European Journal of Cancer (2014) 50, 2714– 2724

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The metabolite 50-methylthioadenosine signals through the adenosine receptor A2B in melanoma Katharina Limm a, Susanne Wallner a, Vladimir M. Milenkovi b, Christian H. Wetzel b, Anja-Katrin Bosserhoff a,⇑ a b

Institute of Pathology, University of Regensburg, Germany Molecular Neurosciences, Department of Psychiatry and Psychotherapy, University of Regensburg, Regensburg, Germany

Received 28 November 2013; received in revised form 6 June 2014; accepted 7 July 2014 Available online 30 July 2014

KEYWORDS 50 -Methylthioadenosine Melanoma MAPK/ERK – PKC signalling Adenosine receptor A2B AP-1-trascription factor CREB-1 pathway

Abstract Several recent studies have shown evidence supporting the general knowledge that tumour cells exhibit changes in metabolism. It is becoming increasingly important to understand how these metabolic changes in tumour cells promote carcinogenesis and disease progression. We recently discovered a lack of methylthioadenosine phosphorylase (MTAP) expression in melanoma, which resulted in an accumulation of the metabolite 50 -methylthioadenosine (MTA) in melanoma cells and in the extracellular environment. MTA was shown to affect cell proliferation of surrounding stroma cells and cell invasiveness and the activation of the transcription factor activator protein-1 (AP-1) in melanoma cells. In this study, we addressed the regulation of cellular signalling by extracellular MTA accumulation. By focusing on putative receptors that could modulate MTA signalling, we identified the adenosine receptor ADORA2B as an important candidate. Knockdown experiments and the use of specific agonists and antagonists confirmed a link between MTA and AP-1 signalling through the ADORA2B receptor. Interestingly, stimulation of the cells with MTA did not result in activation of the classical cyclic adenosine monophosphate (cAMP) signalling cascades or in Ca2+-dependent signalling. We instead showed protein kinase C (PKC) signalling to be involved in MTA-mediated AP-1 activation. In summary, we identified ADORA2B to be the specific receptor and signalling pathway for the metabolite MTA. These findings may influence the use of MTA in a therapeutic manner. Ó 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author: Address: Institute of Pathology, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany. Tel.: +49 941 944 6705; fax: +49 941 944 6602. E-mail address: anja.bosserhoff@klinik.uni-regensburg.de (A.-K. Bosserhoff).

http://dx.doi.org/10.1016/j.ejca.2014.07.005 0959-8049/Ó 2014 Elsevier Ltd. All rights reserved.

K. Limm et al. / European Journal of Cancer 50 (2014) 2714–2724

1. Introduction In addition to transcriptomics, genomics and proteomics, the discipline of metabolomics is becoming an important area of cancer research. Metabolomics enables the analysis of biological fluids, tissues and cell extracts. A number of cellular functions are deregulated in cancer cells in comparison to normal cells, including polyamine metabolism. High polyamine levels were shown to influence tumour growth and cell death regulation [1]. A metabolite that is directly associated with polyamines is 50 -methylthioadenosine (MTA). This molecule is a side product of polyamine biosynthesis and is a substrate of the enzyme methylthioadenosine phosphorylase (MTAP) [2–4]. MTAP is a ubiquitously expressed enzyme that is essential for purine and amino acid metabolism [5]. In many malignant cells, a lack of MTAP activity is caused by chromosomal loss or epigenetic regulation [6]. In malignant melanoma, the loss of MTAP activity is a consequence of promoter hypermethylation [3,7,8]. This loss results in the intracellular and extracellular accumulation of MTA, which has been determined by liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) [9,10]. It was shown that increased extracellular level of MTA affects the stroma cells near the tumour in their proliferation characteristics but further also the invasion behaviour of melanoma cells [4]. MTA has been shown in several studies to be a potent inhibitor of protein arginine methyltransferase (PRMT) activity [11]. We recently showed that high intracellular levels of MTA resulted in an inhibition of PRMT activity in melanoma cells [12] that subsequently altered a number of cellular signalling pathways, including extracellular-signal-regulated kinase (ERK) signalling [12,13]. Additional studies by our group revealed that the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), matrix metalloproteinase 9 (MMP9) and MMP14 was affected by MTA and that MTA induced activator protein-1 (AP-1) activation [4]. These data suggest that MTA directly modulates intracellular signalling. In this study, we characterised MTA-induced signalling pathways and focused on possible extracellular receptors and intracellular pathways that are involved in the modulation of AP-1.

2. Material and methods 2.1. Cell lines and culture conditions The melanoma cell lines Mel Ei, Mel Ho, Mel Juso (derived from primary cutaneous melanomas), HTZ19d, Mel Ju and Mel Im (derived from metastases of malignant melanomas) have been described previously

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[3]. NHEMs (normal human epidermal melanocytes) derived from normal skin of different donors were cultivated in melanocyte growth medium (Promocell, Heidelberg, Germany). Melanoma cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI1640 (Sigma–Aldrich, Deisenhofen, Germany) supplemented with penicillin (400 U/mL), streptomycin (50 lg/mL) (both PAA, Pasching, Austria) and 10% foetal calf serum (FCS; PAN Biotech GmbH, Aidenbach, Germany). All cell lines were incubated in a humidified atmosphere containing 8% CO2 at 37 °C and split at a 1:5 ratio every 4 days. 2.2. RNA isolation and reverse transcription Total cellular RNA was isolated from cultured cells using the E.Z.N.A.Ò Total RNA Kit I (Omega Bio-Tek, VWR, Darmstadt, Germany) according to the manufacturer’s instructions. cDNAs were generated in reverse transcriptase reactions (500 ng of total RNA) using the SuperScript II Reverse Transcriptase Kit (Invitrogen, Groningen, the Netherlands) [12]. 2.3. Analysis of gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) Quantitative real time-PCR analysis of ADORA receptors was performed using specific primers (Sigma–Aldrich, Table 1) and a Lightcycler 480 (Roche, Mannheim, Germany) instrument as described previously. The annealing and melting temperatures were optimised for each primer set. Beta-actin was amplified to ensure cDNA integrity and to normalise expression. Each real-time PCR was performed in duplicates. All experiments were repeated at least three times. 2.4. Transfection experiments and luciferase measurements The reporter plasmids AP-1-Luc and CRE-Luc were used (Promega, Mannheim, Germany) for transient transfections. Cells (2  105 per well) were seeded into 6-well plates and transfected with 0.5 lg of the luciferase reporter construct using Lipofectamine LTX reagent (Life Technologies, Darmstadt, Germany) according to the manufacturer’s instructions. Twenty-four hours later, the cells were lysed, and the lysate luciferase activities were quantified using the Dual-LuciferaseÒ Reporter Assay (Promega, Mannheim, Germany) and a Centro LB 960 Microplate Luminometer (Berthold Technologies, Bad Wildbad, Germany). Transfection efficiency was normalised to the Renilla luciferase activity by co-transfection of 50 ng of the pRL-TK plasmid (Promega, Mannheim, Germany) [14]. Knockdowns were performed using FlexiTube siRNA (Hs_ADORA2B_6 and Hs_ADORA2B_7) for

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K. Limm et al. / European Journal of Cancer 50 (2014) 2714–2724

Table 1 Primers used for quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Name

Forward sequence

Reverse sequence

ADORA1 ADORA2A ADORA2B ADORA3 b-actin Interleukin 6 (IL-6) Vascular endothelial growth factor (VEGF)

50 -CTCGCCATCCTCATCAACATT-30 50 -CTCTTCATTGCCTGCTTCGTC-30 50 -AAGAGTGGGAATGGTCAGGC-30 50 -TGAGCAAAGCGTCAACTCG-30 50 -CTACGTGGCCCTGGACTTCGAGC-30 50 -AAGCGCCTTCGGTCCAGTTGC-30 50 -CAGCGCAGCTACTGCCATCCAATCGAGA-30

50 -GCAGCACCCACACAAAGAA-30 50 -TGGTTCTTGCCCTCCTTTGG-30 50 -AGTGCTCAAGAGAGGCAGTC-30 50 -AGTGGCATAGAGAAGGCTTCG-30 50 -GATGGAGCCGCCGATCCACACGG-30 50 -TGTCTGTGTGGGGCGGCTACA-30 50 -GCTTGTCACATCTGCAAGTACGTTCGTTTA-30

ADORA2B and control siRNA purchased from Qiagen (Hilden, Germany). The siRNA transfection assays were performed using the RNAiMAX reagent (Life Technologies, Darmstadt, Germany) according to the manufacturer’s instructions. 2.5. Chemicals Treatments for analysing signalling cascades via AP-1 luciferase assay: protein kinase C (Ro-31-8220, 1 lM, Calbiochem, Darmstadt, Germany/Bisindolylmaleimide II, 20–80 nM, Santa Cruz, CA/Go¨-6983 5–60 nM, Santa Cruz, CA), Rho (Y-27632, 10 lM, Calbiochem, Billerica, MA), c-Jun N-terminal kinase (JNK) (SP600135, 10 lM, Calbiochem, Darmstadt, Germany), mitogen-activated protein kinase kinase 1/2 (MEK1/2) (UO126, 30 lM, Calbiochem, Darmstadt, Germany), p38 (SB202190, 10 lM, Santa Cruz, CA), protein kinase B (AKT) (AktVI, 1 lM, Calbiochem, Darmstadt, Germany) and tyrosine kinases (Genistein, 10 lM, Calbiochem, Darmstadt, Germany); MTA (100 lM, Sigma–Aldrich, Schnelldorf, Germany), adenosine (Ado, 100 lM, Sigma–Aldrich, Schnelldorf, Germany), Bay60-6385 (Bay60,10 lM) Tocris Bioscience, Bristol, United Kingdom), alloxazine (Allox, 50 lM, Sigma–Aldrich, Schnelldorf, Germany) or forskolin (For, 10 lM, LC Laboratories, Woburn, MA). 2.6. Western blot analysis 3  106 cells were lysed in 200 lL radioimmunoprecipitation assay (RIPA)-buffer (Roche) and incubated for 15 min at 4 °C. Insoluble fragments were removed by centrifugation at 13,000 rpm for 10 min at 4 °C. The supernatant of the lysate was stored at –20 °C. Ten micrograms of RIPA-cell lysates was loaded, separated on 12.75% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gels and subsequently blotted onto a polyvinylidene difluoride (PVDF) membrane (45 min, 15 V). After blocking for 1 h with 3% BSA (bovine serum albumin)/phosphate buffered saline (PBS) the membrane was incubated for 16 h at 4C with the primary antibodies against anti-adenosine A2B (1:3000, AB1589P, Chemicon International, Temecula, CA), which was a generous gift from Prof. Dr. Marina Kreutz (Department of Internal Medicine III, University

Hospital Regensburg, Germany) or anti-b-actin (Sigma– Aldrich). Afterwards the membrane was washed three times in PBS, incubated for 1 h with a horseradish peroxidase-conjugated secondary anti-rabbit or alkaline phosphatase-conjugated secondary anti-mouse antibody (1:3000, Cell Signalling Technology) in PBS and then washed again. Finally, immunoreactions were visualised by Claritye Western ECL Substrate Reagent (Bio-Rad Laboratories GmbH, Munich, Germany) or NBT/BCIP (Invitrogen) staining reaction according to the manufacturer’s instructions. All western blots were repeated three times. 2.7. Cyclic adenosine monophosphate (cAMP)-enzymelinked immunosorbent assay (ELISA) A direct cAMP-ELISA kit (Enzo Life Sciences, Lorrach, Germany) was used for quantification of intracellular concentrations of the secondary messengers. The cells (2.5  106) were seeded in a T25 flask and treated 16 h later with MTA, adenosine, Bay60-6385, alloxazine or forskolin (concentrations are given in the figure legends) for 4 h. The cells were subsequently washed twice with PBS. The cells were lysed in 500 lL of 0.1 M HCl for 15 min and afterwards scraped off of the plate with a cell scraper. Insoluble fragments were removed by centrifugation at 13,200 rpm for 10 min. Aliquots of the supernatants were stored at –80 °C until the measurements were made. The sensitive variant (concentration range 0.078–20 pmol/mL) was used for cAMP detection according to the manufacturer’s instructions. Measurements of optical density values were performed with an ELISA reader (BioRad) at 405 nm. 2.8. Calcium imaging The experiments were performed using a live cell imaging setup based on an inverse microscope (ZEISS Axio Observer Z.1) equipped with Fluar 40/1.3 and EC Plan-Neofluar 20/0.50 Ph2 objective lenses (ZEISS, Jena, Germany). Fura 2-AM (Life Technologies, Darmstadt, Germany)-loaded cells (2 lM, 45 min at 37 °C) were illuminated with light at 340 or 380 nm (BP 340/30 HE, BP 387/15 HE) using a fast wavelength switching and excitation device (Lambda DG-4, Sutter

K. Limm et al. / European Journal of Cancer 50 (2014) 2714–2724

Instrument, Novato, United States of America), and fluorescence was detected at 510 nm (BP 510/90 HE and FT 409) using an AxioCam MRm CCD camera (ZEISS, Jena, Germany). ZEN 2012 software (ZEISS, Jena, Germany) was used to control the hardware and to acquire the data. Ca2+ signals were displayed as the ratio of the fluorescence intensity due to the excitation with 340 nm or 380 nm (F340/F380) [15].

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The enzyme MTAP is deregulated in melanoma cells and results in the intra- and extracellular accumulation of the metabolite MTA [3,4,9,10,12,16]. There are some reports about MTA and its effect on cellular functions [3,12,13,17–19], however the specific mode of action of this metabolite is not clearly understood.

in the range of 0.078–20 pmol/mL using a direct cAMP ELISA assay (Fig. 2A). Stimulation of the tumour cells with MTA, adenosine (Ado), the A2B-specific agonist Bay60, the A2B-specific antagonist Allox or forskolin (For) (adenylate cyclase activator) showed that only forskolin treatment led to an obvious increase in cAMP levels. To further analyse the CREB-1 pathway, cells were transfected with a CRE-luciferase reporter construct, treated with MTA, Ado and Bay60, and compared to control-treated cells (concentrations are given in the figure legends). MTA again had no effect on CREB-1 signalling in both cell lines. Treatment with Ado and Bay60 resulted in an increased cAMP responsive element (CRE) activation (Fig. 2B). Forskolin and TPA, which were used as control treatments in this assay, significantly increased CRE activity. Modulation of the ADORA receptor A2B is also associated with the regulation of Ca2+ signalling [22]. Calcium imaging experiments using Fura-2 were performed to study the effects of MTA on intracellular Ca2+ modulation in HTZ-19d (Fig. 2C, I) and Mel Ju (Fig. 2C, II) melanoma cells. Ionomycin (Santa Cruz) was used as a control. No significant effects of extracellular MTA on intracellular Ca2+ levels were observed. Similar outcomes were detected with Bay60 and Ado (not shown), whereas application of the Ca2+ ionophore ionomycin generated a marked signal.

3.1. Expression of ADORA receptors in melanoma cell lines

3.3. Importance of ADORA2B receptor for AP-1 activation

The structural similarity between adenosine and MTA led us to speculate about a functional implication of adenosine receptors in MTA signalling. Four family members of adenosine receptors are known, including the adenosine receptors ADORA1 (A1) and ADORA3 (A3), which are involved in inhibitory G-protein (Gi)coupled receptor signalling, and the receptors ADORA2A (A2A) and ADORA2B (A2B), which lead to the activation of stimulatory (Gs)-coupled receptor signalling [20]. We therefore analysed the mRNA expression levels of all of the ADORAs in five different melanoma cell lines (Mel Ju, Mel Im, Mel Juso, Mel Ei and HTZ-19d) in comparison to normal melanocytes (NHEM). We found low expression levels of A1 and A3 in all of the melanoma cell lines, and in most of the cell lines only low levels of expression of A2A were observed in comparison to NHEMs. ADORA2B showed the highest mRNA expression level (Fig. 1).

Previous studies showed that MTA treatment led to significantly higher AP-1 activity [3]. Based on these results, we analysed a link between activation of this transcription factor and increased expression of A2B in our melanoma cells. The cells were transfected with an AP-1 luciferase plasmid and then treated with MTA, Ado or Bay60 to identify an association of the adenosine receptor A2B with the activation of the transcription factor AP-1. The results showed that activation of AP-1 signalling occurred with all three compounds (Fig. 3A). Cells were then pre-incubated for 10 min with the specific A2B antagonist Alloxazine before subsequently stimulating with MTA, Ado or Bay60. This pretreatment resulted in a reduced activation of AP-1 in Mel Ju and HTZ-19d cells upon all treatments (concentrations are presented in the figure legends) (Fig. 3A). AP-1 luciferase assays were also performed using a siRNA against the A2B receptor. Knockdown was confirmed by analysis of the mRNA level of the A2B receptor. Reduced mRNA level of ADORA2B was determined by qRT-PCR (Fig. 3B). The knockdown of A2B receptor was detected by western blot analysis (Fig. 3C). The control siRNA- and siA2B-transfected cells were transfected with the AP-1 luciferase plasmid. After treatment with MTA, Ado or Bay60, an increase

2.9. Statistical analysis The results are expressed as either the mean ± S.E.M. (range) or the percent. Comparison between groups was made using paired and unpaired Student’s t-tests. A p-value

The metabolite 5'-methylthioadenosine signals through the adenosine receptor A2B in melanoma.

Several recent studies have shown evidence supporting the general knowledge that tumour cells exhibit changes in metabolism. It is becoming increasing...
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