CTLA-4 Is a Direct Target of Wnt/β-Catenin Signaling and Is Expressed in Human Melanoma Tumors

doi:10.1038/jid.2008.170

J Invest Dermatol. Author manuscript; available in PMC 2011 July 13.

Published in final edited form as:

J Invest Dermatol. 2008 December; 128(12): 2870–2879.

Published online 2008 June 19. doi: 10.1038/jid.2008.170

PMCID: PMC3135173

NIHMSID: NIHMS303888

CTLA-4 Is a Direct Target of Wnt/β-Catenin Signaling and Is Expressed in Human Melanoma Tumors

Kavita V. Shah,¹ Andy J. Chien,² Cassian Yee,^2,³ and Randall T. Moon^1,⁴

¹ Department of Pharmacology, University of Washington, Seattle, Washington, USA

² Department of Medicine, University of Washington, Seattle, Washington, USA

³ Program in Immunology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

⁴ Department of Pharmacology, Howard Hughes Medical Institute and Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA

Correspondence: Dr Randall T. Moon, Department of Pharmacology, University of Washington, Campus Box 357370, Room K336B Health Sciences Building, 1959 NE Pacific Street, Seattle, Washington 98195, USA. Email: rtmoon/at/u.washington.edu

The publisher's final edited version of this article is available free at J Invest Dermatol

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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.

INTRODUCTION

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., 2006), and the incidence of this disease worldwide is increasing faster than that of any other cancer (Diepgen and Mahler, 2002). 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., 2007).

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, 2004; Clevers, 2006). Additionally, several other Wnts appear to act predominantly through a set of β-catenin-independent pathways, which are less well defined (Veeman et al., 2003; Kohn and Moon, 2005). Several studies have implicated both β-catenin-dependent and β-catenin-independent Wnt signaling in melanoma pathogenesis (Weeraratna, 2005; Larue and Delmas, 2006).

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., 2003). β-catenin is increased in several melanoma cell lines (Rubinfeld et al., 1997), 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., 1999; Demunter et al., 2002). In addition, other Wnt pathway components, including adenomatous polyposis coli (Worm et al., 2004), inhibitor of beta-catenin and Tcf-4 (Reifenberger et al., 2002), lymphoid enhancing factor-1 (LEF1) (Murakami et al., 2001), and Dickkopf-1 (DKK) (Kuphal et al., 2006) 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., 2004; Larue and Delmas, 2006; Miller and Mihm, 2006).

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., 2000). 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., 2002; Dissanayake et al., 2007). 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., 2007), 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.

RESULTS

Wnt-3a-treated human melanoma cells exhibit transcriptional changes in known and novel Wnt/β-catenin target genes

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., 2002; Dissanayake et al., 2007). 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.

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 down-regulated, was modulated by 1.5-fold or greater in two independent experiments (Table 1). CTLA-4 increases four-fold 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., 2004), 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., 1998; Dhawan and Richmond, 2002; Chin et al., 2006; Leotlela et al., 2007). 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 1

Genes regulated by Wnt-3a in UACC 1273 human melanoma cells by DNA microarray analysis¹ and real-time quantitative PCR analysis²

CTLA-4 is expressed in primary melanoma tumor cells and primary melanocytes

Although CTLA-4 has been detected in melanoma cell lines (Contardi et al., 2005), 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.

Figure 1

CTLA-4 is expressed in human epidermal melanocytes and primary melanoma tumors

CTLA-4 transcript and protein levels are elevated by Wnt/β-catenin signaling

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., 2006). The full-length and soluble forms may have different functional effects (Pawlak et al., 2005). 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).

Figure 2

CTLA-4 transcript is induced by Wnt/β-catenin signaling in two melanoma cell lines

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.

Figure 3

Wnt-3a increases total CTLA-4 protein levels in UACC 1273 melanoma cells

The CTLA-4 promoter is directly regulated through a single TCF/LEF consensus site

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.

Figure 4

CTLA-4 promoter is directly regulated by Wnt/β-catenin pathway through a single TCF/LEF transcription factor consensus site

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).

DISCUSSION

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., 1996), rejection of tumors (Hurwitz et al., 2000), or reduction of tumors in mice when used in combination with tumor vaccines (van Elsas et al., 1999). 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., 2007). Although recent studies have reported the expression of CTLA-4 (Contardi et al., 2005) and its ligand B7.1 (CD80) in melanoma cell lines (Tirapu et al., 2006), as well as linked CTLA-4 gene polymorphisms with malignancy susceptibility (Zheng et al., 2001; Ghaderi et al., 2004; Monne et al., 2004), the function of CTLA-4 in melanoma is unknown.

We observed that Wnt/β-catenin signaling induces the expression of CTLA-4 in human melanoma cells at both the transcript and protein levels. In both UACC 1273 and A2058 melanoma cell lines, the full-length splice variant of CTLA-4 is elevated by Wnt-3a. This induction is specifically mediated by β-catenin, as it is attenuated by the Wnt/β-catenin pathway inhibitor, DKK-1. The observed increase in CTLA-4 message is accompanied by an increase in total protein levels, although without concomitant changes in cell surface expression.

There may be a few plausible explanations for the discrepancy between low or undetectable surface expression and functional effects. In T cells, CTLA-4 is tightly regulated by restricted trafficking to the cell surface (Iida et al., 2000) and rapid internalization (Chuang et al., 1997). Even during maximal expression of CTLA-4 after T-cell activation, surface CTLA-4 constitutes less than 10% of the total cellular protein, and little can be detected (Alegre et al., 1996; Alegre et al., 1998; Anna et al., 2003). Strikingly, several studies have reported functional effects in T cells even when surface levels of CTLA-4 are undetectable (Krummel and Allison, 1996; Walunas et al., 1996; Fallarino et al., 2003). Similarly, in melanoma cell lines, studies have shown that although low levels of intracellular but not cell surface CTLA-4 are detectable, apoptosis could be induced following treatment with CTLA-4 ligands, B7.1 (CD80) and B7.2 (CD86), suggesting the presence of functional receptor on the surface (Contardi et al., 2005). An alternative possible explanation is that Wnt-mediated induction of CTLA-4 expression in melanoma cells is solely intracellular and has a yet unknown function. These published studies suggest that although we do not detect a change in CTLA-4 cell surface expression, this does not preclude the increase in total expression from having physiological consequences.

Finally, we analyzed a 1.7 kb region of the CTLA-4 promoter containing four putative TCF/LEF binding sites to address whether CTLA-4 is directly or indirectly regulated by the Wnt/β-catenin pathway. We found that Wnt-3a increases CTLA-4 promoter activity through a single TCF/LEF binding site located at −114 to −119 from the transcriptional start site. This result is supported by the finding that LEF1 increases CTLA-4 transcription from a polymorphic promoter variant at position −318 bp, which is located within a TCF/LEF consensus site (Anjos et al., 2004; Chistiakov et al., 2006). This required TCF/LEF site lies within the 335-bp region of the CTLA4 promoter, shown to be essential for the induction of the CTLA4 expression (Perkins et al., 1996), suggesting the importance of Wnt/β-catenin signaling in regulating CTLA-4 expression.

The potential function of CTLA-4 in the activity of regulatory T cells (Treg), a subpopulation of T cells involved in suppressing host immune responses to prevent autoimmune diseases, and alternatively to promote tumor immunity (Wang and Wang, 2007), may offer a possible explanation for the role of Wnt/β-catenin pathway-mediated CTLA-4 induction in melanoma cells. Several studies have suggested that ligation of B7 molecules expressed on dendritic cells with recombinant soluble forms of CTLA-4 or CTLA-4 expressed on Tregs induces tryptophan catabolism by increasing the expression of indoleamine 2,3-dioxygenase in dendritic cells (Mandelbrot et al., 2001; Grohmann et al., 2002; Fallarino et al., 2003). This results in T cell suppression by depleting the essential amino acid and inhibiting growth or by inducing apoptosis from tryptophan metabolites. In support of this idea, CTLA-4 expressed on activated CD4+ T cells can stimulate indoleamine 2,3-dioxygenase in dendritic cells (Munn et al., 2004). Thus, it is interesting to speculate that ligation of CTLA-4, induced by Wnt/β-catenin signaling, in melanoma cells with B7 molecules on dendritic cells in the tumor microenvironment may shield target tumor cells against T-cell-mediated destruction by inducing tryptophan catabolism.

The regulation of CTLA-4 by Wnt/β-catenin signaling and expression of CTLA-4 in melanoma tumors is likely relevant to ongoing clinical trials of CTLA-4 antibody therapy in patients with metastatic melanoma. Although CTLA-4 blockade has been shown to increase autoimmunity and antitumor immunity, the mechanisms underlying these effects remain controversial. As a high level of CTLA-4 is constitutively expressed on Tregs (Takahashi et al., 2000), the effects of CTLA-4 blockade have been attributed to Treg depletion, which may elicit an autoimmune response that leads to an antitumor effect (Attia et al., 2005). A second hypothesis, however, posits that CTLA-4 blockade may not work through Treg depletion, but rather by preventing the inhibitory signals usually triggered by B7-CTLA-4 engagement, with a net consequence of increased activation (Maker et al., 2005). Furthermore, several studies suggest that activation of CTLA-4 in cultured tumor cells has some inhibitory role, as engagement of CTLA-4 by soluble ligand results in apoptosis (Pistillo et al., 2003; Contardi et al., 2005; Laurent et al., 2007). Based on our findings, further studies are needed to address how differences in tumor CTLA-4 levels may relate to differences in patient responses to CTLA-4 blockade. If, indeed, CTLA-4 expression on tumors can affect patient responses to anti-CTLA-4 therapy, then modulation of Wnt/β-catenin signaling by small molecules may provide a potential therapeutic avenue.

MATERIALS AND METHODS

Approval of all described studies by the University of Washington was not necessary.

Cell lines and primary melanoma tumors

UACC 1273 cells were a gift from Dr Ashani T. Weeraratna (National Institute on Aging, Baltimore, MD). Primary melanoma tumors and A2058 human melanoma cells were gifts from Dr Cassian Yee (Fred Hutchinson Cancer Center, Seattle, WA). The tumors were obtained from patients in accordance with IRB guidelines. Mouse L cells or L cells that stably express Wnt3a were a gift from Dr Roel Nusse (Stanford University, Stanford, CA).

Recombinant Wnt and conditioned media

Recombinant Wnt-3a was a generous gift from Dr Steven J. Staats and Dr Irwin D. Bernstein (both from Fred Hutchinson Cancer Center, Seattle, WA). Wnt3a and control-conditioned media were prepared as described (Willert et al., 2003). Preparation of Dkk-1 or green fluorescent protein (control) containing conditioned media is fully detailed in the Supplementary Methods.

Constructs

Full-length CTLA-4 expression construct was created with standard PCR-based cloning strategies. A 1.7 kb region of the CTLA-4 promoter, upstream of the transcriptional start site, was cloned into pGL3 Basic (Promega, Madison, WI) with standard PCR-based cloning strategies. Mutations were made in the TCF/LEF consensus sites of this promoter using site-directed PCR-based mutagenesis followed by cloning into pGL3 Basic. All oligos used are detailed in the supplement (Table S2).

Preparation of cell extracts and western blot analysis

UACC 1273 cells seeded into a 6-well plate were treated with 480 ng ml⁻¹ BSA, 480 ngml⁻¹ recombinant Wnt-3a, DMSO, or 1 uM 6-bromoindirubin-3-oxime (Calbiochem, San Diego, CA) for 72 hours. Total cell lysates were analyzed by western blotting with polyclonal anti-β-catenin (9562; Cell Signaling, Danvers, MA) at 1:5,000, and/or monoclonal anti-β-tubulin antibodies (T7816; Sigma, St Louis, MO) at 1:10,000 as described previously (Angers et al., 2006). Western blots were repeated twice.

RNA isolation, reverse transcription, and quantitative real-time PCR

Total RNA was extracted using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol for all treatment conditions as detailed in the Supplementary Methods. Single-stranded cDNA synthesis and quantitative real-time PCR analysis were performed as described previously (Major et al., 2007). The oligos used for target genes and for the internal controls, GAPDH and 18S, are detailed in the supplement (Table S2). Real-time PCR experiments were conducted in duplicate and repeated 3–5 times.

Transient transfections and luciferase reporter assays

All transient transfections were performed using Lipofectamine 2000 (Invitrogen, Lakewood, NJ) according to the manufacturer’s recommendations. Stable expression of SuperTOPFlash reporter (Kaykas et al., 2004) or TCF/LEF mutated fuBAR reporter (Major et al., 2007) and renilla luciferase reporter (Promega) in UACC 1273 cells was achieved by lentiviral-mediated transduction. TOPFlash, CTLA-4 promoter, and CTLA-4 promoter mutants luciferase assays were performed in accordance with the dual luciferase assay specifications (Promega) using the Mithras LB940 luminometer (Berthold, Bad Wildbad, Germany) and are detailed in the Supplementary Methods. Reporter assays were conducted in duplicate and repeated 3–5 times.

Affymetrix microarray analysis

Total RNA extracted from UACC 1273 cells seeded into a 6-well plate and treated with 480 ng ml⁻¹ BSA or 480 ng ml⁻¹ recombinant Wnt-3a for 6 hours was subject to RNA quality control, target labeling, hybridization to Affymetrix Human Genome U133 Plus 2.0 chips (Affymetrix, Santa Clara, CA), and scanning by the Center for Array Technology (University of Washington, Seattle, WA, http://ra.microslu.washington.edu/). Data were analyzed using GCOS (GeneChip Operating System, Affymetrix, Santa Clara, CA) software as described in the Supplementary Methods.

Flow cytometry for CTLA-4 expression

Wnt-3a or control-conditioned media-treated UACC 1273 cells, human epidermal melanocytes, and dissociated primary melanoma tumor were stained with appropriate antibody (PE-CTLA-4, BD Pharmingen, San Jose, CA, no. 555853; Control for CTLA-4, BD Pharmingen, no. S100; Dako, Carpentaria, CA, no. Z3011; Control for S100, Dako, no. X0936; Alexa Fluor 633, Invitrogen, no. A21070) and analyzed using either BD FACScan flow cytometer (BD Biosciences, San Jose, CA) and Cell Quest software for UACC 1273 cells or Influx flow cytometer (Cytopeia Inc., Seattle, WA) and FCSExpress (Denovo software, Los Angeles, CA) for melanocytes and tumors as detailed in the Supplementary Methods. Antibodies and antibody combinations were extensively tested at several concentrations (data not shown). CTLA-4 expression studies in UACC 1273 cells and melanocytes/tumors were repeated thrice and twice, respectively.

Supplementary Material

Supplemental Figure 1

Table S1. Complete list of overrepresented biological themes following Wnt-3a stimulation of UACC 1273 human melanoma cells.

Table S2. Oligonucleotides used for construction of CTLA-4 expression construct, construction of CTLA-4 wild-type and mutant promoter reporter constructs, and for real-time RT-PCR analysis of putative Wnt-3a target genes and internal controls, GAPDH, and18S.

Figure S1. Wnt/β-catenin signaling is active in UACC 1273 human melanoma cells.

Figure S2. Partial list of overrepresented biological themes following Wnt-3a stimulation of UACC 1273 human melanoma cells.

Figure S3. Real-time RT-PCR validation of putative Wnt-3a target genes in human melanoma cells.

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Supplemental Figure 2

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Supplemental Figure 3

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Supplemental Figure 4

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Supplemental Figure 5

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Supplemental Figure 6

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Acknowledgments

We thank Yong Li for preparation of the human melanoma tumors. We are also grateful to Ashani Weeraratna and to Steven Staats and Irv Bernstein for the UACC 1273 cells and recombinant Wnt-3a, respectively. This work, K.V.S. and R.T.M., were supported by the HHMI. A.J.C. was supported by a career development award from the Dermatology Foundation and a Research Scholar award from the American Skin Association. C.Y. was supported by the Damon Runyon Cancer Research Foundation, the NIH, and the General Clinical Research Center.

Abbreviations


CTLA-4	cytotoxic T-lymphocyte-associated antigen-4

DKK-1	Dickkopf-1

HEM	human epidermal melanocyte

RT-PCR	reverse transcriptase-PCR

TCF/LEF	T-cell factor-1/lymphoid enhancing factor-1

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

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