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Deregulation of protein methylation in melanoma

European Journal of Cancer, 6, 49, pages 1305 - 1313

Abstract

Loss of methylthioadenosine phosphorylase (MTAP) expression and a concomitant accumulation of 5′-methyl-thioadenosine (MTA) characterise several tumour entities including malignant melanoma. MTA affects cellular signalling, proliferation and migration not only of cancer but also surrounding cells including lymphocytes and stromal fibroblasts. The mode of action of MTA is still not known. Interestingly, MTA is a known potent inhibitor of protein arginine methyltransferases (PRMTs) and is used as a tool in studying activity and impact of PRMTs. This study aimed at analysing PRMTs in melanoma and the potential impact of MTA on tumourigenesis.

Our findings demonstrate that expression of PRMT4/CARM1 and PRMT6 is deregulated in melanoma, whereas expression of the remaining PRMTs stays unchanged. General PRMT activity and, consequently, symmetric and asymmetric protein methylation are reduced significantly in melanoma cells and tissues. This is due to a loss of MTAP expression and accumulation of MTA. Reduction of protein methylation by MTA affects cell signalling and leads, for example, to an activation of extracellular signal-regulated kinase (ERK) activity. The effects of endogeneous MTA on PRMTs as presented in this study can strongly support the migratory and invasive phenotype of melanoma cells.

Keywords: PRMT, Methylthioadenosine, Melanoma, MAPK/ERK signalling, Protein arginine methylation.

1. Introduction

Protein methylation is one of the most abundant protein modifications. 1 It has been estimated that up to 3% of arginine residues of cellular proteins can be dimethylated. Arginine methylation is a protein modification catalysed by protein arginine N-methyltransferases (PRMTs). It was thought to be irreversible until the recent discovery of the first protein demethylase JMJD6. 2

Arginine residues can be mono- or dimethylated, and the latter can be asymmetrical (both methyl groups on the same N-atom at the end of the arginine side chain) or symmetrical (one methyl group on each of the two terminal N-atoms), depending on the type of methyltransferase. 3 PRMTs are generally classified as either type I or type II enzymes; type I PRMTs catalyse the formation of both monomethyl arginine (MMA) and asymmetric dimethyl arginine (ADMA), whereas type II PRMTs catalyse the formation of MMA and symmetric dimethyl arginine (SDMA). In humans, nine highly related isoenzymes have been found, five type I enzymes (PRMT1, 3, 4, 6 and 8), three type II (PRMT5, 7 and 9) and one unclassified (PRMT2). 4

Arginine methylation is implicated in signal transduction, RNA transport and RNA splicing and, thereby, influences cellular growth, protein shuttling, differentiation and embryogenesis, and post-transcriptional gene regulation. In addition to the important roles of PRMTs in normal cellular function, the methyltransferase activities of several PRMTs are dysregulated in human disease. 4 For example, PRMT4/CARM1 was shown to be aberrantly expressed in prostate cancer and to contribute to the proliferation of prostate cancer cells.5 and 6 PRMT1 was shown to be associated with a shorter disease free survival 7 and to impact inhibition of apoptosis 8 in breast cancer patients. Further, PRMT5 affects tumour growth of, e.g. lymphoma or mamma carcinoma.9 and 10 Here, regulation of p53, HIF1 or PCD4 was demonstrated.9, 11, 12, and 13

Multiple studies have revealed that the accumulation of MTA is associated with the loss of MTA phosphorylase (MTAP) expression in tumours.14, 16, 17, 18, 19, 20, 21, 22, and 23 Although this relationship is known, this is the first study employing melanoma as a model system to investigate the impact of the reduced MTAP expression on endogenous PRMT activity and protein methylation.

2. Materials and methods

2.1. Cell lines and culture conditions

The melanoma cell lines Mel Ei, Mel Wei, Mel Ho, Mel Juso (derived from primary cutaneous melanomas), HTZ-19d, Mel Ju, SK-MEL-3, SK-MEL-28, Mel Im and A375 (derived from metastases of malignant melanomas) have been described previously. 24 Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (PAA, Pasching, Austria) supplemented with penicillin (400 U/mL), streptomycin (50 μg/mL) (both PAA), and 10% foetal calf serum (FCS; Sigma, Deisenhofen, Germany). Stable or transiently transfected HTZ-19d and Mel Im cell clones were grown in selection medium, containing 50 μg/mL G418 (Sigma-Aldrich, Taufkirchen, Germany).17 and 19 Normal human epidermal melanocytes (NHEM) derived from normal skin of different donors were cultivated in melanocyte growth medium (Promocell, Heidelberg, Germany). All cells were incubated in humidified atmosphere containing 8% CO2 at 37 °C. MTA was obtained from Sigma.

2.2. Tissue samples

Snap-frozen tissue samples of primary tumours (TB62, TB71, TB72, TB97) and metastatic melanomas (TB12, TB43, TB100: all skin metastases; TB50: lung metastasis; TB69: brain metastasis) for qRT-PCR were obtained from the tissue collection of the Institute of Pathology, University of Regensburg, Germany. 25 Sampling and handling of human tissue material was carried out in accordance with the ethical principle of the Declaration of Helsinki.

2.3. RNA isolation and reverse transcription

Total cellular RNA was isolated from cultured cells using the E.Z.N.A.® Total RNA Kit I (Omega Bio-tek, VWR, Darmstadt, Germany) according to the manufacturer’s instructions. cDNAs were generated by reverse transcriptase reaction (500 ng of total RNA) using SuperScript II Reverse Transcriptase Kit (Invitrogen, Groningen, the Netherlands). 26

2.4. Expression analysis

qRT-PCR (quantitative Real Time-PCR) analysis of PRMT1-9 was performed using specific primers ( Table 1 ). The PCR reaction was performed in a 50-μL reaction volume containing 5 μL 10× Taq-buffer, 1 μL of cDNA, 0.5 μL of each primer (20 mM), 0.5 μL of dNTPs (10 mM), 0.5 Units of Taq polymerase and 42 μL of water. The amplification reactions comprised 32 cycles of 1 min at 94 °C, 1 min at 62 °C and a final extension step at 72 °C for 1.5 min. The PCR products were resolved on 1.5% agarose gels. All experiments were repeated at least three times.

Table 1 Primers used for quantitative RT-PCR analysis.

Name Nucleotide sequence
Forward primer reverse primer
β-Actin 5′- CTACGTGGCCCTGGACTTCGAGC-3′ 5′- GATGGAGCCGCCGATCCACACGG-3′
PRMT1 5′- TGTCCTGTGGCCAGGCGGAA-3′ 5′- GAGCCGACGTCCAGCACCAC-3′
PRMT2 5′- TCCAGAGCCTGCAGGAGGGG-3′ 5′- CAGCCCAGCTCAGAGCCACA-3′
PRMT3 5′- CTTCGCCCCAGCTTTA-3′ 5′- TACGGGCATTATGGGAT-3′
PRMT4 5′- CACACCGACTTCAAGGACAA-3′ 5′- AAAAACGACAGGATCCCAGA-3′
PRMT5 5′- TGGCTTTGCCGGCTACTTTGAGAC-3′ 5′- TGCATCGCCAGAAACGCACAC-3′
PRMT6 5′- TCCTGCCGGGACCAGTGGAG-3′ 5′- ACCGCCCTCCTTCAGCCACT-3′
PRMT7 5′- GCTGCCGTGGCACAACCTCT-3′ 5′- GTCACCACAGGGGCTCCGGA-3′
PRMT8 5′- AAGCCCGTGCAATGCGTCCAT-3′ 5′- TCCGGTAAGTGAGAGTCCGCACC-3′
PRMT9 5′- GGGTGACAACTGGCAGCACTCC-3′ 5′- TGTCCTCCCCAGATTTTGTTGCGT-3′

2.5. Transfection experiments

Cells (2 × 105 per well) were seeded into 6-well plates and transfected with 0.5 μg MTAP expression constructs 19 using the lipofectamine method (Invitrogen, Carlsbad, CA, United States of America (USA)) according to the manufacturer’s instructions. Subsequent experiments were performed 48 h after transfection. All transfection experiments were repeated at least three times.

2.6. Western blot analysis

3 × 106 cells were lysed in 200 μL 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 immediately shock frozen and stored at −80 °C. Forty micrograms of RIPA-cell lysates was loaded, separated on 10% SDS–PAGE gels and subsequently blotted onto a PVDF membrane (35 min, 15 V). After blocking for 1 h with 3% BSA (bovine serum albumin)/PBS the membrane was incubated for 16 h at 4 °C with the primary antibodies against dimethyl-arginine, symmetric (SYM10) and asymmetric (ASYM24) (1:80; Millipore, Schwalbach, Germany), MTAP (1:200, Abcam) or pERK1/2/ERK1/2 (1:2000, Sigma) in 3% BSA/PBS. Afterwards the membrane was washed three times in PBS, incubated for 1 h with a horseradish peroxidase-conjugated or alkaline phosphatase-conjugated secondary anti-rabbit antibody (1:4000 or 1:2000, Cell Signaling Technology) in 3% BSA/PBS and then washed again. Finally, immunoreactions were visualised by Amersham ECL Plus™ Western Blotting Detection Reagent (GE Healthcare Europe GmbH, Freiburg, Germany) or NBT/BCIP (Invitrogen) staining reaction according to the manufacturer’s instructions. All western blots were repeated three times.

2.7. Immunohistochemical staining

Paraffin sections of healthy skin, naevi, primary tumours and metastases were screened for protein methylation by immunohistochemistry. 25 The tissues were fixed and, subsequently, incubated with specific primary antibodies Anti-dimethyl-Arginine, symmetric (SYM10) and asymmetric (ASYM24) (1:200; Millipore, Schwalbach, Germany) or anti-MTAP (Abcam, Cambridge, USA) overnight at 4 °C, with the secondary antibody (biotin-labelled anti-rabbit; DAKO, Hamburg, Germany) for 30 min at room temperature, and then with streptavidin-POD (DAKO) for 30 min. Antibody binding was visualised using DAB solution (DAKO). Finally, tissues were counterstained with hemalaun solution (DAKO).

2.8. Statistical analysis

Results are expressed as mean ± SEM (range) or percent. Comparison between groups was made using Student’s unpaired t-test. A p-value <0.05 was considered statistically significant (ns: not significant). All calculations were performed using the GraphPad Prism software (GraphPad software Inc., San Diego, USA).

3. Results

3.1. PRMT4 expression is increased in melanoma development

For malignant melanoma no reports on PRMT expression are available. Therefore, we first established quantitative RT-PCR to determine expression of PRMT1 through 9 in melanoma cell lines and tissues relative to β-actin as housekeeper. RNAs of 10 different melanoma cell lines (Mel Ei, Mel Wei, Mel Ho, Mel Juso, HTZ-19d, Mel Ju, SK-MEL-3, SK-MEL-28, Mel Im and A375) were analysed ( Fig. 1 A). With the exception of PRMT8, all PRMTs were found expressed; PRMT1 and 5 showed strongest mRNA expression. All cell lines except of A375 showed similar expression levels. Next, we compared the expression of PRMTs in tissue samples of primary melanomas (n = 4) and metastases (n = 5) with that in cultured normal human epidermal melanocytes (NHEM) ( Fig. 1 B). Expression of most PRMTs was unchanged in primary tumours and metastases compared to normal melanocytes. Only PRMT4/CARM1 was significantly induced during tumour development, whereas PRMT6 showed reduced expression in primary melanoma and metastases. Analysis of published array-based gene expression data using www.genevestigator.com supports our finding that no major regulation of PRMTs can be found in melanoma ( Fig. 1 C-I). To better illustrate the comparative lack of regulation of PRMTs, the approximate threefold upregulation of MIA (melanoma inhibitory activity), a protein strongly induced in melanoma, 27 is shown in Fig. 1 C-II.

gr1

Fig. 1 Expression of PRMTs in melanoma. Averages and standard errors of mRNA expression ratios of the protein arginine methyltransferases PRMT1 to PRMT9 relative to β-actin were determined by quantitative RT-PCR in n = 10 melanoma cell lines (Mel Ei, Mel Wei, Mel Juso, Mel Ho, Mel Im, Mel Ju, HTZ-19d, SK-MEL-3, SK-MEL-28 (all shown as circles) and A375 (shown as square)) (A), and in tissue samples of primary melanoma (PT, n = 4) and melanoma metastases (MET, n = 5) compared to normal human epidermal melanocytes (NHEM, n = 2) (B). (C) Bioinformatic analysis of published array-based expression data using www.genevestigator.org showed only weak regulation of PRMTs in melanoma samples compared to normal human tissue, while expression of MIA was strongly upregulated in melanoma. (Range of expression: PRMT1: 0.028–0.094, PRMT2: 0.0022–0.011, PRMT3: 0.0017–0.0184, PRMT4: 0.0005–0.0052, PRMT5: 0.014–0.089, PRMT6: 0.00014–0.00187, PRMT7: 0.0006–0.0053, PRMT8: not expressed (all relative to β-actin), p < 0.05, ns: non-significant).

3.2. Symmetric and asymmetric methylation is reduced in melanoma

We then performed immunohistochemistry for symmetric and asymmetric arginine dimethylation using specific antibodies to determine the effect of PRMTs on protein methylation in melanoma in situ ( Fig. 2 ). Interestingly, a reduction in staining for both symmetric and asymmetric methylation was observed in primary melanoma (PT) and metastases (MET) compared to normal skin and naevus. Further, reduction in protein methylation correlated with loss of MTAP expression. This correlation was also found in melanoma cell lines ( Fig. 3 ). Compared to NHEM, reduction in total protein methylation, both symmetric ( Fig. 3 A) and asymmetric ( Fig. 3 B), was observed in 4 of 5 melanoma cell lines, namely Mel Wei, Mel Juso, Mel Ju and Mel Im. The remaining cell, A375, expressed MTAP at a level similar to NHEM and, concordantly, protein methylation was less reduced than in the other cell lines. The bar graph displays the mean ± SEM of densitometric measurements ( Fig. 3 C).

gr2

Fig. 2 Immunohistochemical analysis of protein methylation in melanoma. Immunohistochemistry revealed reduced symmetric (SYM10) and asymmetric (ASYM24) protein methylation in primary melanoma (PT) and metastasis (MET) concomitant with loss of MTAP expression. In healthy skin and nevi protein methylation was strong.

gr3

Fig. 3 Protein methylation in melanocytes and melanoma cell lines. Immunoblotting showed significantly reduced symmetric (SYM10, A) and asymmetric (ASYM24, B) methylation of arginine residues of proteins extracted from melanoma cell lines (Mel Wei, Mel Juso, Mel Im, Mel Ju, A375) compared to NHEM in passage 5 (I) and passage 10 (II). (C) MTAP expression levels were determined in the same samples. β-Actin was used as a loading control. (p < 0.05).

3.3. Induction of methylation after MTAP re-expression

To confirm the apparent correlation between MTAP/MTA and protein methylation, we generated clones of the melanoma cell lines HTZ-19d and Mel Im that were stably or transiently transfected with MTAP and determined the effects of MTAP re-expression on methylation ( Fig. 4 ). A clear induction of symmetric ( Fig. 4 A) and asymmetric ( Fig. 4 B) protein methylation was found upon MTAP re-expression. Effects were more pronounced in the two stably transfected HTZ-19d cell clones ( Fig. 4 A-I and B-I) than in the transiently transfected Mel Im and HTZ-19d melanoma cells ( Fig. 4 A-II and B-II).

gr4

Fig. 4 Protein methylation is dependent on MTAP expression. After re-expression of MTAP, induction of symmetric (SYM10, A) and asymmetric (ASYM24, B) protein methylation was observed in the melanoma cell lines HTZ-19d and Mel Im. For analysis of HTZ-19d, two stable cell clones 19 (A-I and B-I) as well as transiently transfected cells (A-II, B-II) were used. For analysis of Mel Im, only transiently transfected were employed (A-II, B-II). β-Actin was used as a loading control.

3.4. Extracellular signal-regulated kinase (ERK) activity correlates with MTA levels in melanoma

In a recent study, protein methylation was shown to modulate ERK signalling in cells. 28 Therefore, we analysed the effects of re-expression of MTAP and administration of MTA on ERK phosphorylation. Activity of ERK was clearly dependent on MTA levels in melanoma ( Fig. 5 ). Stable ( Fig. 5 A) or transient ( Fig. 5 B and C) re-expression of MTAP resulted in inhibition of ERK phosphorylation. Incubation of Mel Im cells transiently re-expressing MTAP with 0.1 mM MTA resulted in re-induction of ERK phosphorylation and complete omission of the MTAP effect ( Fig. 5 C). These findings are in agreement with effects of MTA on AP1 activity, which have been published previously.

gr5

Fig. 5 ERK activity is dependent on MTAP expression. Strong reduction in ERK activity was observed in the melanoma cell line HTZ-19d after stable (2 cell clones) (A) and transient (B) re-expression of MTAP by Western Blot. (C) Treatment of MTAP re-expressing Mel Im cells with MTA resulted in ERK re-activation. ERK1/2 was used as a loading control. The transfections as well as the validation of the pERK-level were repeated more than three times.

4. Discussion

It is known that arginine methylation exerts a great impact on protein function and, thereby, on cellular processes such as proliferation, apoptosis and migration.1, 4, and 28 Not surprisingly, deregulation of PRMT expression has been shown in tumours of different origin.7, 9, and 10 We revealed differential regulation of 2 of the 9 PRMTs with PRMT4/CARM1 being strongly upregulated and PRMT6 showing reduced expression in melanoma compared to melanocytes. Upregulation of PRMT4/CARM1 was also shown in prostate cancer, breast cancer and colon carcinoma.5, 6, 29, and 30 Downregulation of PRMT6 expression was not previously observed in cancer.

In earlier studies, our group focused on methylthioadenosine phosphorylase (MTAP) in tumourigenesis and revealed strong downregulation of MTAP expression in melanoma and hepatocellular carcinoma.18 and 19 In addition, other groups revealed loss of MTAP in gastric cancer, non-small cell lung carcinoma, pancreatic cancer and several more.22, 31, and 32 Due to lack of MTAP expression, processing of the metabolite MTA is impaired. This leads to the intra- and extracellular accumulation of MTA, with reported intracellular levels of MTA of approx. 100 μM.17 and 33

Interestingly, MTA has been described as a potent inhibitor of PRMT activity with a Ki = 62 μM. 15 Concentrations used to treat cells ranged from 100 μM to 3 mM, albeit intracellular concentrations of MTA under treatment have not been determined. We therefore hypothesised that in MTAP deficient cells activity rather than expression of PRMTs should be strongly reduced due to accumulation of MTA. In agreement with this hypothesis we succeeded in demonstrating that re-expression of MTAP, which had been shown to result in an approximately 5-fold reduction in intracellular MTA concentrations, 17 led to increased protein methylation.

Potentially, strong overexpression of specific PRMTs, such as PRMT4 in melanoma, may also result in increased activity of this PRMT. PRMT4 is known to catalyse methylation of only a few distinct substrates, and it has transcriptional coactivator activity itself. 4 It was shown to methylate mRNA stabilizing proteins having a putative role in post-transcriptional gene regulation. Further, it is involved in STAT5- and NFκB-dependent gene expression,3 and 34 two signalling pathways of great importance in melanoma. However, the effects of PRMT4/CARM1 on NFκB-dependent gene expression are independent of its catalytic activity. 34 As we revealed that activity of PRMTs is reduced in tumour cells by accumulation of MTA, potentially also other PRMTs have functions independent of their catalytic activity and, thus, are still modified by changes of expression.

The activity of ERK can be modulated by protein methylation of CRAF and BRAF. As shown recently, PRMT5-dependent methylation enhances the degradation of activated CRAF and BRAF, thereby reducing their catalytic activity. 28 Interestingly, in our study no induction of PRMT5 expression was found. However, we could show that induction of PRMT activity upon MTAP re-expression results in strong downregulation of ERK phosphorylation. This is in agreement with the important function of BRAF signalling in melanoma. Interestingly, the regulation found seems to be independent of the BRAF mutation V600E as the regulation of ERK activity was found in all melanoma lines independent of BRAF mutations.

In summary, our study demonstrates that loss of MTAP expression in melanoma cells results in a significant general reduction in protein methylation via accumulation of MTA. This exemplifies that changes in metabolism may result in global cellular changes supporting tumour development and aggressiveness.

Conflict of interest statement

None declared.

Acknowledgements

We are indebted to Rudolf Jung for excellent technical assistance. This work was supported by grants from the Medical Faculty of the University of Regensburg (ReForM) and the German Research Foundation (SFB 960 and KFO 262).

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Footnotes

a Institute of Pathology, University of Regensburg, Germany

b Institute of Functional Genomics, University of Regensburg, Germany

c Department of Internal Medicine I, University Hospital of Regensburg, Germany

lowast 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.

d These authors contributed equally to this work.