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• W2025512024 abstract "The NFAT (nuclear factor of activated T cells) family of transcription factors plays a fundamental role in the transcriptional regulation of the immune response. However, NFATs are ubiquitously expressed, and recent evidence points to their important functions in human epithelial cells and carcinomas. Specifically, NFAT has been shown to be active in human breast and colon carcinoma cells and to promote their invasion through Matrigel. The mechanisms by which NFAT promotes invasion have not been defined. To identify NFAT target genes that induce carcinoma invasion, we have established stable breast cancer cell lines that inducibly express transcriptionally active NFAT. Gene expression profiling by cDNA microarray of cells induced to express NFAT revealed up-regulation of cyclooxygenase-2 (COX-2). Increased NFAT expression and activity induced COX-2 expression as well as prostaglandin E2 synthesis. This induction was more prominent when NFAT was activated by phorbol 12-myristate 13-acetate and calcium ionophore ionomycin and was blocked by the NFAT antagonist cyclosporin A. Breast cancer cells with elevated COX-2 expression showed increased invasion through Matrigel, and this was reduced in cells treated with COX-2 inhibitors. Conversely, loss of NFAT1 protein expression using small interfering RNA led to a reduction in COX-2 transcription and reduced invasion. Similarly, Matrigel invasion was reduced in cells in which COX-2 expression was reduced using specific siRNA. These findings demonstrate that NFAT promotes breast cancer cell invasion through the induction of COX-2 and the synthesis of prostaglandins. The NFAT (nuclear factor of activated T cells) family of transcription factors plays a fundamental role in the transcriptional regulation of the immune response. However, NFATs are ubiquitously expressed, and recent evidence points to their important functions in human epithelial cells and carcinomas. Specifically, NFAT has been shown to be active in human breast and colon carcinoma cells and to promote their invasion through Matrigel. The mechanisms by which NFAT promotes invasion have not been defined. To identify NFAT target genes that induce carcinoma invasion, we have established stable breast cancer cell lines that inducibly express transcriptionally active NFAT. Gene expression profiling by cDNA microarray of cells induced to express NFAT revealed up-regulation of cyclooxygenase-2 (COX-2). Increased NFAT expression and activity induced COX-2 expression as well as prostaglandin E2 synthesis. This induction was more prominent when NFAT was activated by phorbol 12-myristate 13-acetate and calcium ionophore ionomycin and was blocked by the NFAT antagonist cyclosporin A. Breast cancer cells with elevated COX-2 expression showed increased invasion through Matrigel, and this was reduced in cells treated with COX-2 inhibitors. Conversely, loss of NFAT1 protein expression using small interfering RNA led to a reduction in COX-2 transcription and reduced invasion. Similarly, Matrigel invasion was reduced in cells in which COX-2 expression was reduced using specific siRNA. These findings demonstrate that NFAT promotes breast cancer cell invasion through the induction of COX-2 and the synthesis of prostaglandins. NFAT 2The abbreviations used are: NFAT, nuclear factor of activated T cells; COX-2, cyclooxygenase-2; PMA, phorbol 12-myristate 13-acetate; siRNA, small interfering RNA; IL-2, interleukin-2; PG, prostaglandin; HA, hemagglutinin; RT, reverse transcription; CsA, cyclosporin A; Dox, doxycycline. was first identified in T cells as a rapidly inducible transcription factor that binds to the distal antigen receptor response element of the human interleukin-2 (IL-2) promoter and is involved in the regulation of inducible genes such as cytokines and cell surface receptors in immune cells (1Shaw J.P. Utz P.J. Durand D.B. Toole J.J. Emmel E.A. Crabtree G.R. Science. 1988; 241: 202-205Crossref PubMed Scopus (10) Google Scholar, 2Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar, 3Serfling E. Berberich-Siebelt F. Chuvpilo S. Jankevics E. Klein-Hessling S. Twardzik T. Avots A. Biochim. Biophys. Acta. 2000; 1498: 1-18Crossref PubMed Scopus (170) Google Scholar). However, subsequent studies have revealed that NFAT is also expressed and is active in many other cell types and tissues, and it regulates the expression of genes related to cell cycle progression, angiogenesis, tumorigenesis, and cell differentiation (4Caetano M.S. Vieira-de-Abreu A. Teixeira L.K. Werneck M.B. Barcinski M.A. Viola J.P. FASEB J. 2002; 16: 1940-1942Crossref PubMed Scopus (71) Google Scholar, 5Baksh S. Widlund H.R. Frazer-Abel A.A. Du J. Fosmire S. Fisher D.E. DeCaprio J.A. Modiano J.F. Burakoff S.J. Mol. Cell. 2002; 10: 1071-1081Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 6Graef I.A. Wang F. Charron F. Chen L. Neilson J. Tessier-Lavigne M. Crabtree G.R. Cell. 2003; 113: 657-670Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 7Neal J.W. Clipstone N.A. J. Biol. Chem. 2003; 278: 17246-17254Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 8Zaichuk T.A. Shroff E.H. Emmanuel R. Filleur S. Nelius T. Volpert O.V. J. Exp. Med. 2004; 199: 1513-1522Crossref PubMed Scopus (117) Google Scholar). The mechanisms by which NFAT controls these cellular processes remain largely undefined. The NFAT family of transcription factors comprises four classical members: NFAT1, NFAT2, NFAT3, and NFAT4 (2Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar). All are calcium-responsive and are regulated by the calcium/calcineurin signaling pathway (9Hogan P.G. Chen L. Nardone J. Rao A. Genes Dev. 2003; 17: 2205-2232Crossref PubMed Scopus (1572) Google Scholar, 10Feske S. Okamura H. Hogan P.G. Rao A. Biochem. Biophys. Res. Commun. 2003; 311: 1117-1132Crossref PubMed Scopus (154) Google Scholar). A recently identified member, NFAT5, is distinct from NFAT1–4 as it is calcium-insensitive and is regulated by osmotic stress and integrins (11Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Copeland N.G. Gilbert D.J. Thomas S. Disteche C. Jenkins N.A. Rao A. Cold Spring Harbor Symp. Quant. Biol. 1999; 64: 517-526Crossref PubMed Scopus (32) Google Scholar). In resting cells, NFATs are phosphorylated at a cluster of serine residues located in the regulatory domain, effectively masking a nuclear localization signal, thereby retaining NFAT in an inactive conformation in the cytoplasm (2Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar, 12Okamura H. Aramburu J. Garcia-Rodriguez C. Viola J.P. Raghavan A. Tahiliani M. Zhang X. Qin J. Hogan P.G. Rao A. Mol. Cell. 2000; 6: 539-550Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Upon stimulation with agonists that elicit an increase in intracellular calcium, NFATs are dephosphorylated by the phosphatase calcineurin and translocate to the nucleus. Here they are transcriptionally active by binding to the promoter regions of target genes (13Shaw K.T. Ho A.M. Raghavan A. Kim J. Jain J. Park J. Sharma S. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11205-11209Crossref PubMed Scopus (318) Google Scholar, 14Loh C. Shaw K.T. Carew J. Viola J.P. Luo C. Perrino B.A. Rao A. J. Biol. Chem. 1996; 271: 10884-10891Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). When the cells return to their unstimulated state, NFAT becomes rephosphorylated and is exported out of the nucleus (2Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar). Classical NFATs typically interact with other transcription factors such as AP-1 (15Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (681) Google Scholar, 16Macian F. Lopez-Rodriguez C. Rao A. Oncogene. 2001; 20: 2476-2489Crossref PubMed Scopus (623) Google Scholar) and GATA4 (17Morimoto T. Hasegawa K. Wada H. Kakita T. Kaburagi S. Yanazume T. Sasayama S. J. Biol. Chem. 2001; 276: 34983-34989Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) to activate transcription. Adjacent NFAT and AP-1 binding sites are present in the promoter region of inducible genes including IL-2 and cyclooxygenase-2 (COX-2) (18Jain J. Miner Z. Rao A. J. Immunol. 1993; 151: 837-848PubMed Google Scholar, 19Iniguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Cyclooxygenases convert arachidonic acid produced from membrane phospholipids by phospholipase A2 to prostaglandin H2 (PGH2). Prostaglandin endoperoxide is then converted to biologically active prostaglandins (PGD2, PGE2, PGF2α), prostacyclin (PGI2), and thromboxanes (TxA2) by tissue-specific synthases or reductases (20Smith W.L. Marnett L.J. DeWitt D.L. Pharmacol. Ther. 1991; 49: 153-179Crossref PubMed Scopus (387) Google Scholar). Following their synthesis, these prostanoids are secreted and bind to G protein-coupled membrane receptors in target cells in an autocrine or paracrine fashion, thereby triggering downstream signaling events (21Smith W.L. Am. J. Physiol. 1992; 263: F181-F191PubMed Google Scholar). Prostaglandins are important regulators of numerous cellular processes including cell proliferation, inflammation, and angiogenesis (22Hla T. Ristimaki A. Appleby S. Barriocanal J.G. Ann. N. Y. Acad. Sci. 1993; 696: 197-204Crossref PubMed Scopus (159) Google Scholar). Cyclooxygenase-2 catalyzes the formation of prostaglandin E2 (PGE2). COX-2 is distinct from the other isoform, COX-1, which is considered a housekeeping enzyme and is expressed constitutively in most tissues. Conversely, COX-2 is normally expressed at very low or undetectable levels and is rapidly induced at sites of inflammation and proliferation in response to stimuli such as growth factors and tumor promoters (23Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2477) Google Scholar). COX-2 expression and PGE2 levels are elevated in a variety of human cancers (24Fujita T. Matsui M. Takaku K. Uetake H. Ichikawa W. Taketo M.M. Sugihara K. Cancer Res. 1998; 58: 4823-4826PubMed Google Scholar, 25Soslow R.A. Dannenberg A.J. Rush D. Woerner B.M. Khan K.N. Masferrer J. Koki A.T. Cancer. 2000; 89: 2637-2645Crossref PubMed Scopus (856) Google Scholar, 26Denkert C. Kobel M. Pest S. Koch I. Berger S. Schwabe M. Siegert A. Reles A. Klosterhalfen B. Hauptmann S. Am. J. Pathol. 2002; 160: 893-903Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) and are associated with increased angiogenesis, tumor invasion, and resistance to apoptosis (27Iniguez M.A. Rodriguez A. Volpert O.V. Fresno M. Redondo J.M. Trends Mol. Med. 2003; 9: 73-78Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 28Chang S.H. Liu C.H. Conway R. Han D.K. Nithipatikom K. Trifan O.C. Lane T.F. Hla T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 591-596Crossref PubMed Scopus (346) Google Scholar, 29Corcoran C.A. He Q. Huang Y. Sheikh M.S. Oncogene. 2005; 24: 1634-1640Crossref PubMed Scopus (63) Google Scholar, 30Ito H. Duxbury M. Benoit E. Clancy T.E. Zinner M.J. Ashley S.W. Whang E.E. Cancer Res. 2004; 64: 7439-7446Crossref PubMed Scopus (127) Google Scholar). Similarly, overexpression of COX-2 has been shown to induce cancer formation in transgenic mice (31Marnett L.J. DuBois R.N. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 55-80Crossref PubMed Scopus (293) Google Scholar, 32Subbaramaiah K. Dannenberg A.J. Trends Pharmacol. Sci. 2003; 24: 96-102Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar). Several epidemiological studies have indicated that continuous users of aspirin and other nonsteroidal anti-inflammatory drugs, which inhibit COX activity, have reduced risk or mortality from cancer (33Rosenberg L. Louik C. Shapiro S. Cancer. 1998; 82: 2326-2333Crossref PubMed Scopus (154) Google Scholar, 34Steinbach G. Lynch P.M. Phillips R.K. Wallace M.H. Hawk E. Gordon G.B. Wakabayashi N. Saunders B. Shen Y. Fujimura T. Su L.K. Levin B. N. Engl. J. Med. 2000; 342: 1946-1952Crossref PubMed Scopus (2299) Google Scholar, 35Jacobs E.J. Rodriguez C. Mondul A.M. Connell C.J. Henley S.J. Calle E.E. Thun M.J. J. Natl. Cancer Inst. 2005; 97: 975-980Crossref PubMed Scopus (148) Google Scholar). Moreover, COX-2-specific inhibitors have been shown to suppress tumor growth in animal models of human cancer (36Kawamori T. Rao C.V. Seibert K. Reddy B.S. Cancer Res. 1998; 58: 409-412PubMed Google Scholar, 37Reddy B.S. Hirose Y. Lubet R. Steele V. Kelloff G. Paulson S. Seibert K. Rao C.V. Cancer Res. 2000; 60: 293-297PubMed Google Scholar). A link between NFAT activity and COX-2 is evident from previous studies. NFAT has been reported to regulate COX-2 expression in human T lymphocytes (19Iniguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Putative NFAT recognition sequences are present in the human COX-2 proximal promoter, and deletion analysis has shown that they are important for its transcriptional activation (19Iniguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). A recent study also demonstrated that these sites are essential for the induction of COX-2 by NFAT in colon carcinoma cells (38Duque J. Fresno M. Iniguez M.A. J. Biol. Chem. 2005; 280: 8686-8693Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The consequence of NFAT-mediated COX-2 induction for cancer cell phenotypes has not been established. The significance of NFAT for cancer development or progression to metastasis has to date not been investigated. Our previous studies on NFAT revealed that it plays an essential role in promoting migration and invasion of breast and colon carcinoma cells (39Jauliac S. Lopez-Rodriguez C. Shaw L.M. Brown L.F. Rao A. Toker A. Nat. Cell Biol. 2002; 4: 540-544Crossref PubMed Scopus (349) Google Scholar). To identify the downstream NFAT target genes that are important for invasion, we have analyzed the gene expression profile of breast cancer cells that express NFAT1. We detected a significant up-regulation of COX-2 in these cells. We show that activation of NFAT increases COX-2 expression and PGE2 synthesis. Inactivation of NFAT by cyclosporin A (CsA) or siRNA significantly diminished COX-2 expression. Expression of COX-2 promoted invasion through Matrigel, and this was reduced by the COX-2 inhibitor NS-398 or with siRNA. Together, these results provide the first direct evidence that NFAT promotes breast cancer cell invasion through the induction of COX-2. Cell Lines and Reagents—The human cell lines MDA-MB-435 and MDA-MB-231 were obtained from American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium with 1 g/ml glucose, l-glutamine, and sodium pyruvate (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Nova-Tech, Grand Island, NE). The estrogen-independent breast cancer cell line SUM-159-PT has been described (40Flanagan L. Van Weelden K. Ammerman C. Ethier S.P. Welsh J. Breast Cancer Res. Treat. 1999; 58: 193-204Crossref PubMed Scopus (63) Google Scholar) and was maintained in Ham's F-12 medium with l-glutamine (Cambrex, Walkersville, MD) supplemented with 5% fetal bovine serum, 1 μg/ml hydrocortisone, and 5 μg/ml insulin (Sigma). SUM.N1-16 and 435.N1-23 cells with inducible NFAT1 expression were derived from SUM-159-PT and MDA-MB-435 cells, respectively, and generated using the tetracycline-regulated expression system from Invitrogen. HA-tagged NFAT1 was subcloned into pcDNA4/TO/Myc-His and was co-transfected into SUM-159-PT cells with pcDNA6/TR. Positive clones were selected and maintained in medium with 20 μg/ml blasticidin and 0.1 mg/ml zeocin (InvivoGen, San Diego, CA). 435.N1-23 cells were prepared by transfecting HA-NFAT1 in pcDNA4/TO/Myc-His into MDA-MB-435 cells that have constitutive expression of tetracycline repressors from pcDNA6/TR. Positive clones were selected and maintained in culture medium containing 10 μg/ml blasticidin and 0.4 mg/ml zeocin. Expression of NFAT1 was induced with 1 μg/ml doxycycline (Clontech) for 16–24 h at 37 °C. SUM.N1 siRNA-4 and SUM.N1 siRNA-17 cells that express NFAT1 siRNA were prepared by transfecting SUM-159-PT cells with NFAT1 siRNA (see below). Stable clones were selected with 20 μg/ml blasticidin and 0.5 mg/ml Geneticin (Invitrogen). Cells were treated with PMA (100 nm; Alexis Biochemicals, San Diego, CA) and ionomycin (100 nm; CalBiochem) for 16–24 h. Cyclosporin A (CalBiochem) was used at 10 μm and NS-398 (Cayman Chemical Co., Ann Arbor, MI) at 50–100 μm. Anti-HA antibody was purified in-house from the 12CA5 hybridoma. Monoclonal anti-COX-2 was from Cayman Chemical Co., and anti-NFAT1 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin antibody was from Sigma. Plasmid Constructs—HA-NFAT1 was prepared by cloning HA-tagged murine NFAT1 cDNA into pcDNA4/TO/Myc-His (Invitrogen). The pIL2-Luc luciferase reporter plasmid has been described (41Durand D.B. Shaw J.P. Bush M.R. Replogle R.E. Belagaje R. Crabtree G.R. Mol. Cell. Biol. 1988; 8: 1715-1724Crossref PubMed Scopus (375) Google Scholar). The pCS2-(n)-β-gal plasmid was from Promega (Madison, WI). The pCMV-COX-2 expression plasmid and the COX-2 promoter luciferase reporter P2-274 have been described (19Iniguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) and were provided by Dr. Miguel Iñiguez. COX-2 siRNA was generated by cloning the annealed oligonucleotides with sequences 5′-GAT CCC CAA CCG AGG TGT ATG TAT GAG TGT TTC AAG AGA ACA CTC ATA CAT ACA CCT CGG TTT TTT TGG AAA-3′ and 5′-AGC TTT TCC AAA AAA ACC GAG GTG TAT GTA TGA GTG TTC TCT TGA AAC ACT CAT ACA TAC ACC TCG GTT GGG-3′ into the BglII/HindIII sites of the pSUPER vector (Oligoengine, Seattle, WA). To silence NFAT1 expression, SUM.N1 siRNA-4 and SUM.N1 siRNA-17 cells were generated with the siRNA plasmid constructed with the following sequences: 5′-GAT CCC CTC CTT AAG CCG CAC GCC TTT TCA AGA GAA AGG CGT GCG GCT TAA GGA TTT TTG GAA A-3′ and 5′-AGC TTT TCC AAA AAT CCT TAA GCC GCA CGC CTT TCT CTT GAA AAG GCG TGC GGC TTA AGG AGG G-3′. Immunoblotting—Total cell lysates were prepared in ice-cold radioimmune precipitation assay lysis buffer (50 mm Tris HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.2 mm phenylmethylsulfonyl fluoride, and 2 mm sodium orthovanadate) supplemented with protease inhibitor mixture from Sigma. The lysates were clarified by centrifugation at 12,000 × g for 10 min, and protein concentration was determined by the Bradford assay (Bio-Rad). Protein lysates were denatured, resolved by SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes. The protein blots were blocked with 5% nonfat milk and incubated with the appropriate primary antibodies for 2 h at room temperature or overnight at 4 °C. Signals were developed by using the SuperSignal West Pico chemiluminescent substrate from Pierce. Real-time RT-PCR—Total RNA samples were extracted from the cultured cells using TRIzol (Invitrogen) and were reverse transcribed into cDNA using Taqman reverse transcriptase and oligo(dT)16 (Roche Applied Science) according to the manufacturer's instructions. Quantitative real-time PCR was performed using the SYBR Green PCR master mix in an ABI Prism 7700 sequence detector (both from Applied Biosystems, Foster City, CA). The reactions were carried out with a polymerase-activating step of 95 °C for 10 min followed by 40 cycles of a two-step cycling program (95 °C for 15 s; 60 °C for 1 min) for NFAT1 detection or a three-step cycling program (95 °C for 15 s; 57 °C for 30 s, 72 °C for 45 s) for analyzing COX-2 transcription. For murine NFAT1, the primers were 5′-CGG AGT CCA AGG TTG TGT TCA-3′ (sense) and 5′-TGT GGC TGA CTT CGT TTC CTC-3′ (antisense). For human NFAT1, the primers were 5′-TGC ATC TAA CCC CAT CGA GTG-3′ (sense) and 5′-TGA GGA TCA TTT GCT GGC C-3′ (antisense). For glyceraldehyde-3-phosphate dehydrogenase, the primers were 5′-GCA AAT TCC ATG GCA CCG T-3′ (sense) and 5′-TCG CCC CAC TTG ATT TTG G-3′ (antisense). For COX-2, the primers were 5′-CAA AAG CTG GGA AGC CTT CTC TAA CC-3′ (sense) and 5′-GCC CAG CCC GTT GGT GAA AG-3′ (antisense). The PCR products were analyzed on 1 or 1.5% agarose gels to ensure the specificity of amplification. Transfection and Luciferase Assays—All cell lines were transfected using the TransIT-LT1 transfection reagent from Mirus Bio Corporation (Madison, WI). Luciferase reporter constructs were transiently co-transfected into the cells with pCS2-(n)-β-gal. Cells were then left untreated or treated overnight with PMA/ionomycin with or without cyclosporin A. Total cell lysates were prepared 24 h after transfection. Luciferase and β-galactosidase activities were determined using Promega's luciferase assay system and Galacton-Plus from Tropix (Bedford, MA), respectively, in a MicroLumat LB 96 P luminometer (Berthold Analytical Instruments, Nashua, NH). Matrigel Invasion Assays—Invasion assays were performed essentially as described previously (39Jauliac S. Lopez-Rodriguez C. Shaw L.M. Brown L.F. Rao A. Toker A. Nat. Cell Biol. 2002; 4: 540-544Crossref PubMed Scopus (349) Google Scholar). Briefly, Transwell chambers with 8-μm pore filters (Corning, Acton, MA) were coated with 1–5 μgof Matrigel (BD Biosciences). Cells were harvested by trypsinization, resuspended in serum-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin, and added (1.0–1.5 × 105 cells/assay) in triplicates to Transwell chambers. The cells were allowed to invade the Matrigel-coated filters at 37 °C toward NIH 3T3-conditioned medium in the lower compartment. After 4–8 h, cells that had invaded to the lower surface of the filter were fixed and stained with crystal violet or 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) and counted. PGE2 Measurements—PGE2 levels in the culture medium of treated cells were determined using a PGE2-monoclonal enzyme immunoassay kit from Cayman Chemical Co. according to the manufacturer's protocol. To collect culture medium supernatants for the assay, growth medium of the cells was replaced with serum-free medium, and after overnight incubation at 37 °C, cell culture medium was spiked with 10 μg/ml arachidonic acid and collected 30 min later. The samples were clarified by centrifugation and analyzed in triplicates. The amount of PGE2 in the culture medium was determined by referring to the signal intensities obtained from a set of PGE2 standards. Our previous study demonstrated that the α6β4 integrin, a tumor-associated antigen, up-regulates NFAT activity in breast carcinoma. We showed that although NFAT5 increases cell migration, NFAT1 promotes both migration and invasion (39Jauliac S. Lopez-Rodriguez C. Shaw L.M. Brown L.F. Rao A. Toker A. Nat. Cell Biol. 2002; 4: 540-544Crossref PubMed Scopus (349) Google Scholar). To identify the target genes induced by NFAT that are responsible for promoting migration and invasion, we established clones of MDA-MB-435 and SUM-159-PT cells that inducibly express NFAT1 upon stimulation with tetracycline or the analog doxycycline. As shown in Fig. 1A, NFAT1 expression was induced in doxycycline-treated clones of MDA-MB-435 (435.N1-23) and SUM-159-PT (SUM.N1-16) cells. Increased NFAT1 expression induction was also confirmed at the mRNA level by real-time RT-PCR (Fig. 1B). To investigate whether the induced NFAT1 was transcriptionally active, we transfected an IL-2 luciferase reporter plasmid. As predicted, the induced NFAT1 was functional at driving NFAT-dependent transcription of IL-2 (Fig. 1C). When the stable transfectants were allowed to invade Matrigel in an in vitro invasion assay, doxycycline-treated cells were significantly more invasive compared with their untreated controls (Fig. 1D), consistent with previous data (39Jauliac S. Lopez-Rodriguez C. Shaw L.M. Brown L.F. Rao A. Toker A. Nat. Cell Biol. 2002; 4: 540-544Crossref PubMed Scopus (349) Google Scholar). Using cDNA prepared from untreated and doxycycline-treated 435.N1-23 and SUM.N1-16 cells, we analyzed the gene expression profile induced subsequent to NFAT1 expression. A 6.06-fold (435.N1-23) and 3.05-fold (SUM.N1-16) induction of COX-2 was observed (data not shown). NFAT has been shown to bind to the COX-2 promoter and regulate its transcription in immune cells (19Iniguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), and a recent study has reported the regulation of COX-2 by NFAT in human colon carcinoma (38Duque J. Fresno M. Iniguez M.A. J. Biol. Chem. 2005; 280: 8686-8693Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). However, the relevance of NFAT in COX-2 regulation and function in breast cancer cell signaling or responses has not been determined. To address this question, we analyzed the expression of COX-2 in doxycycline-treated cells. COX-2 transcription measured by RT-PCR was increased upon NFAT1 expression induced by doxycycline (Fig. 2A). At the protein level, untreated SUM.N1-16 cells revealed low to undetectable levels of both NFAT1 and COX-2. After doxycycline treatment, NFAT1 expression significantly increased and was accompanied by a small but reproducible elevation in COX-2 protein (Fig. 2B). Stimulation with the NFAT agonists PMA and ionomycin led to a dramatic increase in COX-2 expression, likely because of the activation of endogenous NFAT. Moreover, combined treatment of doxycycline and PMA/ionomycin led to an even greater increase in NFAT1 and COX-2 expression (Fig. 2B). Next we determined whether the induction of COX-2 by NFAT1 is mediated through the transcriptional activation of the COX-2 promoter. SUM.N1-16 and MDA-MB-231 cells were transfected with the pIL2-luc or the human COX-2 promoter luciferase reporter construct P2-274. Cells stimulated with PMA/ionomycin showed a basal low level of NFAT activity when compared with untreated control (Fig. 3A, left panel). In contrast, induction of NFAT1 expression with doxycycline significantly increased the transcription of both IL-2 (Fig. 3A, left panel) and COX-2 (right panel). Maximum induction of IL-2 and COX-2 transcription was revealed when induced NFAT1 was activated by PMA/ionomycin. Blocking the activation of NFAT with CsA significantly diminished transcription from both reporters (Fig. 3A). As MDA-MB-231 cells have high levels of endogenous NFAT, we also examined the NFAT-driven IL-2 and COX-2 transcription in these cells. Activation of endogenous NFAT by PMA/ionomycin significantly increased transcriptions from both the IL-2 and COX-2 promoters (Fig. 3B). Again, NFAT inhibition by CsA reduced the induction of transcriptional activation of both reporters. To further investigate the regulation of COX-2 by NFAT, we have developed clones of SUM-159-PT cells in which NFAT expression is reduced by siRNA. Real-time RT-PCR analysis demonstrated that two distinct clones expressed significantly less NFAT1 transcripts compared with parental SUM-159-PT cells. This was confirmed by immunoblotting with anti-NFAT1 (Fig. 4A). Reduced NFAT1 expression with siRNA also markedly attenuated transcription from the IL-2 promoter in cells treated with PMA/ionomycin compared with cells transfected with control siRNA (Fig. 4B). Most importantly, reduced expression of NFAT by siRNA also led to a quantitative reduction of COX-2 promoter luciferase activity (Fig. 4C). Finally, to demonstrate that loss of NFAT1 translates into loss of invasion, we measured Matrigel invasion in control SUM-159-PT cells compared with the stable siRNA clones. As predicted, reduced expression of NFAT1 by siRNA resulted in not only a loss of COX-2 expression but also a marked loss of invasion (Fig. 4D). To extend the above findings, we transiently transfected COX-2 into MDA-MB-435 and SUM-159-PT cells and measured Matrigel invasion. In both cell lines, COX-2 expression increased invasion (Fig. 5A). COX-2 expression was confirmed by immunoblotting. We also used a loss-of-function approach and constructed COX-2 siRNA to silence COX-2 expression. The efficacy of silencing endogenous COX-2 was determined by immunoblotting (Fig. 5B). Next, SUM.N1-16 cells were transfected with either control or COX-2 siRNA, induced with doxycycline to express NFAT1, followed by Matrigel assays. When NFAT1 expression was induced in control transfected cells, as already demonstrated, this led to a reproducible increase in invasion. However, in the presence of COX-2 siRNA, invasion was reduced to control levels (Fig. 5C, left panel). COX-2 siRNA also significantly blunted Matrigel invasion of MDA-MB-231 cells (Fig. 5C, right panel). These results demonstrate directly that at least one mechanism by which NFAT promotes invasion is through the induction of COX-2. PGE2 is the major product of COX-2. To further define the role of COX-2 induction by NFAT1 in the invasion phenotype, we assayed PGE2 levels in the culture medium of SUM.N1 cells. In control cells, low levels of PGE2 were detected, but these levels significantly increased when cells were stimulated by PMA/ionomycin to activate endogenous NFAT (Fig. 6A). Moreover, treatment with NS-398, a potent and specific COX-2 inhibitor, blocked PGE" @default.
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• W2025512024 date "2006-05-01" @default.
• W2025512024 modified "2023-10-03" @default.
• W2025512024 title "NFAT Induces Breast Cancer Cell Invasion by Promoting the Induction of Cyclooxygenase-2" @default.
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• W2025512024 doi "https://doi.org/10.1074/jbc.m600184200" @default.
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