatory factor phosphatidylinostiol 3-kinase cAMP-responsive element-binding protein-binding protein. IFNγ initiates signaling by binding to its receptor, which results in transphosphorylation and activation of JAK1/JAK2 and subsequent phosphorylation of STAT1. Upon phosphorylation at Tyr701, STAT1 is released from the IFNγ receptor and forms STAT1 homodimers. Dimers translocate to the nucleus and induce transcription of target genes which harbor GAS (gamma-activated site) sites in their promoter region (reviewed in Refs. 13Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (820) Google Scholar and 14Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4974) Google Scholar). Binding of IFN α/β to the type I receptor results in transphosphorylation and activation of the Jak1/Tyk 2 kinase (15Kisseleva T. Bhattacharya S. Braunstein J. Schindler C.W. Gene (Amst.). 2002; 285: 1-24Crossref PubMed Scopus (898) Google Scholar, 16Jove R. Oncogene. 2000; 19: 2466-2467Crossref PubMed Scopus (53) Google Scholar). The activated kinases elicit tyrosine phosphorylation and dimerization of STAT1 and STAT2, which subsequently recruit the DNA-binding protein subunit, p48 (IRF-9) (17Bluyssen A.R. Durbin J.E. Levy D.E. Cytokine Growth Factor Rev. 1996; 7: 11-17Crossref PubMed Scopus (118) Google Scholar, 18Bluyssen H.A. Levy D.E. J. Biol. Chem. 1997; 272: 4600-4605Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). STAT1, STAT2, and p48 form a heterotrimeric transcription factor complex, ISGF-3, which activates genes that contain an ISRE (interferon-stimulated response element) site (19Dale T.C. Rosen J.M. Guille M.J. Lewin A.R. Porter A.G. Kerr I.M. Stark G.R. EMBO J. 1989; 8: 831-839Crossref PubMed Scopus (86) Google Scholar, 20Veals S.A. Schindler C. Leonard D. Fu X.Y. Aebersold R. Darnell Jr., J.E. Levy D.E. Mol. Cell. Biol. 1992; 12: 3315-3324Crossref PubMed Scopus (344) Google Scholar). Although STAT1 was thought to be required for the expression of the IFNγ target genes, IFNγ has recently been shown to modulate the expression of its target genes in STAT1 deficient cells (21Ramana C.V. Gil M.P. Schreiber R.D. Stark G.R. Trends Immunol. 2002; 23: 96-101Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 22Ramana C.V. Gil M.P. Han Y. Ransohoff R.M. Schreiber R.D. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6674-6679Crossref PubMed Scopus (208) Google Scholar, 23Gil M.P. Bohn E. O'Guin A.K. Ramana C.V. Levine B. Stark G.R. Virgin H.W. Schreiber R.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6680-6685Crossref PubMed Scopus (293) Google Scholar), demonstrating that IFNγ modulates the expression of a subset of its target genes in the absence of functional STAT1. The importance of intact IFNγ signaling and STAT1 in tumor surveillance has been established also in vivo. Mice with a targeted deletion of the IFNγ receptor and STAT1–/– mice display increased incidence of tumor formation in response to methylcholanthrene, and STAT-1 deficiency accelerated spontaneous tumor formation in mice with a p53 null background (24Kaplan D.H. Shankaran V. Dighe A.S. Stockert E. Aguet M. Old L.J. Schreiber R.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7556-7561Crossref PubMed Scopus (1156) Google Scholar). Similarly, mouse embryonal fibroblasts with a targeted deletion of the IFNαR1 undergo spontaneous transformation, and mutant mice develop papilloma of the skin at a high incidence when treated with a chemical carcinogen (25Takaoka A. Hayakawa S. Yanai H. Stoiber D. Negishi H. Kikuchi H. Sasaki S. Imai K. Shibue T. Honda K. Taniguchi T. Nature. 2003; 424: 516-523Crossref PubMed Scopus (732) Google Scholar). The ability of IFNs to regulate cell cycle progression, to induce cell differentiation, and to modulate the immune response has also led to intensive investigation of interferons as potential antitumor agents (26Vilcek J. Science. 1970; 168: 398-399Crossref PubMed Scopus (5) Google Scholar, 27Sun M. Science. 1981; 212: 141-142Crossref PubMed Scopus (11) Google Scholar, 28Marx J.L. Science. 1979; 204: 1183-1186Crossref PubMed Scopus (18) Google Scholar). The majority of cancer cell lines and primary tumors, however, are resistant to IFNs (24Kaplan D.H. Shankaran V. Dighe A.S. Stockert E. Aguet M. Old L.J. Schreiber R.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7556-7561Crossref PubMed Scopus (1156) Google Scholar). Cancer cells have been shown to acquire resistance to IFN through deletion of the IFNα/β locus (29Colamonici O.R. Domanski P. Platanias L.C. Diaz M.O. Blood. 1992; 80: 744-749Crossref PubMed Google Scholar) or down-regulation or alterations of the IFNα receptor (30Billard C. Sigaux F. Castaigne S. Valensi F. Flandrin G. Degos L. Falcoff E. Aguet M. Blood. 1986; 67: 821-826Crossref PubMed Google Scholar). Several cancers resistant to the anti-proliferative action of IFNs have been shown to have defects in the expression of transcription factors required for IFN signaling (31Abril E. Real L.M. Serrano A. Jimenez P. Garcia A. Canton J. Trigo I. Garrido F. Ruiz-Cabello F. Cancer Immunol. Immunother. 1998; 47: 113-120Crossref PubMed Scopus (61) Google Scholar, 32Clifford J.L. Walch E. Yang X. Xu X. Alberts D.S. Clayman G.L. El-Naggar A.K. Lotan R. Lippman S.M. Clin. Cancer Res. 2002; 8: 2067-2072PubMed Google Scholar, 33Landolfo S. Guarini A. Riera L. Gariglio M. Gribaudo G. Cignetti A. Cordone I. Montefusco E. Mandelli F. Foa R. Hematol. J. 2000; 1: 7-14Crossref PubMed Scopus (52) Google Scholar, 34Wong L.H. Krauer K.G. Hatzinisiriou I. 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Here we demonstrate that the expression of STAT1 and STAT2 is negatively regulated by an activating Ki-ras mutation, suggesting that tumors harboring activating Ki-ras mutations may escape tumor surveillance mechanisms due to reduced IFNγ sensitivity. Cells and Western Blot Analysis—The HCT116 colorectal carcinoma cell line, and its two clonal derivatives Hkh2 and Hke-3 that lack the mutant Ki-ras allele (11Shirasawa S. Furuse M. Yokoyama N. Sasazuki T. Science. 1993; 260: 85-88Crossref PubMed Scopus (598) Google Scholar), were cultured under standard conditions in minimal essential medium, supplemented with 10% fetal calf serum and antibiotics. Cell lysates were prepared and Western blot analysis performed using standard procedures. Briefly, 50 μg of total cell lysates were fractionated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membrane was incubated with antibodies for 3 h at room temperature and ECL (Amersham Biosciences) was used for visualization of immune complexes. The surface expression of the IFNγRα chain was determined by immunofluorescence. Cells were incubated with an anti IFNγRα antibody for 30 min on ice and washed three times with phosphate-buffered saline containing 1% fetal calf serum. After incubation, cells were stained with anti-mouse secondary antibody conjugated to fluorescein isothiocyanate for 30 min. Transient Transfections and Reporter Gene Assay—Plasmid 8×GAS-p36LUC was kindly provided to us by Dr. C. Glass. It contains eight GAS sites cloned upstream of the luciferase reporter gene. Cells were transfected with 1 μg of DNA per 12-well plate using the calcium phosphate method. Transfection efficiency was normalized by cotransfection with pTK-renilla (Promega, dual-luciferase reporter assay system). Cells were either left untreated or were treated with IFNγ (100 ng/ml) or IFNβ (100 units/ml). The basal transcriptional activity of STAT1, as well as IFN inducible activity, was determined 48 h after transfection (e.g. 24 h after treatment). The expression vector CMV-RASV12 was purchased from Clontech. cDNA Microarray Analysis—We used microarray slides produced by the Albert Einstein College of Medicine, containing 9216 or 27,224 sequences and identified sequences that are differentially expressed in HCT116 cells compared with the two clones with a disrupted mutant Ki-Ras allele. Only genes with a signal/background ratio > 1.25 were further analyzed. Using these criteria, on average 70% of sequences were included in the analysis. We considered sequences as potential Ki-Ras target genes if the ratio of the signals between HCT116 and either Hkh2 or Hke-3 cells was greater than 1.5 (Ki-Ras up-regulated genes) or less than 0.6 (Ki-Ras down-regulated genes). Total RNA was isolated using the RNeasy Midi kit (Quiagen) as suggested by the manufacturer. 100 μg of RNA from HCT116 cells was labeled with Cy3 (channel 1) and RNA from Hke-3 or Hkh2 cells with Cy5 (channel 2) fluorescent dye. After hybridization and washing (36Mariadason J.M. Corner G.A. Augenlicht L.H. Cancer Res. 2000; 60: 4561-4572PubMed Google Scholar), slides were scanned (532 nm/Cy3 and 633 nm/Cy5), and the two images were superimposed and quantified using Scanalyze 1.41 software. Data were imported into an Access data base for further analysis. The expression profile for HCT116 cells is presented as the intensity of channel 1 corrected for the background (ch1I/B), and expression data for Hkh2 or Hke-3 cells as the intensity of channel 2 corrected for the background (ch2I/B). Only genes with a signal/background ratio > 1.25 in both Hkh2 and Hke-3 clones were further analyzed. Results are expressed as the ratio between intensity of signal in HCT116 and Hke-3 cells or between HCT116 and Hkh2 cells. The expression level of the IFN target genes was similarly assayed by array analysis in a panel of 12 colon carcinoma cell lines and compared with reference RNA, representing a pool of RNA isolated from a panel of 12 colorectal cancer cell lines. cDNA microarray analysis was performed using microarray slides comprising 9216 cloned sequences produced by the Microarray Facility at the Albert Einstein College of Medicine. Microarray experiments were repeated twice and the data shown in Table II represent the average gene expression. For detailed analysis of the entire data base, see Mariadason et al. (79Mariadason J. Arango D. Wilson A.J. Corner G.A. Nicholas C. Aranes M.J. Schwartz E.L. Shi Q. Lesser M. Augenlicht L. Cancer Res. 2003; (in press)Google Scholar).Table IIDifferential expression of interferon-inducible genes in isogenic cell lines HCT116, Hke-2, and Hke-3Accession no.GeneRatioHCT116/Hkh2HCT116/Hke-3R82716IFN-15/170.120.09 (0.12)0.3 (0.3)0.3AA419251IFN-9-270.40.13 (0.11)0.3 (0.3)0.4AA058323IFN-1-8U0.610.280.3AA489743IFN-56K0.30.230.5NDAA187365IFN-1-8DND0.120.28NDW49768IFN-6-16ND0.220.30.2AA160539IFN-30ND0.20.51.3AA489743IFN/tetra1ND0.80.5 (0.6)0.1T97408Bag-1ND0.20.5NDBF525953p480.70.20.4 (0.8)0.6AA210708STAT20.60.20.3NDW90777IRF-20.50.30.50.8AA478043IRF-10.80.60.7 (0.8)NDN46151IFN-PKR0.40.50.70.9R722442′-5′ A synthetase0.50.80.5NDAA447481Nuclear antigen SP1000.5ND0.70.7AA456886Myx-10.6ND0.4 (0.2)NDAA286908Myx-20.40.60.3NDR23341β2MND0.10.3NDAA464246MHC class I0.60.19 (0.4)0.4 (0.5)0.4W00378MHC class I, B0.4ND0.5NDR96664MHC class, IIDR20.15ND0.20.2H50622MHC class, DR70.16ND0.3NDT82054MHC class, IIDPb0.3ND0.5NDR72489MHC class, IIDG0.4ND0.8NDNM_001223Caspase 10.50.5 (1)0.5 (0.8)0.6R14760Caspase 30.70.7 (1)0.5 (0.5)1.0 Open table in a new tab Quantitative RT-PCR Analysis—The differential expression level of 4 genes selected from Table II was confirmed by quantitative real-time RT-PCR analysis. RNA was isolated from HCT116, HKh2, and Hke-3 cells that were either left untreated or were treated with IFNγ (10 ng/ml) for 1, 3, or 6 h. 1 μg of RNA was reversed transcribed using TaqMan reverse transcription reagents (Applied Biosystems). cDNA was amplified using specific primers for IRF-9, β2-microglobulin (β2M), PKR and IFN/TETRA1, and c-myc. Primer sequences were as follows: IFN/TETRA1 (forward), 5′-TTCCACTATGGTCGGTTTCA-3′; IFN/TETRA1 (reverse), 5′-GAGGCTCAAGCTTTCCAGAT-3′; β2M (forward), 5′-TGTGCTCGCGCTACTCTCTCT-3′; β2M (reverse), 5′-GTCAACTTCAATGTCGGATGGA-3′; IRF-9 (forward), 5′-CCGTGATAATCGTGTCCTGAAA-3′; IRF-9 (reverse), 5′-CCTGGGTTCACACCATTTGG-3′; PKR (forward), 5′-ATGATGGAAAGCGAACAAGGA-3′; PKR (reverse), 5′-GTTTCAAAAGCAGTGTCACATACATG-3′; c-myc (forward), 5′-CGTCTCCACACATCAGAGCACAA-3′; c-myc (reverse), 5′-TTGGCAGCAGGATAGTCCTT-3′. Experiments were done in triplicate and the expression of each gene was standardized using glyceraldehyde-3-phosphate dehydrogenase as a reference. Amplification reactions were run using a 7900HT real-time PCR instrument (Applied Biosystems, Foster City, CA). The results were expressed as the ratio between the expression of the IFN target gene and the expression of glyceraldehyde-3-phosphate dehydrogenase. Expression of IFN-responsive Genes Is Repressed in a Panel of Colon Cancer Cell Lines That Harbor a Ki-Ras Mutation—To identify profiles of gene expression that predict the response of colon cancer cells to chemotherapeutic drugs, we performed genome-wide analysis of 30 colorectal cell lines. A detailed statistical analysis of the entire data base has been published elsewhere (79Mariadason J. Arango D. Wilson A.J. Corner G.A. Nicholas C. Aranes M.J. Schwartz E.L. Shi Q. Lesser M. Augenlicht L. Cancer Res. 2003; (in press)Google Scholar), and data can be found on our website: sequence.aecom.yu.edu/bioinf/Augenlicht/default.html. To study the mechanism whereby activating mutations of Ki-ras promote tumorigenesis we selected from the data base 12 cells lines with diverse Ki-ras status and compared their gene expression profile. Six of these cell lines harbor an oncogenic mutation in the Ki-ras protooncogene (Dld1 (G13D), HCT15 (G13D), HCT116 (G13D), LoVo (G12D), SW480 (G12V), and SW620 (G12V)) and six cell lines have wild type (WT) Ki-ras (Caco-2, Colo201, HT29, RKO, T84, and WiDr). cDNA microarray analysis was performed as described under "Experimental Procedures," and the expression of IFN-dependent genes was compared with reference RNA, prepared from a panel of 12 colon carcinoma cell lines. The experiments were performed twice and the average level of gene expression was used to present the data in Table I. The data for the 12 arrays were analyzed by unsupervised clustering, using the Cluster and Treeview programs. The duplicates clustered together, illustrating the high degree of reproducibility of the microarray data. In addition, Dld1 and HCT15, cell lines derived from the same colon carcinoma by two independent laboratories, clustered closely together. Likewise, SW480 and SW620, cell lines generated from a primary and metastatic cancer from the same patient, also clustered together, demonstrating a high degree of sensitivity and reproducibility of the gene expression profiling (79Mariadason J. Arango D. Wilson A.J. Corner G.A. Nicholas C. Aranes M.J. Schwartz E.L. Shi Q. Lesser M. Augenlicht L. Cancer Res. 2003; (in press)Google Scholar).Table IThe expression of IFN-responsive genes in colon cancer cell lines with diverse Ki-ras statusAccession no.GeneMean expression/standard RNAMT/WT ratioWT Ki-rasMT Ki-rasAA419251IFN-9-272.120.500.23AA479795IFN-202.300.600.26ap < 0.05, Mann-Whitney nonparametric test.AA157813IFN-271.161.020.88AA406020IFN-151.980.860.43AA491191IFN-162.670.310.11ap < 0.05, Mann-Whitney nonparametric test.AA630800IFN-IP 300.960.920.95W24246IFN/RA-581.050.900.85W77927GBP21.220.450.37AA489743IFN/tetra15.490.740.13ap < 0.05, Mann-Whitney nonparametric test.AA448478IFN-6-162.700.500.18ap < 0.05, Mann-Whitney nonparametric test.AI017240Bag-11.281.000.77AA291577p482.110.800.37AA454813IRF-21.180.860.73T62627IFN-75/SP1002.130.800.37ap < 0.05, Mann-Whitney nonparametric test.W42587IFN-PKR2.591.060.40AA456886Myx-17.430.710.09ap < 0.05, Mann-Whitney nonparametric test.AA286908Myx-22.910.750.25ap < 0.05, Mann-Whitney nonparametric test.AA644657MHC class I, A1.771.200.67T63324MHC class II, Dqα11.180.820.69AA486072A51.200.850.71Mean: 2.27 ± 0.35Mean: 0.78 ± 0.05Mean: 0.34, p = 0.0002a p < 0.05, Mann-Whitney nonparametric test. Open table in a new tab When we analyzed the data base generated from the 12 cell lines with diverse Ki-ras status, we noticed that the expression of 20 IFN-responsive genes was reduced in six cell lines that harbor activating mutation of Ki-ras, compared with the six cell lines with WT Ki-ras. 20 genes were included in our analysis based on two criteria: 1) the gene had to be described as an IFN-responsive gene, 2) the signal from the microarray experiment had to be larger than the background + 1 S.D. in at least 11 of the 12 cell lines examined. The expression of IFN-responsive genes varied among the cell lines. Cell lines used in the experiment were grouped based solely on the Ki-ras status; however, they also differ in other genetic markers, such as mutations in p53, Apc, β-catenin. Such changes are likely to contribute to heterogeneity in the expression of these genes. However, despite the heterogeneity in the genetic background, the mean expression of the IFN-responsive genes in the six cell lines harboring mutant (MT) Ki-ras was significantly lower than in the cell lines with wt Ki-ras. The average ratio between the expression of all 20 IFN-responsive genes and the reference RNA in the six cell lines with WT Ki-ras was 2.27, compared with 0.78 in the six cell lines with a mutant Ki-ras, a highly statistically significant difference (p = 0.0002, Mann-Whitney test). In addition, the mean expression of individual IFN-dependent genes was markedly lower in cell lines containing MT Ki-ras than in cell lines containing WT Ki-ras. The differences were statistically significant (p < 0.05, Mann-Whitney test) for 35% of the individual genes examined (Table I). When we compared the expression of the 20 IFN-dependent genes in cell lines with WT p53 (HCT116, Lovo, LS174T, RKO, and SW48) and MT p53 (16E, 19A, Colo205, HCT15, HT29, KM12, SW480, and SW620), we did not detect any significant differences in the expression of IFN-responsive genes between the two groups (ratio MTp53/WTp53 = 1.1, p = 0.42, data not shown), suggesting that the expression of IFN-dependent genes is not under major regulatory control of p53. These results demonstrated that the effect of mutant Ras on the expression of interferon responsive genes is sufficiently strong to be detected in a population of cell lines that differ in a number of other genetic determinants. In addition, they suggested that oncogenic Ki-Ras interferes with the expression of IFN-responsive genes. Targeted Inactivation of Mutant Ki-ras in Colon Cancer Cells Is Sufficient to Restore the Expression of IFN-responsive Genes—To further determine the role of oncogenic Ras in IFN signaling, we utilized a well defined system of isogenic cell lines that differ only in the presence of the mutant Ki-ras allele. The HCT116 cell line harbors an activating mutation in codon 13 of the Ki-ras protooncogene, and the Hkh2 and Hke-3 clones were generated by the targeted deletion of the mutant Ki-ras allele (11Shirasawa S. Furuse M. Yokoyama N. Sasazuki T. Science. 1993; 260: 85-88Crossref PubMed Scopus (598) Google Scholar). Inactivation of the mutant Ki-ras allele in HCT116 cells resulted in reduced proliferation of cells, reduced tumorigenicity in vivo, and reduced capacity for anchorage-independent growth (11Shirasawa S. Furuse M. Yokoyama N. Sasazuki T. Science. 1993; 260: 85-88Crossref PubMed Scopus (598) Google Scholar). We performed cDNA microarray analysis on the isogenic cell lines as described under "Experimental Procedures." Experiments were performed twice in Hke-3 cells and twice in Hkh2 cells (Table II). The genes shown in Table I and Table II overlap only partially, because different cDNA microarray slides were used in the experiments (see "Experimental Procedures"). Consistent with our hypothesis that mutant Ras interferes with the expression of IFN responsive genes, we found that a cluster of IFN-responsive genes included on the array was down-regulated in HCT116 cells, compared with the two isogenic clones with a targeted deletion of the mutant Ki-ras allele, Hkh2 and Hke-3 (Table II). The expression of IFN-responsive genes is normally induced in response to IFNs and several of them harbor a STAT1 binding site in their regulatory regions (37Ramana C.V. Chatterjee-Kishore M. Nguyen H. Stark G.R. Oncogene. 2000; 19: 2619-2627Crossref PubMed Scopus (246) Google Scholar). Their basal level of expression may be due to production of low endogenous levels of IFN (38Taniguchi T. Takaoka A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 378-386Crossref PubMed Scopus (401) Google Scholar, 39Hata N. Sato M. Takaoka A. Asagiri M. Tanaka N. Taniguchi T. Biochem. Biophys. Res. Commun. 2001; 285: 518-525Crossref PubMed Scopus (89) Google Scholar). Although IFN-responsive genes represent a hitherto unrecognized group of Ras target genes, the expression of the IFN-9–27
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