Top

BMC Cancer

Published in:

Open Access 01-12-2011 | Research article

EFS shows biallelic methylation in uveal melanoma with poor prognosis as well as tissue-specific methylation

Authors: Lisa C Neumann, Andreas Weinhäusel, Stefanie Thomas, Bernhard Horsthemke, Dietmar R Lohmann, Michael Zeschnigk

Published in: BMC Cancer | Issue 1/2011

Abstract

Background

Uveal melanoma (UM) is a rare eye tumor. There are two classes of UM, which can be discriminated by the chromosome 3 status or global mRNA expression profile. Metastatic progression is predominantly originated from class II tumors or from tumors showing loss of an entire chromosome 3 (monosomy 3). We performed detailed EFS (embryonal Fyn-associated substrate) methylation analyses in UM, cultured uveal melanocytes and normal tissues, to explore the role of the differentially methylated EFS promoter region CpG island in tumor classification and metastatic progression.

Methods

EFS methylation was determined by direct sequencing of PCR products from bisulfite-treated DNA or by sequence analysis of individual cloned PCR products. The results were associated with clinical features of tumors and tumor-related death of patients.

Results

Analysis of 16 UM showed full methylation of the EFS CpG island in 8 (50%), no methylation in 5 (31%) and partial methylation in 3 (19%) tumors. Kaplan-Meier analysis revealed a higher risk of metastatic progression for tumors with EFS methylation (p = 0.02). This correlation was confirmed in an independent set of 24 randomly chosen tumors. Notably, only UM with EFS methylation gave rise to metastases. Real-time quantitative RT-PCR expression analysis revealed a significant inverse correlation of EFS mRNA expression with EFS methylation in UM. We further found that EFS methylation is tissue-specific with full methylation in peripheral blood cells, and no methylation in sperm, cultured primary fibroblasts and fetal muscle, kidney and brain. Adult brain samples, cultured melanocytes from the uveal tract, fetal liver and 3 of 4 buccal swab samples showed partial methylation. EFS methylation always affects both alleles in normal and tumor samples.

Conclusions

Biallelic EFS methylation is likely to be the result of a site-directed methylation mechanism. Based on partial methylation as observed in cultured melanocytes we hypothesize that there might be methylated and unmethylated precursor cells located in the uveal tract. The EFS methylation of a UM may depend on which type of precursor cell the tumor originated from.

Available only for authorised users

Onken MD, Worley LA, Ehlers JP, Harbour JW: Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res. 2004, 64: 7205-7209. 10.1158/0008-5472.CAN-04-1750.CrossRefPubMed

Tschentscher F, Hüsing J, Hölter T, Kruse E, Dresen IG, Jöckel KH, Anastassiou G, Schilling H, Bornfeld N, Horsthemke B, Lohmann DR, Zeschnigk M: Tumor classification based on gene expression profiling shows that uveal melanomas with and without monosomy 3 represent two distinct entities. Cancer Res. 2003, 63: 2578-2584.PubMed

Prescher G, Bornfeld N, Hirche H, Horsthemke B, Jökel KH, Becher R: Prognostic implications of monosomy 3 in uveal melanoma. Lancet. 1996, 347: 1222-1225. 10.1016/S0140-6736(96)90736-9.CrossRefPubMed

Damato B, Duke C, Coupland SE, Hiscott P, Smith PA, Campbell I, Douglas A, Howard P: Cytogenetics of uveal melanoma: a 7-year clinical experience. Ophthalmology. 2007, 114: 1925-1931. 10.1016/j.ophtha.2007.06.012.CrossRefPubMed

Harbour JW, Onken MD, Roberson ED, Duan S, Cao L, Worley LA, Council ML, Matatall KA, Helms C, Bowcock AM: Frequent mutation of BAP1 in metastasizing uveal melanomas. Science. 2010, 330: 1410-1413. 10.1126/science.1194472.CrossRefPubMedPubMedCentral

Chang SH, Worley LA, Onken MD, Harbour JW: Prognostic biomarkers in uveal melanoma: evidence for a stem cell-like phenotype associated with metastasis. Melanoma Res. 2008, 18: 191-200. 10.1097/CMR.0b013e3283005270.CrossRefPubMed

Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, Schlawe K, Chang HD, Bopp T, Schmitt E, Klein-Hessling S, Serfling E, Hamann A, Huehn J: Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol. 2007, 5: e38-10.1371/journal.pbio.0050038.CrossRefPubMedPubMedCentral

Takai D, Jones PA: Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci USA. 2002, 99: 3740-3745. 10.1073/pnas.052410099.CrossRefPubMedPubMedCentral

Zeschnigk M, Martin M, Betzl G, Kalbe A, Sirsch C, Buiting K, Gross S, Fritzilas E, Frey B, Rahmann S, Horsthemke B: Massive parallel bisulfite sequencing of CG-rich DNA fragments reveals that methylation of many X-chromosomal CpG islands in female blood DNA is incomplete. Hum Mol Genet. 2009, 18: 1439-1448. 10.1093/hmg/ddp054.CrossRefPubMed

10.

Greger V, Passarge E, Höping W, Messmer E, Horsthemke B: Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum Genet. 1989, 83: 155-158. 10.1007/BF00286709.CrossRefPubMed

11.

Shen L, Kondo Y, Guo Y, Zhang J, Zhang L, Ahmed S, Shu J, Chen X, Waterland RA, Issa JP: Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters. PLoS Genet. 2007, 3: 2023-2036.CrossRefPubMed

12.

Deb-Rinker P, Ly D, Jezierski A, Sikorska M, Walker PR: Sequential DNA methylation of the Nanog and Oct-4 upstream regions in human NT2 cells during neuronal differentiation. J Biol Chem. 2005, 280: 6257-6260. 10.1074/jbc.C400479200.CrossRefPubMed

13.

Illingworth R, Kerr A, Desousa D, Jorgensen H, Ellis P, Stalker J, Jackson D, Clee C, Plumb R, Rogers J, Humphray S, Cox T, Langford C, Bird A: A novel CpG island set identifies tissue-specific methylation at developmental gene loci. PLoS Biol. 2008, 6: e22-10.1371/journal.pbio.0060022.CrossRefPubMedPubMedCentral

14.

Straussman R, Nejman D, Roberts D, Steinfeld I, Blum B, Benvenisty N, Simon I, Yakhini Z, Cedar H: Developmental programming of CpG island methylation profiles in the human genome. Nat Struct Mol Biol. 2009, 16: 564-571. 10.1038/nsmb.1594.CrossRefPubMed

15.

Ohm JE, McGarvey KM, Yu X, Cheng L, Schuebel KE, Cope L, Mohammad HP, Chen W, Daniel VC, Yu W, Berman DM, Jenuwein T, Pruitt K, Sharkis SJ, Watkins DN, Herman JG, Baylin SB: A stem cell-like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing. Nat Genet. 2007, 39: 237-242. 10.1038/ng1972.CrossRefPubMedPubMedCentral

16.

Widschwendter M, Fiegl H, Egle D, Mueller-Holzner E, Spizzo G, Marth C, Weisenberger DJ, Campan M, Young J, Jacobs I, Laird PW: Epigenetic stem cell signature in cancer. Nat Genet. 2007, 39: 157-158. 10.1038/ng1941.CrossRefPubMed

17.

Maat W, van der Velden PA, Out-Luiting C, Plug M, Dirks-Mulder A, Jager MJ, Gruis NA: Epigenetic inactivation of RASSF1a in uveal melanoma. Invest Ophthalmol Vis Sci. 2007, 48: 486-490. 10.1167/iovs.06-0781.CrossRefPubMed

18.

Merhavi E, Cohen Y, Avraham BC, Frenkel S, Chowers I, Pe'er J, Goldenberg-Cohen N: Promoter methylation status of multiple genes in uveal melanoma. Invest Ophthalmol Vis Sci. 2007, 48: 4403-4406. 10.1167/iovs.07-0272.CrossRefPubMed

19.

Moulin AP, Clement G, Bosman FT, Zografos L, Benhattar J: Methylation of CpG island promoters in uveal melanoma. Br J Ophthalmol. 2008, 92: 281-285. 10.1136/bjo.2007.127035.CrossRefPubMed

20.

Zeschnigk M, Tschentscher F, Lich C, Brandt B, Horsthemke B, Lohmann DR: Methylation Analysis of Several Tumour Suppressor Genes Shows a Low Frequency of Methylation of CDKN2A and RARB in Uveal Melanomas. Comp Funct Genomics. 2003, 4: 329-336. 10.1002/cfg.295.CrossRefPubMedPubMedCentral

21.

Tschentscher F, Prescher G, Zeschnigk M, Horsthemke B, Lohmann DR: Identification of chromosomes 3, 6, and 8 aberrations in uveal melanoma by microsatellite analysis in comparison to comparative genomic hybridization. Cancer Genet Cytogenet. 2000, 122: 13-17. 10.1016/S0165-4608(00)00266-1.CrossRefPubMed

22.

Hu DN, McCormick SA, Ritch R, Pelton-Henrion K: Studies of human uveal melanocytes in vitro: isolation, purification and cultivation of human uveal melanocytes. Invest Ophthalmol Vis Sci. 1993, 34: 2210-2219.PubMed

23.

Fischer S, Lüdecke HJ, Wieczorek D, Böhringer S, Gillessen-Kaesbach G, Horsthemke B: Histone acetylation dependent allelic expression imbalance of BAPX1 in patients with the oculo-auriculo-vertebral spectrum. Hum Mol Genet. 2006, 15: 581-587. 10.1093/hmg/ddi474.CrossRefPubMed

24.

Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B: A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet. 1997, 5: 94-98.PubMed

25.

Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS: 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991, 19: 4008-10.1093/nar/19.14.4008.CrossRefPubMedPubMedCentral

26.

Han W, Cauchi S, Herman JG, Spivack SD: DNA methylation mapping by tag-modified bisulfite genomic sequencing. Anal Biochem. 2006, 355: 50-61. 10.1016/j.ab.2006.05.010.CrossRefPubMed

27.

Rohde C, Zhang Y, Jurkowski TP, Stamerjohanns H, Reinhardt R, Jeltsch A: Bisulfite sequencing Data Presentation and Compilation (BDPC) web server--a useful tool for DNA methylation analysis. Nucleic Acids Res. 2008, 36: e34-10.1093/nar/gkn083.CrossRefPubMedPubMedCentral

28.

Ronan A, Buiting K, Dudding T: Atypical Angelman syndrome with macrocephaly due to a familial imprinting center deletion. Am J Med Genet A. 2008, 146A: 78-82. 10.1002/ajmg.a.31952.CrossRefPubMed

29.

Ghosh S, Yates AJ, Fruhwald MC, Miecznikowski JC, Plass C, Smiraglia D: Tissue specific DNA methylation of CpG islands in normal human adult somatic tissues distinguishes neural from non-neural tissues. Epigenetics. 2010, 5: 527-538. 10.4161/epi.5.6.12228.CrossRefPubMedPubMedCentral

30.

Ishino M, Ohba T, Sasaki H, Sasaki T: Molecular cloning of a cDNA encoding a phosphoprotein, Efs, which contains a Src homology 3 domain and associates with Fyn. Oncogene. 1995, 11: 2331-2338.PubMed

31.

Alexandropoulos K, Baltimore D: Coordinate activation of c-Src by SH3- and SH2-binding sites on a novel p130Cas-related protein, Sin. Genes Dev. 1996, 10: 1341-1355. 10.1101/gad.10.11.1341.CrossRefPubMed

32.

Tikhmyanova N, Little JL, Golemis EA: CAS proteins in normal and pathological cell growth control. Cell Mol Life Sci. 2010, 67: 1025-1048. 10.1007/s00018-009-0213-1.CrossRefPubMed

33.

Danzl NM, Donlin LT, Alexandropoulos K: Regulation of medullary thymic epithelial cell differentiation and function by the signaling protein Sin. J Exp Med. 2010, 207: 999-1013. 10.1084/jem.20092384.CrossRefPubMedPubMedCentral

34.

Donlin LT, Danzl NM, Wanjalla C, Alexandropoulos K: Deficiency in expression of the signaling protein Sin/Efs leads to T-lymphocyte activation and mucosal inflammation. Mol Cell Biol. 2005, 25: 11035-11046. 10.1128/MCB.25.24.11035-11046.2005.CrossRefPubMedPubMedCentral

The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/380/prepub

Title: EFS shows biallelic methylation in uveal melanoma with poor prognosis as well as tissue-specific methylation
Authors: Lisa C Neumann
Andreas Weinhäusel
Stefanie Thomas
Bernhard Horsthemke
Dietmar R Lohmann
Michael Zeschnigk
Publication date: 01-12-2011
Publisher: BioMed Central
Published in: BMC Cancer / Issue 1/2011
Electronic ISSN: 1471-2407
DOI: https://doi.org/10.1186/1471-2407-11-380

Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras

Prof. Fabrice Barlesi

Developed by: Springer Medicine

At a glance: The STEP trials

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

Please log in to get access to this content

Other articles of this Issue 1/2011

ABI3 ectopic expression reduces in vitro and in vivocell growth properties while inducing senescence

Genistein inhibits proliferation of colon cancer cells by attenuating a negative effect of epidermal growth factor on tumor suppressor FOXO3 activity

Clinical management of gastric cancer: results of a multicentre survey

Mental adjustment to cancer and its relation to anxiety, depression, HRQL and survival in patients with laryngeal cancer - A longitudinal study

Biomarker analysis of cetuximab plus oxaliplatin/leucovorin/5-fluorouracil in first-line metastatic gastric and oesophago-gastric junction cancer: results from a phase II trial of the Arbeitsgemeinschaft Internistische Onkologie (AIO)

Fatty acid binding proteins (FABPs) in prostate, bladder and kidney cancer cell lines and the use of IL-FABP as survival predictor in patients with renal cell carcinoma

Keynote webinar | Spotlight on antibody–drug conjugates in cancer