Expression of DNA Methyltransferase 1 Is a Hallmark of Melanoma, Correlating with Proliferation and Response to B-Raf and Mitogen-Activated Protein Kinase Inhibition in Melanocytic Tumors
2020; Elsevier BV; Volume: 190; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2020.07.002
ISSN1525-2191
AutoresMaximilian Gassenmaier, Maximilian Rentschler, Birgit Fehrenbacher, Thomas Eigentler, Kristian Ikenberg, Corinna Kosnopfel, Tobias Sinnberg, Heike Niessner, Hans Bösmüller, Nikolaus B. Wagner, Martin Schaller, Claus Garbe, Martin Röcken,
Tópico(s)Genomics and Chromatin Dynamics
ResumoAberrant DNA methylation is an epigenetic hallmark of melanoma, but the expression of DNA methyltransferase (Dnmt)-1 in melanocytic tumors is unknown. Dnmt1 expression was analyzed in primary melanocytes, melanoma cell lines, and 83 melanocytic tumors, and its associations with proliferation, mutational status, and response to B-Raf and mitogen-activated protein kinase kinase (MEK) inhibition were explored. Dnmt1 expression was increased incrementally from nevi [mean fluorescence intensity (MFI), 48.1; interquartile range, 41.7 to 59.6] to primary melanomas (MFI, 68.8; interquartile range, 58.4 to 77.0) and metastatic melanomas (MFI, 87.5; interquartile range, 77.1 to 114.5) (P < 0.001). Dnmt1 expression was correlated with Ki-67 expression (Spearman correlation, 0.483; P < 0.001) and was independent of BRAF mutation status (P = 0.55). In BRAF-mutant melanoma, Dnmt1 was down-regulated during response to B-Raf and MEK inhibition and was again up-regulated on drug resistance in vitro and in vivo. Degradation of Dnmt1 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid was associated with decreased cell viability in B-Raf inhibitor–sensitive and –resistant cell lines. This study demonstrates that Dnmt1 expression is correlated with proliferation in melanocytic tumors, is increased with melanoma progression, and is associated with response to B-Raf and MEK inhibition. Given its strong expression in metastatic melanoma, Dnmt1 may be a promising target for combined epigenetic and immunotherapy. Aberrant DNA methylation is an epigenetic hallmark of melanoma, but the expression of DNA methyltransferase (Dnmt)-1 in melanocytic tumors is unknown. Dnmt1 expression was analyzed in primary melanocytes, melanoma cell lines, and 83 melanocytic tumors, and its associations with proliferation, mutational status, and response to B-Raf and mitogen-activated protein kinase kinase (MEK) inhibition were explored. Dnmt1 expression was increased incrementally from nevi [mean fluorescence intensity (MFI), 48.1; interquartile range, 41.7 to 59.6] to primary melanomas (MFI, 68.8; interquartile range, 58.4 to 77.0) and metastatic melanomas (MFI, 87.5; interquartile range, 77.1 to 114.5) (P < 0.001). Dnmt1 expression was correlated with Ki-67 expression (Spearman correlation, 0.483; P < 0.001) and was independent of BRAF mutation status (P = 0.55). In BRAF-mutant melanoma, Dnmt1 was down-regulated during response to B-Raf and MEK inhibition and was again up-regulated on drug resistance in vitro and in vivo. Degradation of Dnmt1 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid was associated with decreased cell viability in B-Raf inhibitor–sensitive and –resistant cell lines. This study demonstrates that Dnmt1 expression is correlated with proliferation in melanocytic tumors, is increased with melanoma progression, and is associated with response to B-Raf and MEK inhibition. Given its strong expression in metastatic melanoma, Dnmt1 may be a promising target for combined epigenetic and immunotherapy. Epigenetic modifications refer to heritable changes in gene expression that occur independent of changes in the primary DNA sequence.1Sharma S. Kelly T.K. Jones P.A. Epigenetics in cancer.Carcinogenesis. 2010; 31: 27-36Crossref PubMed Scopus (1620) Google Scholar DNA methylation is an essential component of epigenetic modification and provides a stable gene-silencing mechanism that plays an important role in regulating gene expression and chromatin architecture.1Sharma S. Kelly T.K. Jones P.A. Epigenetics in cancer.Carcinogenesis. 2010; 31: 27-36Crossref PubMed Scopus (1620) Google Scholar In mammals, DNA methylation occurs primarily by the covalent addition of methyl groups at cytosine residues in CpG dinucleotides. Site-specific hypermethylation can contribute to tumor development and progression by silencing tumor suppressor genes and has been found in many cancers.1Sharma S. Kelly T.K. Jones P.A. Epigenetics in cancer.Carcinogenesis. 2010; 31: 27-36Crossref PubMed Scopus (1620) Google Scholar,2Roh M.R. Gupta S. Park K.H. Chung K.Y. Lauss M. Flaherty K.T. Jonsson G. Rha S.Y. Tsao H. Promoter methylation of PTEN is a significant prognostic factor in melanoma survival.J Invest Dermatol. 2016; 136: 1002-1011Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar There are significant differences in methylation patterns between melanocytic nevi and melanoma, highlighting that aberrant DNA methylation is an epigenetic hallmark of melanoma.3Conway K. Edmiston S.N. Khondker Z.S. Groben P.A. Zhou X. Chu H. Kuan P.F. Hao H. Carson C. Berwick M. Olilla D.W. Thomas N.E. DNA-methylation profiling distinguishes malignant melanomas from benign nevi.Pigment Cell Melanoma Res. 2011; 24: 352-360Crossref PubMed Scopus (53) Google Scholar, 4Gao L. Smit M.A. van den Oord J.J. Goeman J.J. Verdegaal E.M. van der Burg S.H. Stas M. Beck S. Gruis N.A. Tensen C.P. Willemze R. Peeper D.S. van Doorn R. Genome-wide promoter methylation analysis identifies epigenetic silencing of MAPK13 in primary cutaneous melanoma.Pigment Cell Melanoma Res. 2013; 26: 542-554Crossref PubMed Scopus (36) Google Scholar, 5Micevic G. Theodosakis N. Bosenberg M. Aberrant DNA methylation in melanoma: biomarker and therapeutic opportunities.Clin Epigenetics. 2017; 9: 34Crossref PubMed Scopus (55) Google Scholar DNA methyltransferase (Dnmt)-1 is the major enzyme involved in maintaining methylation patterns after cellular replication, but its expression in melanocytic tumors in unknown.6Bestor T.H. Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain.EMBO J. 1992; 11: 2611-2617Crossref PubMed Scopus (369) Google Scholar Clinical trials of hypomethylating drugs that target Dnmt1 in patients with metastatic melanoma are currently being conducted, and these drugs have recently shown promising immunomodulatory and antitumor activity.7Di Giacomo A.M. Covre A. Finotello F. Rieder D. Danielli R. Sigalotti L. Giannarelli D. Petitprez F. Lacroix L. Valente M. Cutaia O. Fazio C. Amato G. Lazzeri A. Monterisi S. Miracco C. Coral S. Anichini A. Bock C. Nemc A. Oganesian A. Lowder J. Azab M. Fridman W.H. Sautes-Fridman C. Trajanoski Z. Maio M. Guadecitabine plus ipilimumab in unresectable melanoma: the NIBIT-M4 clinical trial.Clin Cancer Res. 2019; 25: 7351-7362PubMed Google Scholar Dnmt1 depletion has been reported to overcome resistance of interferon-induced apoptosis in melanoma cells, to lead to reactivation of cyclin-dependent kinase inhibitor 2A protein in cancer cells, and to trigger senescence in fibroblasts.8Cruickshanks H.A. McBryan T. Nelson D.M. Vanderkraats N.D. Shah P.P. van Tuyn J. Singh Rai T. Brock C. Donahue G. Dunican D.S. Drotar M.E. Meehan R.R. Edwards J.R. Berger S.L. Adams P.D. Senescent cells harbour features of the cancer epigenome.Nat Cell Biol. 2013; 15: 1495-1506Crossref PubMed Scopus (191) Google Scholar, 9Fournel M. Sapieha P. 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Mailhammer R. Schaak K. Ghoreschi K. Yazdi A. Haubner R. Sander C.A. Mocikat R. Schwaiger M. Forster I. Huss R. Weber W.A. Kneilling M. Rocken M. TNFR1 signaling and IFN-gamma signaling determine whether T cells induce tumor dormancy or promote multistage carcinogenesis.Cancer Cell. 2008; 13: 507-518Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 13Braumuller H. Wieder T. Brenner E. Assmann S. Hahn M. Alkhaled M. Schilbach K. Essmann F. Kneilling M. Griessinger C. Ranta F. Ullrich S. Mocikat R. Braungart K. Mehra T. Fehrenbacher B. Berdel J. Niessner H. Meier F. van den Broek M. Haring H.U. Handgretinger R. Quintanilla-Martinez L. Fend F. Pesic M. Bauer J. Zender L. Schaller M. Schulze-Osthoff K. Rocken M. T-helper-1-cell cytokines drive cancer into senescence.Nature. 2013; 494: 361-365Crossref PubMed Scopus (409) Google Scholar, 14Brenner E. Schorg B.F. Ahmetlic F. Wieder T. Hilke F.J. Simon N. Schroeder C. Demidov G. Riedel T. Fehrenbacher B. Schaller M. Forschner A. Eigentler T. Niessner H. Sinnberg T. Bohm K.S. Homberg N. Braumuller H. Dauch D. Zwirner S. Zender L. Sonanini D. Geishauser A. Bauer J. Eichner M. Jarick K.J. Beilhack A. Biskup S. Docker D. Schadendorf D. Quintanilla-Martinez L. Pichler B.J. Kneilling M. Mocikat R. Rocken M. Cancer immune control needs senescence induction by interferon-dependent cell cycle regulator pathways in tumours.Nat Commun. 2020; 11: 1335Crossref PubMed Scopus (20) Google Scholar the role of Dnmt1 in melanocytic tumors was investigated in the present study. In this study, Dnmt1 expression was strongly correlated with the proliferation of melanocytic tumors and was associated with the response of melanoma cells to B-Raf and mitogen-activated protein kinase kinase (MEK) inhibitors. Finally, the translational and therapeutic implications of the findings are discussed. Statistical calculations were performed with SPSS statistics software version 23.0 (IBM, Armonk, NY). Numeric variables are described by median values and interquartile ranges (IQRs) or means ± SD. Graphs were built with JMP software version 14.2.0 (SAS, Cary, NC) and Prism software version 8.4 (GraphPad, San Diego, CA). Throughout the analysis, P values of <0.05 were considered as statistically significant. Melanoma cell lines were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA), which was supplemented with 10% fetal calf serum (Merck, Darmstadt, Germany) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Melanoma cell lines were kindly provided by Meenhard Herlyn (451LU, 1205LU, SbCl2, WM35, WM793, WM1346, WM1366, WM1552, WM115, and Mel1617), by Léon C. van Kempen (MV3), and by Claus Garbe (SKMel19) or were purchased from the ATCC (Manassas, VA; SKMel28, A375, and MeWo). Vemurafenib-resistant cell lines were generated by continuous treatment with an increasing concentration of vemurafenib, up to 2 μmol/L, for several months.15Sinnberg T. Makino E. Krueger M.A. Velic A. Macek B. Rothbauer U. Groll N. Potz O. Czemmel S. Niessner H. Meier F. Ikenberg K. Garbe C. Schittek B. A nexus consisting of beta-catenin and Stat3 attenuates BRAF inhibitor efficacy and mediates acquired resistance to vemurafenib.EBioMedicine. 2016; 8: 132-149Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar The effect of targeted therapy on Dnmt1 expression in vitro was evaluated after treatment with 2 μmol/L vemurafenib and/or 0.1 μmol/L cobimetinib or 2 μmol/L suberoylanilide hydroxamic acid (SAHA) for 24 hours. Stock solutions of the B-Raf inhibitor (B-Rafi) vemurafenib (LC Laboratories, Woburn, MA), the MEK inhibitor (MEKi) cobimetinib (Hycultec, Beutelsbach, Germany), and the histone deacetylase inhibitor SAHA (InvivoGen, San Diego, CA) were prepared in dimethyl sulfoxide. Cell viability was assessed using the 4-methylumbelliferyl heptanoate assay. In brief, 2.5 × 103 cells were seeded into 96-well plate cavities in six replicates 24 hours before treatment with vemurafenib or SAHA (both up to 25 μmol/L for 72 hours). Viability was analyzed after a washing step with phosphate-buffered saline and subsequent incubation of the cells in 100 μg/mL 4-methylumbelliferyl heptanoate (Merck) diluted in phosphate-buffered saline for 1 hour at 37°C. The fluorescence (ex = 355 nm, em = 460 nm) was detected in a Tristar fluorescence microplate reader (Berthold Technologies, Bad Wildbad, Germany). Human primary melanocytes were isolated from human foreskin samples as described16Mancianti M.L. Herlyn M. Weil D. Jambrosic J. Rodeck U. Becker D. Diamond L. Clark W.H. Koprowski H. Growth and phenotypic characteristics of human nevus cells in culture.J Invest Dermatol. 1988; 90: 134-141Crossref PubMed Scopus (44) Google Scholar and cultivated in Dermalife M medium (Cell Systems, Troisdorf, Germany). Melanocytes were expanded until confluency. Cells were lysed in RIPA buffer (50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail (cOmplete and PhosSTOP; Roche Diagnostics, Rotkreuz, Switzerland). After the determination of protein content by the Pierce BCA protein assay kit (Thermo Fisher Scientific), proteins were resolved by 10% SDS-PAGE, transferred onto an Immobilon-FL PVDF transfer membrane (Merck), and blocked with Odyssey or Intercept blocking buffer (LI-COR Biosciences, Lincoln, NE). The membrane was then incubated overnight at 4°C with primary antibodies anti–β-actin (1:5000; catalog number MAB1501R; Merck) and anti-Dnmt1 (1:500; catalog number ab13537; Abcam, Cambridge, UK), or anti–β-actin (1:1000; catalog number 4970; Cell Signaling Technology, Danvers, MA) and anti-proliferating cell nuclear antigen (PCNA; 1:2000; catalog number 2586; Cell Signaling Technology), respectively. After washing with Tris-buffered saline with Tween 20 and subsequent blocking, the blots were incubated for 1 hour at room temperature with fluorescent dye–conjugated secondary antibodies IRDye 800CW goat anti-mouse IgG (1:15,000; catalog number 926-32210) and IRDye 680RD goat anti-rabbit IgG (1:20,000; catalog number 926-68071) (both, LI-COR Biosciences) and washed again. The antibody binding was then detected with the Odyssey Sa Imager (LI-COR Biosciences). Immunoreactive bands were quantified using Image Studio Lite software version 3.1 (LI-COR Biosciences), and the target/reference protein ratio of the samples was calculated. Gene expression information was retrieved from publicly accessible data sets and analyzed with the web-based genomics analysis and visualization platform R2 (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi, last accessed March 30, 2020). In each analysis, DNMT1 expression values were centered around 0 (log2 centered). The following data sets from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) were used: accession numbers GSE3189 (nevi, n = 18; melanoma, n = 45)17Talantov D. Mazumder A. Yu J.X. Briggs T. Jiang Y. Backus J. Atkins D. Wang Y. Novel genes associated with malignant melanoma but not benign melanocytic lesions.Clin Cancer Res. 2005; 11: 7234-7242Crossref PubMed Scopus (377) Google Scholar and GSE116237 (patient-derived xenograft model under treatment with dabrafenib and trametinib; cells analyzed pretreatment, n = 172; 4 days on treatment, n = 155; 28 days on treatment, n = 199; and 57 days on treatment, n = 148).18Rambow F. Rogiers A. Marin-Bejar O. Aibar S. Femel J. Dewaele M. Karras P. Brown D. Chang Y.H. Debiec-Rychter M. Adriaens C. Radaelli E. Wolter P. Bechter O. Dummer R. Levesque M. Piris A. Frederick D.T. Boland G. Flaherty K.T. van den Oord J. Voet T. Aerts S. Lund A.W. Marine J.C. Toward minimal residual disease-directed therapy in melanoma.Cell. 2018; 174: 843-55 e19Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar Formalin-fixed, paraffin-embedded (FFPE) tissue was cut into 2.5-μm–thick sections, deparaffinized, and stained with an anti-Dnmt1 antibody (1:1500; catalog number ab13537; Abcam) on an automated immunostainer (Leica Bond-MAX; Leica Biosystems, Wetzlar, Germany). For immunofluorescence, the following antibodies were used: anti-Dnmt1 (1:100; catalog number ab19905; Abcam), anti-melanoma (1:200; catalog number ab733; Abcam), anti–Ki-67 (1:75; catalog number 14-5698; Thermo Fisher Scientific), Alexa Fluor 488 donkey anti-mouse (1:250; catalog number 715-546-151; Dianova, Hamburg, Germany), Alexa Fluor 647 donkey anti-rat (1:250; catalog number 712-606-153; Dianova), and Cy3 donkey anti-rabbit (1:250; catalog number 711-166-152; Dianova). Nuclei were stained with DAPI (1:2000; catalog number D9542; Sigma-Aldrich/Merck). Immunofluorescence images were obtained with a confocal laser scanning microscope (LSM 800; Carl Zeiss, Jena, Germany), and images were processed and analyzed with ZEN Blue software version 2.3 (Carl Zeiss). At least two representative areas of each melanocytic tumor were examined under the microscope at 250× or 400× magnification, with a median (IQR) of 268 (143 to 450) cells analyzed. The nuclear mean fluorescence intensity values of Dnmt1 and Ki-67 in anti-melanoma antibody–positive cells were measured in a computerized manner (Supplemental Figure S1 and Supplemental Table S1) and were calculated for each individual tumor. Epidermal progenitor cells in the basal and suprabasal layers of the epidermis were used as an internal positive control for the quantification of Dnmt1- and Ki-67–positive cells.19Sen G.L. Reuter J.A. Webster D.E. Zhu L. Khavari P.A. DNMT1 maintains progenitor function in self-renewing somatic tissue.Nature. 2010; 463: 563-567Crossref PubMed Scopus (305) Google Scholar FFPE tissue was retrieved from the archives of the Department of Dermatology and Institute of Pathology, University Hospital Tübingen (Tübingen, Germany). Dnmt1 and Ki-67 expression levels were analyzed in 13 nevi, 35 primary melanomas, and 3 melanoma metastases on a tissue microarray. A total of 32 metastases with known BRAF-mutation status and 12 matched metastases (before treatment, n = 6; time of progression/response, n = 6) from six patients treated with the B-Rafi vemurafenib or the B-Rafi/MEKi combination vemurafenib/cobimetinib were investigated. The study protocol was reviewed and approved by the ethics committee of the University Hospital Tübingen (project number 888/2017BO2). On comparison of Dnmt1 protein expression in melanoma cell lines (n = 12) versus melanocytes isolated from human foreskin samples (n = 3), Dnmt1 expression was significantly higher in the melanoma cell lines [Dnmt1/β-actin ratios (IQR), 0.12 (0.10 to 0.21) versus 0.06 (0.02 to 0.08); P = 0.009 (U-test)] (Figure 1A). To validate this finding in melanocytic tumors, data from available microarray data sets from 18 benign nevi and 45 primary melanomas were analyzed (Figure 1B). DNMT1 RNA expression was up-regulated in the melanomas as compared with the nevi [0.19 (−0.02 to 0.38) versus −0.47 (−0.75 to −0.05) (log2 centered); P < 0.001 (U-test)]. As Dnmt1 is the key enzyme determining DNA methylation after cellular replication, the expression of Dnmt1 during melanoma progression and its association with cellular proliferation were explored. The expression levels of Dnmt1 and Ki-67 in melanocytes from FFPE nevi (n = 13), thin primary melanomas [median (IQR) Breslow thickness, 0.67 mm (0.47 to 0.86); n = 35], and metastases (n = 35) were first determined (Figure 2). Dnmt1 protein levels increased incrementally from nevi to primary melanomas and metastatic melanomas, almost uniformly showing a strong expression [median (IQR) mean fluorescence intensity, 48.1 (41.7 to 59.6), 68.8 (58.4 to 77.0), and 87.5 (77.1 to 114.5), respectively] (Figure 2, A and B). A similar trend was found in the protein expression of the proliferation marker Ki-67, which was increased from nevi to primary melanomas and metastases [mean fluorescence intensity,13.8 (11.9 to 18.0), 23.6 (20.1 to 33.9), and 32.4 (24.1 to 44.0), respectively] (Figure 2C) and showed a strong positive correlation with Dnmt1 expression (Spearman correlation, 0.483; P < 0.001) (Figure 2D). At the single-cell level, most (92.1%) Ki-67–positive melanocytes were also positive for Dnmt1 (Figure 2E). It has been shown that the expression of Dnmt1 is down-regulated by knockdown of BRAF V600E in melanoma cell lines.20Hou P. Liu D. Dong J. Xing M. The BRAF(V600E) causes widespread alterations in gene methylation in the genome of melanoma cells.Cell Cycle. 2012; 11: 286-295Crossref PubMed Scopus (66) Google Scholar Therefore, the Dnmt1 and Ki-67 expression levels in BRAF-mutant and BRAF–wild-type melanoma were analyzed. Both Dnmt1 expression [median (IQR) mean fluorescence intensity, 95.9 (78.8 to 115.7) versus 86.1 (78.1 to 112.3); P = 0.55] and Ki-67 expression [37.1 (24.1 to 45.7) versus 28.0 (24.0 to 42.3); P = 0.69] were similar between BRAF-mutant and BRAF–wild-type metastatic melanoma (U-test) (Figure 3, A and B ). To determine whether Dnmt1 expression could be modulated by pharmacologic inhibition of the mitogen-activated protein kinase (MAPK) pathway, BRAF-mutant melanoma cell lines were treated with the B-Rafi vemurafenib and/or the MEKi cobimetinib in vitro. With both drugs, Dnmt1 and the proliferation marker PCNA were markedly down-regulated in B-Rafi–sensitive but not in B-Rafi–resistant cell lines (Figure 3, C–E). To explore whether B-Rafi and MEKi also affect Dnmt1 expression in vivo, single-cell transcriptome data published by Rambow et al18Rambow F. Rogiers A. Marin-Bejar O. Aibar S. Femel J. Dewaele M. Karras P. Brown D. Chang Y.H. Debiec-Rychter M. Adriaens C. Radaelli E. Wolter P. Bechter O. Dummer R. Levesque M. Piris A. Frederick D.T. Boland G. Flaherty K.T. van den Oord J. Voet T. Aerts S. Lund A.W. Marine J.C. Toward minimal residual disease-directed therapy in melanoma.Cell. 2018; 174: 843-55 e19Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar were analyzed. In that study, patient-derived xenograft models from patients with BRAF V600E/K–mutant melanoma were exposed to B-Raf and MEK inhibition. In brief, 250,000 melanoma cells were injected s.c. into 8- to 16-week–old female NMRI nude mice. Mice with tumors reaching 1000 mm3 were treated daily with the B-Rafi/MEKi combination dabrafenib/trametinib, and tumor volume was monitored with a caliper. All treated lesions shrank (phase 1, day 4) and reached an impalpable size (phase 2, day 28). However, continuous treatment led to the development of drug resistance and regrowth of the implanted tumors (phase 3, day 57). Tumor cells from each phase were isolated, and single-cell RNA sequencing of 674 cells was performed. Single-cell transcriptomics allowed for the analysis of DNMT1 and PCNA expression under treatment. A significant down-regulation of DNMT1 was induced in phases 1 and 2, followed by an up-regulation in the resistance phase 3 (Figure 4A). The same pattern was observed in six patients with Stage IV melanoma treated with targeted therapy in the Department of Dermatology, University of Tübingen (Figure 4B). All of the patients with progression on vemurafenib treatment (n = 3) showed a strong expression of DNMT1 before treatment and at the time of progression. In contrast, all patients with a response to vemurafenib/cobimetinib therapy (n = 3) showed a strong expression of DNMT1 before treatment but a marked decrease at the time of response. The xenograft model also showed that the DNMT1 expression started to increase again earlier (phase 2, day 28) than did PCNA expression (phase 3, day 57) (Figure 4C). This finding suggests that the DNMT1 expression precedes the proliferative activity in melanoma and is not the secondary result of the proliferative state. It has been shown that histone deacetylase inhibitor increases Dnmt1 acetylation and stimulates its degradation.21Cheng J. Yang H. Fang J. Ma L. Gong R. Wang P. Li Z. Xu Y. Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation.Nat Commun. 2015; 6: 7023Crossref PubMed Scopus (75) Google Scholar To investigate whether Dnmt1 is essential for the survival of melanoma cells, B-Rafi–sensitive and –resistant cell lines were treated with the histone deacetylase inhibitor SAHA, and cell viability was measured after 72 hours (Figure 5A). SAHA was associated with impaired cell viability in both B-Rafi–sensitive and –resistant cell lines in a dose-dependent manner. Histone deacetylase inhibitor–mediated Dnmt1 down-regulation was not the consequence of reduced proliferation, as a significant Dnmt1 degradation was observed after 24 hours, when PCNA protein levels were still unchanged (Figure 5, B and C). Dnmt1 maintains methylation patterns after cellular replication and is essential for progenitor cell function and enriched in undifferentiated cells, where it is required for retaining proliferative stamina and the suppression of differentiation.19Sen G.L. Reuter J.A. Webster D.E. Zhu L. Khavari P.A. DNMT1 maintains progenitor function in self-renewing somatic tissue.Nature. 2010; 463: 563-567Crossref PubMed Scopus (305) Google Scholar In line with this finding, Dnmt1 expression was increased in primary and metastatic melanoma, and the correlation between Dnmt1 and the proliferation marker Ki-67 was strong. The close link between Dnmt1 and Ki-67 raises an important question: Is the up-regulation of Dnmt1 of pathogenic relevance for melanoma progression, or the mere reflection of an increased proliferation rate? Studies have reported that Dnmt1 was needed during the S phase in order to maintain the methylation patterns of the newly synthesized strand, but that it is additionally associated with transcription factors during the G0/G1 and G2/M phases.22Sharif J. Muto M. Takebayashi S. Suetake I. Iwamatsu A. Endo T.A. Shinga J. Mizutani-Koseki Y. Toyoda T. Okamura K. Tajima S. Mitsuya K. Okano M. Koseki H. The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA.Nature. 2007; 450: 908-912Crossref PubMed Scopus (828) Google Scholar, 23Easwaran H.P. Schermelleh L. Leonhardt H. Cardoso M.C. Replication-independent chromatin loading of Dnmt1 during G2 and M phases.EMBO Rep. 2004; 5: 1181-1186Crossref PubMed Scopus (129) Google Scholar, 24Hervouet E. Nadaradjane A. Gueguen M. Vallette F.M. Cartron P.F. Kinetics of DNA methylation inheritance by the Dnmt1-including complexes during the cell cycle.Cell Div. 2012; 7: 5Crossref PubMed Google Scholar In 1991, El-Deiry et al25El-Deiry W.S. Nelkin B.D. Celano P. Yen R.W. Falco J.P. Hamilton S.R. Baylin S.B. High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer.Proc Natl Acad Sci U S A. 1991; 88: 3470-3474Crossref PubMed Scopus (289) Google Scholar described that increased Dnmt1 expression characterized human cancer cells and may play a role in the pathogenesis of colon cancer. These findings were disputed a few years later by a group that reported normalized Dnmt1 expression to an S phase–specific proliferation marker and concluded that increased DNMT1 mRNA levels in colon cancer are nonspecific and reflect increased cell proliferation.26Lee P.J. Washer L.L. Law D.J. Boland C.R. Horon I.L. Feinberg A.P. Limited up-regulation of DNA methyltransferase in human colon cancer reflecting increased cell proliferation.Proc Natl Acad Sci U S A. 1996; 93: 10366-10370Crossref PubMed Scopus (119) Google Scholar However, more recent publications have demonstrated several proliferation-independent roles of Dnmt1 in cancer cells: Epigenetic editing of gene expression by Dnmt1 can help tumor cells to escape attacks from the immune system, and it has been shown that Dnmt1 depletion in melanoma and renal cell carcinoma can overcome resistance to interferon-induced apoptosis.11Reu F.J. Bae S.I. Cherkassky L. Leaman D.W. Lindner D. Beaulieu N. MacLeod A.R. Borden E.C. Overcoming resistance to interferon-induced apoptosis of renal carcinoma and melanoma cells by DNA demethylation.J Clin Oncol. 2006; 24: 3771-3779Crossref PubMed Scopus (111) Google Scholar Moreover, the inhibition of Dnmt1 and DNA methylation has been reported to render tumors more immunogenic by increasing both cancer testis antigens and antigen presentation.11Reu F.J. Bae S.I. Cherkassky L. Leaman D.W. Lindner D. Beaulieu N. MacLeod A.R. Borden E.C. Overcoming resistance to interferon-induced apoptosis of renal carcinoma and melanoma cells by DNA demethylation.J Clin Oncol. 2006; 24: 3771-3779Crossref PubMed Scopus (111) Google Scholar,27Siebenkas C. Chiappinelli K.B. Guzzetta A.A. Sharma A. Jeschke J. Vatapalli R. Baylin S.B. Ahuja N. Inhibiting DNA methylation activates cancer testis antigens and expression of the antigen processing and presentation machinery in colon and ovarian cancer cells.PLoS One. 2017; 12: e0179501Crossref PubMed Scopus (44) Google Scholar,28Haffner M.C. 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Cutaia O. Fazio C. Amato G. Lazzeri A. Monterisi S. Miracco C. Coral S. Anichini A. Bock C. Nemc A. Oganesian A. Lowder J. Azab M. Fridman W.H. Sautes-Fridman C. Trajanoski Z. Maio M. Guadecitabine plus ipilimumab in unresectable melanoma: the NIBIT-M4 clinical trial.Clin Cancer Res. 2019; 25: 7351-7362PubMed Google Scholar,38Burton E.M. Woody T. Glitza I.C. Amaria R.N. Keung E.Z. Diab A. Patel S.P. Wong M.K.K. Yee C. Hwu P. McQuade J.L. Woodman S.E. Tetzlaff M.T. Davies M.A. Wargo J.A. Rai K. Tawbi H.A. A phase II study of oral azacitidine (CC-486) in combination with pembrolizumab (PEMBRO) in patients (pts) with metastatic melanoma (MM).J Clin Oncol. 2019; 37 (Abstract 9560)Crossref Google Scholar BRAF V600E is the most common driver mutation in cutaneous melanoma and has been reported to promote aberrant gene methylation by the overexpression of Dnmt1 and enhancer of zeste homolog 2.20Hou P. Liu D. Dong J. Xing M. The BRAF(V600E) causes widespread alterations in gene methylation in the genome of melanoma cells.Cell Cycle. 2012; 11: 286-295Crossref PubMed Scopus (66) Google Scholar Dnmt1 is down-regulated by knockdown of BRAF V600E in melanoma cell lines, and it has been shown in a series of nonmelanoma cancer cell lines that the MEK/ERK pathway was essential for the overexpression of Dnmt1.39Li J. Wang R. Hu X. Gao Y. Wang Z. Li J. Wong J. Activated MEK/ERK pathway drives widespread and coordinated overexpression of UHRF1 and DNMT1 in cancer cells.Sci Rep. 2019; 9: 907Crossref PubMed Scopus (9) Google Scholar Our results add to the growing body of evidence that Dnmt1 expression depends on the MAPK pathway in cutaneous melanoma. However, the epigenetic impact of targeted therapies on melanoma cells is unclear at the moment and remains to be defined in future studies. In conclusion, the findings from the present study show, for the first time, a correlation between Dnmt1 expression and cellular proliferation in melanocytic tumors. Given its strong expression in metastatic melanoma, as well as its known immunomodulatory role and association with the response to B-Rafi/MEKi, Dnmt1 may be a promising target in combination with immunotherapy or targeted therapy. We thank Renate Nordin, Monika Beer, Letehawariat Haile, Sybille Kohler, Angelika Koj-Kurzynski, Petra Leber, Margareta Leippert, Simon Riel, and Karen Greif for excellent technical assistance and Ulrike Keim for statistical advice. Download .pdf (2.43 MB) Help with pdf files Supplemental Figure S1Quantification of DNA methyltransferase 1 (Dnmt1) and Ki-67 expression in melanocytic tumors. A: Nuclei of anti-melanoma–positive cells were selected. The boxed area highlights an exemplary cell (brown line through the nucleus). B: Mean fluorescence intensity values of nuclear Dnmt1 (red) and Ki-67 (blue) expression (green, melanoma; black, DAPI). Download .docx (.01 MB) Help with docx files Supplemental Table S1
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