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• W2091250703 abstract "Our goal was to identify genes regulated by Wnt/β-catenin signaling in melanoma cells, as this pathway has been implicated in melanocyte development and in melanoma biology. We therefore undertook transcriptional profiling of UACC 1273 human melanoma cells following treatment with recombinant Wnt-3a and found that cytotoxic T-lymphocyte antigen-4 (CTLA-4) was the most highly induced gene. We observed CTLA-4 expression in human epidermal melanocytes and in patient-derived primary melanoma tumors and found that Wnt/β-catenin signaling elevates CTLA-4 expression in two cultured melanoma cell lines. CTLA-4 is likely a direct target of Wnt/β-catenin signaling, as the β-catenin responsiveness of a 1.7 kb region of the CTLA-4 promoter requires a T-cell factor-1/lymphoid enhancing factor-1 consensus site present at -114 to -119 bp from the transcriptional start site. These findings are the initial demonstration that CTLA-4 is a direct target of Wnt/β-catenin signaling and the first report of its expression in primary melanoma tumors and melanocytes. Given the described role of CTLA-4 in inhibiting the immune response, these findings may shed light on the role of Wnt/β catenin signaling in melanoma and on the mechanism of action of human anti-CTLA-4 antibody, currently in phase III clinical trials for the treatment of melanoma. Our goal was to identify genes regulated by Wnt/β-catenin signaling in melanoma cells, as this pathway has been implicated in melanocyte development and in melanoma biology. We therefore undertook transcriptional profiling of UACC 1273 human melanoma cells following treatment with recombinant Wnt-3a and found that cytotoxic T-lymphocyte antigen-4 (CTLA-4) was the most highly induced gene. We observed CTLA-4 expression in human epidermal melanocytes and in patient-derived primary melanoma tumors and found that Wnt/β-catenin signaling elevates CTLA-4 expression in two cultured melanoma cell lines. CTLA-4 is likely a direct target of Wnt/β-catenin signaling, as the β-catenin responsiveness of a 1.7 kb region of the CTLA-4 promoter requires a T-cell factor-1/lymphoid enhancing factor-1 consensus site present at -114 to -119 bp from the transcriptional start site. These findings are the initial demonstration that CTLA-4 is a direct target of Wnt/β-catenin signaling and the first report of its expression in primary melanoma tumors and melanocytes. Given the described role of CTLA-4 in inhibiting the immune response, these findings may shed light on the role of Wnt/β catenin signaling in melanoma and on the mechanism of action of human anti-CTLA-4 antibody, currently in phase III clinical trials for the treatment of melanoma. cytotoxic T-lymphocyte-associated antigen-4 Dickkopf-1 human epidermal melanocyte reverse transcriptase-PCR T-cell factor-1/lymphoid enhancing factor-1 Malignant melanoma is a highly aggressive cancer derived from melanocytes found primarily in the epidermis. With a dismal 5-year survival rate of 5–15%, the outlook for patients with metastatic melanoma remains quite bleak (Cummins et al., 2006Cummins D.L. Cummins J.M. Pantle H. Silverman M.A. Leonard A.L. Chanmugam A. Cutaneous malignant melanoma.Mayo Clin Proc. 2006; 81: 500-507Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), and the incidence of this disease worldwide is increasing faster than that of any other cancer (Diepgen and Mahler, 2002Diepgen T.L. Mahler V. The epidemiology of skin cancer.Br J Dermatol. 2002; 146: 1-6Crossref PubMed Google Scholar). The exact molecular mechanisms underlying this complex disease remain unresolved, although recent discoveries have identified some of the crucial cell-signaling pathways involved in melanoma development and progression, including the Wnt signaling pathways (Fecher et al., 2007Fecher L.A. Cummings S.D. Keefe M.J. Alani R.M. Toward a molecular classification of melanoma.J Clin Oncol. 2007; 25: 1606-1620Crossref PubMed Scopus (185) Google Scholar). Wnts are a family of highly conserved cysteine-rich proteins that signal through multiple distinct but overlapping pathways. In the Wnt/β-catenin pathway, the binding of Wnts to Frizzled receptors and LRP5/6 coreceptors activates a cascade of events resulting in the accumulation and nuclear translocation of β-catenin. Nuclear β-catenin interacts with multiple proteins, including T-cell factor-1/lymphoid enhancing factor-1 (TCF/LEF) transcription factors, to regulate gene transcription (Logan and Nusse, 2004Logan C.Y. Nusse R. The Wnt signaling pathway in development and disease.Annu Rev Cell Dev Biol. 2004; 20: 781-810Crossref PubMed Scopus (3969) Google Scholar; Clevers, 2006Clevers H. Wnt/beta-catenin signaling in development and disease.Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4165) Google Scholar). Additionally, several other Wnts appear to act predominantly through a set of β-catenin-independent pathways, which are less well defined (Veeman et al., 2003Veeman M.T. Axelrod J.D. Moon R.T. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling.Dev Cell. 2003; 5: 367-377Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar; Kohn and Moon, 2005Kohn A.D. Moon R.T. Wnt and calcium signaling: beta-catenin-independent pathways.Cell Calcium. 2005; 38: 439-446Crossref PubMed Scopus (535) Google Scholar). Several studies have implicated both β-catenin-dependent and β-catenin-independent Wnt signaling in melanoma pathogenesis (Weeraratna, 2005Weeraratna A.T. A Wnt-er wonderland—the complexity of Wnt signaling in melanoma.Cancer Metastasis Rev. 2005; 24: 237-250Crossref PubMed Scopus (59) Google Scholar; Larue and Delmas, 2006Larue L. Delmas V. The WNT/Beta-catenin pathway in melanoma.Front Biosci. 2006; 11: 733-742Crossref PubMed Scopus (196) Google Scholar). Wnt/β-catenin signaling is known to play a critical role in melanocyte development, raising the question of its role in melanoma tumorigenesis. A pilot study of Wnt production in melanoma reported that Wnt-2, Wnt-5, Wnt-7b and Wnt-10b are synthesized by a subset of melanoma cells (Pham et al., 2003Pham K. Milovanovic T. Barr R.J. Truong T. Holcombe R.F. Wnt ligand expression in malignant melanoma: pilot study indicating correlation with histopathological features.Mol Pathol. 2003; 56: 280-285Crossref PubMed Scopus (45) Google Scholar). β-catenin is increased in several melanoma cell lines (Rubinfeld et al., 1997Rubinfeld B. Robbins P. El-Gamil M. Albert I. Porfiri E. Polakis P. Stabilization of beta-catenin by genetic defects in melanoma cell lines.Science. 1997; 275: 1790-1792Crossref PubMed Scopus (1112) Google Scholar), and although mutations in β-catenin are rare in primary melanomas, nuclear localization of this protein, an indicator of β-catenin signaling, is frequently observed (Rimm et al., 1999Rimm D.L. Caca K. Hu G. Harrison F.B. Fearon E.R. Frequent nuclear/cytoplasmic localization of beta-catenin without exon 3 mutations in malignant melanoma.Am J Pathol. 1999; 154: 325-329Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar; Demunter et al., 2002Demunter A. Libbrecht L. Degreef H. De Wolf-Peeters C. van den Oord J.J. Loss of membranous expression of beta-catenin is associated with tumor progression in cutaneous melanoma and rarely caused by exon 3 mutations.Mod Pathol. 2002; 15: 454-461Crossref PubMed Scopus (63) Google Scholar). In addition, other Wnt pathway components, including adenomatous polyposis coli (Worm et al., 2004Worm J. Christensen C. Gronbaek K. Tulchinsky E. Guldberg P. Genetic and epigenetic alterations of the APC gene in malignant melanoma.Oncogene. 2004; 23: 5215-5226Crossref PubMed Scopus (81) Google Scholar), inhibitor of beta-catenin and Tcf-4 (Reifenberger et al., 2002Reifenberger J. Knobbe C.B. Wolter M. Blaschke B. Schulte K.W. Pietsch T. et al.Molecular genetic analysis of malignant melanomas for aberrations of the WNT signaling pathway genes CTNNB1, APC, ICAT and BTRC.Int J Cancer. 2002; 100: 549-556Crossref PubMed Scopus (95) Google Scholar), lymphoid enhancing factor-1 (LEF1) (Murakami et al., 2001Murakami T. Toda S. Fujimoto M. Ohtsuki M. Byers H.R. Etoh T. et al.Constitutive activation of Wnt/beta-catenin signaling pathway in migration-active melanoma cells: role of LEF-1 in melanoma with increased metastatic potential.Biochem Biophys Res Commun. 2001; 288: 8-15Crossref PubMed Scopus (64) Google Scholar), and Dickkopf-1 (DKK) (Kuphal et al., 2006Kuphal S. Lodermeyer S. Bataille F. Schuierer M. Hoang B.H. Bosserhoff A.K. Expression of Dickkopf genes is strongly reduced in malignant melanoma.Oncogene. 2006; 25: 5027-5036Crossref PubMed Scopus (144) Google Scholar) are modified in melanoma tumors and cell lines. Microphthalmia-associated transcription factor (MITF) and other known Wnt/β-catenin target genes, including CCND1, c-MYC, Brn-2, and Nr-CAM, have also been implicated in melanoma biology (Rodolfo et al., 2004Rodolfo M. Daniotti M. Vallacchi V. Genetic progression of metastatic melanoma.Cancer Lett. 2004; 214: 133-147Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar; Larue and Delmas, 2006Larue L. Delmas V. The WNT/Beta-catenin pathway in melanoma.Front Biosci. 2006; 11: 733-742Crossref PubMed Scopus (196) Google Scholar; Miller and Mihm, 2006Miller A.J. Mihm Jr, M.C. Melanoma.N Engl J Med. 2006; 355: 51-65Crossref PubMed Scopus (1070) Google Scholar). Interestingly, β-catenin-independent Wnt signaling has been implicated in melanoma progression. Gene array expression profiling of melanomas identified Wnt-5a as a robust marker of highly aggressive behavior (Bittner et al., 2000Bittner M. Meltzer P. Chen Y. Jiang Y. Seftor E. Hendrix M. et al.Molecular classification of cutaneous malignant melanoma by gene expression profiling.Nature. 2000; 406: 536-540Crossref PubMed Scopus (1660) Google Scholar). Subsequent studies have correlated Wnt-5a and activation of a β-catenin-independent pathway that involves protein kinase C with increased cell motility and invasiveness of UACC 1273 human melanoma cells (Weeraratna et al., 2002Weeraratna A.T. Jiang Y. Hostetter G. Rosenblatt K. Duray P. Bittner M. et al.Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma.Cancer Cell. 2002; 1: 279-288Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar; Dissanayake et al., 2007Dissanayake S.K. Wade M. Johnson C.E. O’Connell M.P. Leotlela P.D. French A.D. et al.The Wnt5A/protein kinase C pathway mediates motility in melanoma cells via the inhibition of metastasis suppressors and initiation of an epithelial to mesenchymal transition.J Biol Chem. 2007; 282: 17259-17271Crossref PubMed Scopus (277) Google Scholar). In summary, the development of malignant melanoma, an aggressive and increasingly common cancer, is associated with both β-catenin-dependent and β-catenin-independent Wnt signaling, although the molecular and cellular mechanisms involved are unclear. To better understand the role of Wnt signaling in melanoma, we undertook transcriptional profiling of UACC 1273 human melanoma cells following treatment with recombinant Wnt-3a. We identified cytotoxic T-lymphocyte antigen-4 (CTLA-4), a potential therapeutic target for melanoma (Kasper et al., 2007Kasper B. D’Hondt V. Vereecken P. Awada A. Novel treatment strategies for malignant melanoma: a new beginning?.Crit Rev Oncol Hematol. 2007; 62: 16-22Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), as the gene most strongly induced by Wnt-3a. We then determined that Wnt/β-catenin signaling directly induces CTLA-4 in human melanoma cells and that CTLA-4 is expressed in primary melanoma tumors and melanocytes. These results are relevant to the ongoing development of human anti-CTLA-4 antibody for the treatment of metastatic melanoma. To identify genes regulated by the Wnt/β-catenin pathway in melanoma, we focused on the UACC 1273 human melanoma cell line, which has previously been studied in the context of Wnt-5a signaling (Weeraratna et al., 2002Weeraratna A.T. Jiang Y. Hostetter G. Rosenblatt K. Duray P. Bittner M. et al.Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma.Cancer Cell. 2002; 1: 279-288Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar; Dissanayake et al., 2007Dissanayake S.K. Wade M. Johnson C.E. O’Connell M.P. Leotlela P.D. French A.D. et al.The Wnt5A/protein kinase C pathway mediates motility in melanoma cells via the inhibition of metastasis suppressors and initiation of an epithelial to mesenchymal transition.J Biol Chem. 2007; 282: 17259-17271Crossref PubMed Scopus (277) Google Scholar). To evaluate the β-catenin-mediated Wnt responsiveness of these cells, we tested for two hallmarks of Wnt/β-catenin signaling: (1) activation of a luciferase reporter vector containing an TCF promoter (TOPFlash); and (2) an increase in total cellular β-catenin protein following treatment with recombinant Wnt-3a. Wnt-3a increases TOPFlash activity in a dose-dependent manner and at a concentration of 500 ng ml−1 for 6 hours and activates TOPFlash 26-fold relative to the FOPFlash control reporter, which contains mutant TCF sites (Figure S1a). Furthermore, after 72 hours of treatment with 480 ng ml−1 of recombinant Wnt-3a, the total amount of β-catenin protein increases relative to control (Figure S1b, left panel). In support of this result, 6-bromoindirubin-3-oxime, a small molecule activator of Wnt signaling that works by inhibiting glycogen synthase kinase 3-beta, also increases β-catenin protein levels (Figure S1b, right panel). These results suggest that UACC 1273 human melanoma cells can actively signal via the Wnt/β-catenin pathway by upregulating the levels of β-catenin and activating TCF-mediated transcription. Download .pdf (.27 MB) Help with pdf files Supplementary Figure 1Wnt/β-catenin signaling is active in UACC 1273 human melanoma cells. (a) UACC 1273 cells stably expressing TOPFlash or FOPFlash luciferase reporter and pRLTK renilla luciferase reporter were treated with 0.12, 0.24, 0.48, and 0.96 mg/ml of recombinant Wnt-3a or vehicle (BSA) for 6 h. Fold change of normalized luciferase units is calculated relative to untreated reporter. Data represent one of three similar experiments. (b) Western blot probing with anti-β-catenin antibody of cell lysate from UACC 1273 cells treated with 0.48 μg/ml of recombinant Wnt-3a or vehicle (BSA) and 1uM BIO or vehicle (DMSO) for 72 h. Equal loading of protein amount for each lane was determined by probing with anti-β-tubulin antibody. This experiment was done in duplicate. Having determined that the cells were responsive to recombinant Wnt-3a, we next investigated the gene expression profiles of UACC 1273 cells using the Affymetrix Human U133 plus 2.0 microarray system, following 6 hours of treatment with recombinant Wnt-3a. We found that the expression of 79 genes, 58 upregulated and 21 downregulated, was modulated by 1.5-fold or greater in two independent experiments (Table 1). CTLA-4 increases fourfold following Wnt-3a stimulation, which is the greatest fold change observed in our microarray results. Wnt-5a, which predominantly activates β-catenin-independent Wnt signaling, does not change CTLA-4 levels in similar array studies using recombinant Wnt-5a (data not shown). Several of the microarray targets, including c-Jun, LEF1, MITF, BAMBI (Sekiya et al., 2004Sekiya T. Adachi S. Kohu K. Yamada T. Higuchi O. Furukawa Y. et al.Identification of BMP and activin membrane-bound inhibitor (BAMBI), an inhibitor of transforming growth factor-beta signaling, as a target of the beta-catenin pathway in colorectal tumor cells.J Biol Chem. 2004; 279: 6840-6846Crossref PubMed Scopus (124) Google Scholar), ID2, and CLDN1, are known Wnt/β-catenin targets genes (http://www.stanford.edu/~rnusse/pathways/targets.html). In addition to MITF, four upregulated genes in our array, MC1R, MARCKS, TRPM1, NEDD9, and two downregulated ones, CLDN1 and CXCL1, have been implicated in melanoma progression (Manenti et al., 1998Manenti S. Malecaze F. Chap H. Darbon J.M. Overexpression of the myristoylated alanine-rich C kinase substrate in human choroidal melanoma cells affects cell proliferation.Cancer Res. 1998; 58: 1429-1434PubMed Google Scholar; Dhawan and Richmond, 2002Dhawan P. Richmond A. Role of CXCL1 in tumorigenesis of melanoma.J Leukoc Biol. 2002; 72: 9-18PubMed Google Scholar; Chin et al., 2006Chin L. Garraway L.A. Fisher D.E. Malignant melanoma: genetics and therapeutics in the genomic era.Genes Dev. 2006; 20: 2149-2182Crossref PubMed Scopus (395) Google Scholar; Leotlela et al., 2007Leotlela P.D. Wade M.S. Duray P.H. Rhode M.J. Brown H.F. Rosenthal D.T. et al.Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility.Oncogene. 2007; 26: 3846-3856Crossref PubMed Scopus (126) Google Scholar). Functional categorization of the entire set of putative Wnt-3a target genes shows that most of the overrepresented biological responses are consistent with previous knowledge about Wnt/β-catenin pathway functions (Figure S2, Table S1). To verify the microarray results, the induction/repression of 17 genes was assessed by quantitative real-time PCR analysis. There is a high degree (77%) of concordance between the microarray results and these studies (Table 1, Figure S3). Taken together, these findings confirm the validity of our microarray screen.Table 1Genes regulated by Wnt-3a in UACC 1273 human melanoma cells by DNA microarray analysis1 and real-time quantitative PCR analysis2 Open table in a new tab Download .pdf (.2 MB) Help with pdf files Supplementary Figure 2Partial list of overrepresented biological themes following Wnt-3a stimulation of UACC 1273 human melanoma cells. The 79 putative Wnt-3a target genes were classified into biological pathways using DAVID (http://david.abcc.ncifcrf.gov/home.jsp), based on GO ontology. The statistically significant subset (p<0.05), containing 7 functional groups, is shown. Consistent with previous knowledge about Wnt/β-catenin pathway functions, many of the regulated genes are involved in regulation of signal transduction, development, transcription, cell communication, and cell growth. Our analysis also showed that induction of Wnt signaling in melanoma led to functional overrepresentation of metabolism and induction of apoptosis. Download .pdf (.3 MB) Help with pdf files Supplementary Table 1Complete list of overrepresented biological themes following Wnt-3a stimulation of UACC 1273 human melanoma cells. The biological pathways were identified using DAVID and are listed with the corresponding p-values. “Count” and “%” represent the number of genes and the percent of 79 total genes, respectively, falling within a given category. Each gene may be listed in more than one category. Download .pdf (.25 MB) Help with pdf files Supplementary Figure 3Real-time RT-PCR validation of putative Wnt-3a target genes in human melanoma cells. RNA was extracted from UACC 1273 human melanoma cells 6 h after treatment with 480 ng/ml recombinant Wnt-3a or vehicle (BSA) for 6 h and subjected to real-time quantitative RT-PCR analysis. Expression of these putative Wnt-3a target genes was normalized by comparison to GAPDH and 18S (not shown) as internal controls. Fold change, calculated relative to BSA, for the validated genes is shown (shaded black bar). For comparison, real time PCR data is plotted beside the average fold change, relative to BSA, from the array data (white bar). Four genes, CDC42EP3, FBXO32, PMAIP1, KIT were not validated (not shown). Experiments were done in duplicate. Although CTLA-4 has been detected in melanoma cell lines (Contardi et al., 2005Contardi E. Palmisano G.L. Tazzari P.L. Martelli A.M. Fala F. Fabbi M. et al.CTLA-4 is constitutively expressed on tumor cells and can trigger apoptosis upon ligand interaction.Int J Cancer. 2005; 117: 538-550Crossref PubMed Scopus (182) Google Scholar), its presence in primary melanoma tumors and melanocytes has not been reported. We investigated the expression of CTLA-4 in two primary tumors and human epidermal melanocytes by flow cytometry using antibodies for CTLA-4 and S100, a marker of melanocytes and melanoma cells. In all the three samples, the isotype control antibodies for CTLA-4 and S100 showed low background staining and were used to set gates (Figures 1a–c). S100 costaining of the samples with the CTLA-4 isotype control showed strong expression of S100 in tumors and less intense staining of melanocytes, confirming the presence of melanoma and melanocyte cells, respectively (Figures 1d–f). CTLA-4 is expressed in the strongly S100-positive population of all the three samples (Figure 1g–i versus d–f see upper right quadrant). These data are summarized in histograms comparing CTLA-4-isotype control with CTLA-4 staining in the S100-positive cells (Figures 1j–l). This provides the first report of CTLA-4 expression from patient melanoma tumor samples. Human CTLA-4 undergoes alternative splicing to yield three transcripts, including full-length mRNA, a transcript coding for soluble CTLA-4 that does not contain the transmembrane region, and a short transcript coding only for the leader peptide sequence and cytoplasmic tail (Teft et al., 2006Teft W.A. Kirchhof M.G. Madrenas J. A molecular perspective of CTLA-4 function.Annu Rev Immunol. 2006; 24: 65-97Crossref PubMed Scopus (390) Google Scholar). The full-length and soluble forms may have different functional effects (Pawlak et al., 2005Pawlak E. Kochanowska I.E. Frydecka I. Kielbinski M. Potoczek S. Bilinska M. The soluble CTLA-4 receptor: a new marker in autoimmune diseases.Arch Immunol Ther Exp. 2005; 53: 336-341PubMed Google Scholar). Therefore, identification of the form of CTLA-4 induced by Wnt-3a may help to elucidate its function in melanoma. To this end, the mRNA transcripts were analyzed in UACC 1273 cells as well as the commonly studied A2058 human melanoma cell line by reverse transcriptase-PCR (RT-PCR) amplification using primers that flank the ATG initiation and translation termination codons. Only full-length CTLA-4 was detected in UACC1273 and A2058 cell lines following 6 hours of recombinant Wnt-3a stimulation (Figure 2a). To validate CTLA-4 induction by Wnt-3a in melanoma, we measured CTLA-4 transcript by real-time quantitative RT-PCR using two distinct sets of primers. Using primers that detect any isoform of CTLA-4, we found that CTLA-4 is elevated 5.9-fold and 4.4-fold in UACC 1273 and A2058 cells, respectively, following Wnt-3a treatment (Figure 2b). To confirm that the full-length form of CTLA-4 is induced by Wnt-3a specifically through Wnt/β-catenin signaling, primers specific for this splice variant were used to analyze CTLA-4 transcript by real-time RT-PCR in both cell lines following 6 hours treatment with Wnt-3a-conditioned media in the presence or absence of DKK-1, a secreted protein that inhibits the Wnt/β-catenin pathway by direct binding to the LRP5/6 Wnt coreceptor. DKK-1 blocks Wnt-3a induction of full-length CTLA-4 message in both cell lines (Figure 2c). Next, we evaluated CTLA-4 protein expression in UACC 1273 cells by flow cytometry following treatment with Wnt-3a conditioned media for 72 hours. In fixed and permeabilized cells, levels of total CTLA-4 increased in Wnt-3a relative to control-conditioned media-treated cells (Figure 3a); however, surface expression, in live nonpermeabilized cells, remained unchanged (Figure 3b). There was no change in surface expression levels at 4, 12, 24, and 48 hours (data not shown). We also observed that low but detectable levels of CTLA-4 were expressed in both control-treated and untreated cells (data not shown), suggesting that UACC 1273 cells constitutively express CTLA-4. Expression was also observed in untreated A2058 and Mel 375 melanoma cell lines (data not shown). In summary, our results demonstrate that Wnt/β-catenin signaling induces the expression of CTLA-4 in human melanoma cells at both the transcript and protein levels. We next investigated whether CTLA-4 is regulated by the Wnt/β-catenin pathway directly or indirectly. Inspection of the 1704 bp region immediately 5′ of the transcriptional start site of CTLA-4 revealed the presence of four potential TCF/LEF transcriptional response elements (Figure 4a). To test whether these sites are important for the regulation of CTLA-4 expression, we isolated this region by PCR and subcloned it upstream of a luciferase reporter (WT). In A2058 cells, Wnt-3a increased CTLA-4 promoter activity relative to the control in a dose-dependent manner, up to 4.5-fold (Figure 4b). Wnt-3a activation of this promoter was blocked by DKK-1-conditioned media, a known inhibitor of Wnt signaling (Figure 4b). These results support the hypothesis that CTLA-4 is transcriptionally regulated by the Wnt/β-catenin pathway. To test whether any of the four potential TCF/LEF binding sites are required for the observed Wnt-3a-responsiveness of the WT reporter, we introduced site-specific mutations into each of these sites by PCR (Figure 4a) and constructed a set of luciferase reporter vectors, each containing one mutant site. The ability of Wnt-3a to increase CTLA-4 promoter-driven luciferase expression was attenuated by an A-to-G mutation at position -116 in the second TCF-binding element (at -114 to -119) within the CTLA-4 promoter (Mut 2), strongly suggesting that the induction of CTLA-4 transcription by Wnt signaling is dependent upon the activity of TCF/LEF transcription factors (Figure 4c). Although it is clear that activation of Wnt/β-catenin signaling can be important in melanoma progression, the molecular details remain unclear. In this study, we investigated the transcription profile of human melanoma cells following Wnt-3a treatment and identified CTLA-4 as a direct transcriptional target of the Wnt/β-catenin signaling pathway. Furthermore, we made the initial observation that CTLA-4 is expressed in patient-derived melanoma tumors. Given the pivotal role of CTLA-4 in inhibiting immune responses, these results may offer insight into the regulation of CTLA-4 in several autoimmune diseases and malignancies. The predominant role of CTLA-4 as a negative regulator of T cell-mediated immune responses has led to widespread interest in making it a target of mAb therapy to boost antitumor immunity. CTLA-4 blockade leads to enhancement of immune response (Leach et al., 1996Leach D.R. Krummel M.F. Allison J.P. Enhancement of antitumor immunity by CTLA-4 blockade.Science. 1996; 271: 1734-1736Crossref PubMed Scopus (2400) Google Scholar), rejection of tumors (Hurwitz et al., 2000Hurwitz A.A. Foster B.A. Kwon E.D. Truong T. Choi E.M. Greenberg N.M. et al.Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade.Cancer Res. 2000; 60: 2444-2448PubMed Google Scholar), or reduction of tumors in mice when used in combination with tumor vaccines (van Elsas et al., 1999van Elsas A. Hurwitz A.A. Allison J.P. Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation.J Exp Med. 1999; 190: 355-366Crossref PubMed Scopus (820) Google Scholar). Promising preclinical results have led to the development of fully humanized anti-CTLA-4 antibodies that are currently being tested in over 10 clinical trials, including a phase III trial for the treatment of metastatic melanoma (Kasper et al., 2007Kasper B. D’Hondt V. Vereecken P. Awada A. Novel treatment strategies for malignant melanoma: a new beginning?.Crit Rev Oncol Hematol. 2007; 62: 16-22Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Although recent studies have reported the expression of CTLA-4 (Contardi et al., 2005Contardi E. Palmisano G.L. Tazzari P.L. Martelli A.M. Fala F. Fabbi M. et al.CTLA-4 is constitutively expressed on tumor cells and can trigger apoptosis upon ligand interaction.Int J Cancer. 2005; 117: 538-550Crossref PubMed Scopus (182) Google Scholar) and its ligand B7.1 (CD80) in melanoma cell lines (Tirapu et al., 2006Tirapu I. Huarte E. Guiducci C. Arina A. Zaratiegui M. Murillo O. et al.Low surface expression of B7-1 (CD80) is an immunoescape mechanism of colon carcinoma.Cancer Res. 2006; 66: 2442-2450Crossref PubMed Scopus (98) Google Scholar), as well as linked CTLA-4 gene polymorphisms with malignancy susceptibility (Zheng et al., 2001Zheng C. Huang D. Liu L. Bjorkholm M. Holm G. Yi Q. et al.Cytotoxic T-lymphocyte antigen-4 microsatellite polymorphism is associated with multiple myeloma.Br J Haematol. 2001; 112: 216-218Crossref PubMed Scopus (37) Google Scholar; Ghaderi et al., 2004Ghaderi A. Yeganeh F. Kalantari T. Talei A.R. Pezeshki A.M. Doroudchi M. et al.Cytotoxic T lymphocyte antigen-4 gene in" @default.
• W2091250703 created "2016-06-24" @default.
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• W2091250703 date "2008-12-01" @default.
• W2091250703 modified "2023-10-17" @default.
• W2091250703 title "CTLA-4 Is a Direct Target of Wnt/β-Catenin Signaling and Is Expressed in Human Melanoma Tumors" @default.
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