BMP Inhibition in Seminomas Initiates Acquisition of Pluripotency via NODAL Signaling Resulting in Reprogramming to an Embryonal Carcinoma

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The understanding of germ cell cancer pathogenesis is based on a linear model, where seminomas and non-seminomas represent distinct entities, although originating from a common precursor lesion, the carcinoma in situ. We demonstrate that germ cell cancer development is a microenvironment-dependent plastic process that allows latent pluripotent seminomas /TCam-2 to acquire primed pluripotency and transit into an EC. For the first time, we show that this plasticity is initiated after interference with BMP signaling and driven by NODAL signaling, which is accompanied by considerable remodeling of the methylome. In conclusion, our data strongly suggest that ECs might also be able to transit into a seminoma upon interference with the drivers of reprogramming identified in this study.

Vyšlo v časopise: BMP Inhibition in Seminomas Initiates Acquisition of Pluripotency via NODAL Signaling Resulting in Reprogramming to an Embryonal Carcinoma. PLoS Genet 11(7): e32767. doi:10.1371/journal.pgen.1005415
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1005415

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1. Oosterhuis JW, Looijenga LH. Testicular germ-cell tumours in a broader perspective. Nat Rev Cancer. 2005;5: 210–222. 15738984

2. Kristensen DG, Skakkebaek NE, Rajpert-de-Meyts E, Almstrup K. Epigenetic features of testicular germ cell tumours in relation to epigenetic characteristics of foetal germ cells. Int J Dev Biol. 2013;57: 309–317. doi: 10.1387/ijdb.130142ka 23784842

3. de Jong J, Stoop H, Gillis AJ, van Gurp RJ, van de Geijn GJ, Boer M, et al. Differential expression of SOX17 and SOX2 in germ cells and stem cells has biological and clinical implications. J Pathol. 2008;215: 21–30. doi: 10.1002/path.2332 18348160

4. Gillis AJM, Stoop H, Biermann K, van Gurp RJHLM, Swartzman E, Cribbes S, et al. Expression and interdependencies of pluripotency factors LIN28, OCT3/4, NANOG and SOX2 in human testicular germ cells and tumours of the testis. International Journal of Andrology. 2011;34: e160–74. doi: 10.1111/j.1365-2605.2011.01148.x 21631526

5. Biermann K, Heukamp LC, Steger K, Zhou H, Franke FE, Sonnack V, et al. Genome-wide expression profiling reveals new insights into pathogenesis and progression of testicular germ cell tumors. Cancer Genomics Proteomics. 2007;4: 359–367. 17993720

6. Wermann H, Stoop H, Gillis AJ, Honecker F, van Gurp RJ, Ammerpohl O, et al. Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance. J Pathol. 221: 433–442. doi: 10.1002/path.2725 20593487

7. Nettersheim D, Heukamp LC, Fronhoffs F, Grewe MJ, Haas N, Waha A, et al. Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development. Deb S, editor. PLoS ONE. 2013;8: e82881. doi: 10.1371/journal.pone.0082881 24386123

8. McGlynn KA. Increasing Incidence of Testicular Germ Cell Tumors Among Black Men in the United States. Journal of Clinical Oncology. 2005;23: 5757–5761. 16110032

9. Kao C-S, Ulbright TM, Young RH, Idrees MT. Testicular embryonal carcinoma: a morphologic study of 180 cases highlighting unusual and unemphasized aspects. Am J Surg Pathol. 2014;38: 689–697. doi: 10.1097/PAS.0000000000000171 24503753

10. Ulbright TM. Germ cell tumors of the gonads: a selective review emphasizing problems in differential diagnosis, newly appreciated, and controversial issues. Modern Pathology. 2005;18 Suppl 2: S61–79. 15761467

11. Looijenga LHJ, Gillis AJM, Stoop H, Biermann K, Oosterhuis JW. Dissecting the molecular pathways of (testicular) germ cell tumour pathogenesis; from initiation to treatment-resistance. International Journal of Andrology. Blackwell Publishing Ltd; 2011;34: e234–51. doi: 10.1111/j.1365-2605.2011.01157.x 21564133

12. Ulbright TM. The most common, clinically significant misdiagnoses in testicular tumor pathology, and how to avoid them. Adv Anat Pathol. 2008;15: 18–27. 18156809

13. Looijenga LH, Stoop H, de Leeuw HP, de Gouveia Brazao CA, Gillis AJ, van Roozendaal KE, et al. POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors. Cancer Res. 2003;63: 2244–2250. 12727846

14. Irie N, Weinberger L, Tang WWC, Kobayashi T, Viukov S, Manor YS, et al. SOX17 Is a Critical Specifier of Human Primordial Germ Cell Fate. Cell. 2015;160: 253–268. doi: 10.1016/j.cell.2014.12.013 25543152

15. Eckert D, Nettersheim D, Heukamp LC, Kitazawa S, Biermann K, Schorle H. TCam-2 but not JKT-1 cells resemble seminoma in cell culture. Cell and Tissue Research. 2008;331: 529–538. 18008088

16. Biermann K, Heukamp LC, Steger K, Zhou H, Franke FE, Guetgemann I, et al. Gene expression profiling identifies new biological markers of neoplastic germ cells. Anticancer Res. 2007;27: 3091–3100. 17970049

17. Lim LS, Loh Y-H, Zhang W, Li Y, Chen X, Wang Y, et al. Zic3 is required for maintenance of pluripotency in embryonic stem cells. Mol Biol Cell. American Society for Cell Biology; 2007;18: 1348–1358.

18. Jia Y, Yang Y, Liu S, Herman JG, Lu F, Guo M. SOX17 antagonizes WNT/β-catenin signaling pathway in hepatocellular carcinoma. Epigenetics. 2010;5: 743–749. doi: 10.4161/epi.5.8.13104 20716954

19. Yin D, Jia Y, Yu Y, Brock MV, Herman JG, Han C, et al. SOX17 methylation inhibits its antagonism of Wnt signaling pathway in lung cancer. Discov Med. 2012;14: 33–40. 22846201

20. Sperger JM, Chen X, Draper JS, Antosiewicz JE, Chon CH, Jones SB, et al. Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc Natl Acad Sci USA. National Acad Sciences; 2003;100: 13350–13355.

21. Moustakas A, Pardali K, Gaal A, Heldin CH. Mechanisms of TGF-beta signaling in regulation of cell growth and differentiation. Immunol Lett. 2002;82: 85–91. 12008039

22. Wrana JL, Attisano L, Cárcamo J, Zentella A, Doody J, Laiho M, et al. TGF beta signals through a heteromeric protein kinase receptor complex. Cell. 1992;71: 1003–1014. 1333888

23. Moustakas A, Souchelnytskyi S, Heldin CH. Smad regulation in TGF-beta signal transduction. Journal of Cell Science. 2001;114: 4359–4369.

24. Shen MM. Nodal signaling: developmental roles and regulation. Development. 2007;134: 1023–1034. 17287255

25. Strizzi L, Postovit L-M, Margaryan NV, Seftor EA, Abbott DE, Seftor REB, et al. Emerging roles of nodal and Cripto-1: from embryogenesis to breast cancer progression. Breast Dis. 2008;29: 91–103. 19029628

26. Schier AF. Nodal morphogens. Cold Spring Harb Perspect Biol. Cold Spring Harbor Lab; 2009;1: a003459–a003459. doi: 10.1101/cshperspect.a003459

27. Schier AF. Nodal signaling in vertebrate development. Annu Rev Cell Dev Biol. Annual Reviews 4139 El Camino Way, P.O. Box 10139, Palo Alto, CA 94303–0139, USA; 2003;19: 589–621.

28. Spiller CM, Feng CW, Jackson A, Gillis AJM, Rolland AD, Looijenga LHJ, et al. Endogenous Nodal signaling regulates germ cell potency during mammalian testis development. Development. 2012;139: 4123–4132. doi: 10.1242/dev.083006 23034635

29. Wu Q, Fukuda K, Weinstein M, Graff JM, Saga Y. SMAD2 and p38 signaling pathways act in concert to determine XY primordial germ cell fate in mice. Development. The Company of Biologists Limited; 2015;142: 575–586.

30. Souquet B, Tourpin S, Messiaen S, Moison D, Habert R, Livera G. Nodal signaling regulates the entry into meiosis in fetal germ cells. Endocrinology. Endocrine Society Chevy Chase, MD; 2012;153: 2466–2473.

31. Spiller CM, Bowles J, Koopman P. Nodal/Cripto signaling in fetal male germ cell development: implications for testicular germ cell tumors. Int J Dev Biol. 2013;57: 211–219. doi: 10.1387/ijdb.130028pk 23784832

32. James D, Levine AJ, Besser D, Hemmati-Brivanlou A. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development. 2005;132: 1273–1282. 15703277

33. Hollnagel A, Oehlmann V, Heymer J, Rüther U, Nordheim A. Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells. Journal of Biological Chemistry. 1999;274: 19838–19845. 10391928

34. Lewis TC, Prywes R. Serum regulation of Id1 expression by a BMP pathway and BMP responsive element. Biochim Biophys Acta. 2013;1829: 1147–1159. doi: 10.1016/j.bbagrm.2013.08.002 23948603

35. Yang J, Li X, Li Y, Southwood M, Ye L, Long L, et al. Id proteins are critical downstream effectors of BMP signaling in human pulmonary arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2013;305: L312–21. doi: 10.1152/ajplung.00054.2013 23771884

36. Aramaki S, Hayashi K, Kurimoto K, Ohta H, Yabuta Y, Iwanari H, et al. A Mesodermal Factor, T, Specifies Mouse Germ Cell Fate by Directly Activating Germline Determinants. Developmental Cell. Elsevier Inc; 2013;27: 516–529.

37. Saitou M, Barton SC, Surani MA. A molecular programme for the specification of germ cell fate in mice. Nature. Nature Publishing Group; 2002;418: 293–300. 12124616

38. Cantú AV, Laird DJ. Wnt and Bmp Fit Germ Cells to a T. Developmental Cell. Elsevier Inc; 2013;27: 485–487.

39. Dudley B, Palumbo C, Nalepka J, Molyneaux K. BMP signaling controls formation of a primordial germ cell niche within the early genital ridges. Developmental Biology. Elsevier B.V; 2010;: 1–10.

40. Neumann JC, Chandler GL, Damoulis VA, Fustino NJ, Lillard K, Looijenga L, et al. Mutation in the type IB bone morphogenetic protein receptor Alk6b impairs germ-cell differentiation and causes germ-cell tumors in zebrafish. Proc Natl Acad Sci USA. National Acad Sciences; 2011;108: 13153–13158.

41. Neumann JC, Lillard K, Damoulis V, Amatruda JF. Zebrafish models of germ cell tumor. Methods Cell Biol. Elsevier; 2011;105: 3–24.

42. Neumann JC, Dovey JS, Chandler GL, Carbajal L, Amatruda JF. Identification of a heritable model of testicular germ cell tumor in the zebrafish. Zebrafish. Mary Ann Liebert, Inc. 140 Huguenot Street, 3rd Floor New Rochelle, NY 10801 USA; 2009;6: 319–327.

43. Fustino N, Rakheja D, Ateek CS, Neumann JC, Amatruda JF. Bone morphogenetic protein signalling activity distinguishes histological subsets of paediatric germ cell tumours. International Journal of Andrology. Blackwell Publishing Ltd; 2011;34: e218–33. doi: 10.1111/j.1365-2605.2011.01186.x 21696393

44. Sablitzky F, Moore A, Bromley M, Deed RW, Newton JS, Norton JD. Stage- and subcellular-specific expression of Id proteins in male germ and Sertoli cells implicates distinctive regulatory roles for Id proteins during meiosis, spermatogenesis, and Sertoli cell function. Cell Growth Differ. 1998;9: 1015–1024. 9869302

45. Pereira PNG, Dobreva MP, Maas E, Cornelis FM, Moya IM, Umans L, et al. Antagonism of Nodal signaling by BMP/Smad5 prevents ectopic primitive streak formation in the mouse amnion. Development. 2012;139: 3343–3354. doi: 10.1242/dev.075465 22912414

46. Yeo C, Whitman M. Nodal signals to Smads through Cripto-dependent and Cripto-independent mechanisms. Molecular Cell. 2001;7: 949–957. 11389842

47. Nettersheim D, Gillis AJ, Looijenga LH, Schorle H. TGF-beta1, EGF and FGF4 synergistically induce differentiation of the seminoma cell line TCam-2 into a cell type resembling mixed non-seminoma. Int J Androl. 2011;34: e189–203. doi: 10.1111/j.1365-2605.2011.01172.x 21649665

48. Nettersheim D, Westernströer B, Haas N, Leinhaas A, Brüstle O, Schlatt S, et al. Establishment of a versatile seminoma model indicates cellular plasticity of germ cell tumor cells. Genes Chromosom Cancer. 2012;51: 717–726. doi: 10.1002/gcc.21958 22489004

49. Josephson R, Ording CJ, Liu Y, Shin S, Lakshmipathy U, Toumadje A, et al. Qualification of embryonal carcinoma 2102Ep as a reference for human embryonic stem cell research. STEM CELLS. 2007;25: 437–446. 17284651

50. International Stem Cell Initiative, Adewumi O, Aflatoonian B, Ahrlund-Richter L, Amit M, Andrews PW, et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nature Biotechnology. Nature Publishing Group; 2007;25: 803–816. 17572666

51. Differentiation of Human Embryonal Carcinomas In vitro and In vivo Reveals Expression Profiles Relevant to Normal Development. 2005;: 1–11.

52. Honecker F, Rohlfing T, Harder S, Braig M, Gillis AJM, Glaesener S, et al. Proteome analysis of the effects of all-trans retinoic acid on human germ cell tumor cell lines. Journal of Proteomics. Elsevier B.V; 2013;: 1–14.

53. Mostert MM, van de Pol M, Olde Weghuis D, Suijkerbuijk RF, Geurts van Kessel A, van Echten J, et al. Comparative genomic hybridization of germ cell tumors of the adult testis: confirmation of karyotypic findings and identification of a 12p-amplicon. Cancer Genet Cytogenet. 1996;89: 146–152. 8697422

54. Mavilio F, Simeone A, Boncinelli E, Andrews PW. Activation of four homeobox gene clusters in human embryonal carcinoma cells induced to differentiate by retinoic acid. Differentiation. 1988;37: 73–79. 2898410

55. Andrews PW, Fenderson B, Hakomori S. Human embryonal carcinoma cells and their differentiation in culture. International Journal of Andrology. 1987;10: 95–104. 2438234

56. de Jong J, Stoop H, Gillis AJM, Hersmus R, van Gurp RJHLM, van de Geijn G-JM, et al. Further characterization of the first seminoma cell line TCam-2. Genes Chromosom Cancer. 2008;47: 185–196. 18050305

57. Eckert D, Biermann K, Nettersheim D, Gillis AJ, Steger K, Jack HM, et al. Expression of BLIMP1/PRMT5 and concurrent histone H2A/H4 arginine 3 dimethylation in fetal germ cells, CIS/IGCNU and germ cell tumors. BMC Dev Biol. 2008;8: 106. doi: 10.1186/1471-213X-8-106 18992153

58. Bahrami AR, Matin MM, Andrews PW. The CDK inhibitor p27 enhances neural differentiation in pluripotent NTERA2 human EC cells, but does not permit differentiation of 2102Ep nullipotent human EC cells. Mech Dev. 2005;122: 1034–1042. 16023837

59. Clark AT, Rodriguez RT, Bodnar MS, Abeyta MJ, Cedars MI, Turek PJ, et al. Human STELLAR, NANOG, and GDF3 genes are expressed in pluripotent cells and map to chromosome 12p13, a hotspot for teratocarcinoma. STEM CELLS. John Wiley & Sons, Ltd; 2004;22: 169–179. 14990856

60. Bowles J, Teasdale RP, James K, Koopman P. Dppa3 is a marker of pluripotency and has a human homologue that is expressed in germ cell tumours. Cytogenet Genome Res. 2003;101: 261–265. 14684992

61. Gashaw I, Dushaj O, Behr R, Biermann K, Brehm R, Rübben H, et al. Novel germ cell markers characterize testicular seminoma and fetal testis. Mol Hum Reprod. 2007;13: 721–727. 17785371

62. Assou S, Le Carrour T, Tondeur S, Ström S, Gabelle A, Marty S, et al. A meta-analysis of human embryonic stem cells transcriptome integrated into a web-based expression atlas. STEM CELLS. John Wiley & Sons, Ltd; 2007;25: 961–973.

63. Ezeh UI, Turek PJ, Reijo RA, Clark AT. Human embryonic stem cell genesOCT4, NANOG, STELLAR, andGDF3 are expressed in both seminoma and breast carcinoma. Cancer. 2005;104: 2255–2265. 16228988

64. Yamamoto T, Komatsubara S, Suzuki T, Oboshi S. In vitro cultivation of human testicular embryonal carcinoma and establishment of a new cell line. Gan. 1979;70: 677–680. 520758

65. Motoyama T, Watanabe H, Yamamoto T, Sekiguchi M. Human testicular germ cell tumors in vitro and in athymic nude mice. Acta Pathol Jpn. 1987;37: 431–448. 3618218

66. Bronson DL, Andrews PW, Solter D, Cervenka J, Lange PH, Fraley EE. Cell line derived from a metastasis of a human testicular germ cell tumor. Cancer Res. 1980;40: 2500–2506. 7388807

67. Zimmerman LB, De Jesús-Escobar JM, Harland RM. The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell. 1996;86: 599–606. 8752214

68. Dias V, Meachem S, Rajpert-De Meyts E, McLachlan R, Manuelpillai U, Loveland KL. Activin receptor subunits in normal and dysfunctional adult human testis. Hum Reprod. Oxford University Press; 2008;23: 412–420. 18077314

69. Dias VL, Rajpert-de-Meyts E, McLachlan R, Loveland KL. Analysis of activin/TGFB-signaling modulators within the normal and dysfunctional adult human testis reveals evidence of altered signaling capacity in a subset of seminomas. Reproduction. Society for Reproduction and Fertility; 2009;138: 801–811.

70. Jorgensen A, Young J, Nielsen JE, Joensen UN, Toft BG, Rajpert-De Meyts E, et al. Hanging drop cultures of human testis and testis cancer samples: a model used to investigate activin treatment effects in a preserved niche. British Journal of Cancer. Nature Publishing Group; 2014;110: 2604–2614. doi: 10.1038/bjc.2014.160 24781282

71. Korkola JE, Houldsworth J, Chadalavada RSV, Olshen AB, Dobrzynski D, Reuter VE, et al. Down-regulation of stem cell genes, including those in a 200-kb gene cluster at 12p13.31, is associated with in vivo differentiation of human male germ cell tumors. Cancer Research. 2006;66: 820–827. 16424014

72. Aruga J, Mikoshiba K. Role of BMP, FGF, calcium signaling, and Zic proteins in vertebrate neuroectodermal differentiation. Neurochem Res. 2011;36: 1286–1292. doi: 10.1007/s11064-011-0422-5 21336820

73. Marchal L, Luxardi G, Thomé V, Kodjabachian L. BMP inhibition initiates neural induction via FGF signaling and Zic genes. Proc Natl Acad Sci USA. National Acad Sciences; 2009;106: 17437–17442.

74. Ware SM, Harutyunyan KG, Belmont JW. Heart defects in X-linked heterotaxy: evidence for a genetic interaction of Zic3 with the nodal signaling pathway. Dev Dyn. 2006;235: 1631–1637. 16496285

75. Morkel M, Huelsken J, Wakamiya M, Ding J, van de Wetering M, Clevers H, et al. Beta-catenin regulates Cripto- and Wnt3-dependent gene expression programs in mouse axis and mesoderm formation. Development. 2003;130: 6283–6294. 14623818

76. Aksoy I, Jauch R, Chen J, Dyla M, Divakar U, Bogu GK, et al. Oct4 switches partnering from Sox2 to Sox17 to reinterpret the enhancer code and specify endoderm. The EMBO Journal. Nature Publishing Group; 2013;32: 938–953. doi: 10.1038/emboj.2013.31 23474895

77. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell. 2006;127: 469–480. 17081971

78. Barker N, van den Born M. Detection of beta-catenin localization by immunohistochemistry. Methods Mol Biol. Totowa, NJ: Humana Press; 2008;468: 91–98. doi: 10.1007/978-1-59745-249-6_7 19099248

79. Katoh M, Katoh M. Comparative genomics on Wnt5a and Wnt5b genes. Int J Mol Med. 2005;15: 749–753. 15754042

80. Samavarchi-Tehrani P, Golipour A, David L, Sung H-K, Beyer TA, Datti A, et al. Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell. 2010;7: 64–77. doi: 10.1016/j.stem.2010.04.015 20621051

81. Hansson J, Rafiee MR, Reiland S, Polo JM, Gehring J, Okawa S, et al. Highly coordinated proteome dynamics during reprogramming of somatic cells to pluripotency. CellReports. 2012;2: 1579–1592.

82. Mizuno Y, Gotoh A, Kamidono S, Kitazawa S. [Establishment and characterization of a new human testicular germ cell tumor cell line (TCam-2)]. Nippon Hinyokika Gakkai Zasshi. 1993;84: 1211–1218. 8394948

83. Andrews PW. Human embryonal carcinoma cells in culture do not synthesize fibronectin until they differentiate. Int J Cancer. 1982;30: 567–571.

84. Andrews PW, Damjanov I, Simon D, Banting GS, Carlin C, Dracopoli NC, et al. Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro. Lab Invest. 1984;50: 147–162. 6694356

85. Damjanov I, Horvat B, Gibas Z. Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, NCCIT. Lab Invest. 1993;68: 220–232. 7680083

86. Hiraoka A, Vogelzang NJ, Rosner MC, Golomb HM. Ultrastructure of four human germ cell tumor-derived cell lines: effect of 12-O-tetradecanoyl phorbol-13-acetate. Cancer Invest. 1988;6: 393–402. 2846127

87. Casper J, Schmoll HJ, Schnaidt U, Fonatsch C. Cell lines of human germinal cancer. International Journal of Andrology. 1987;10: 105–113. 3034787

88. Schlappack OK, Bush C, Delic JI, Steel GG. Growth and chemotherapy of a human germ-cell tumour line (GCT 27). Eur J Cancer Clin Oncol. 1988;24: 777–781. 3383978

89. Nettersheim D, Biermann K, Gillis AJM, Steger K, Looijenga LHJ, Schorle H. NANOG promoter methylation and expression correlation during normal and malignant human germ cell development. Epigenetics. 2011;6: 114–122. doi: 10.4161/epi.6.1.13433 20930529

90. Touleimat N, Tost J. Complete pipeline for Infinium(®) Human Methylation 450K BeadChip data processing using subset quantile normalization for accurate DNA methylation estimation. Epigenomics. Future Medicine Ltd London, UK; 2012;4: 325–341. doi: 10.2217/epi.12.21 22690668

91. Du P, Zhang X, Huang C-C, Jafari N, Kibbe WA, Hou L, et al. Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis. BMC Bioinformatics. BioMed Central Ltd; 2010;11: 587. doi: 10.1186/1471-2105-11-587 21118553

92. 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 Research. Oxford University Press; 2008;36: e34–e34. doi: 10.1093/nar/gkn083 18296484

93. Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, et al. STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Research. Oxford University Press; 2015;43: D447–52. doi: 10.1093/nar/gku1003 25352553

94. Backes C, Keller A, Kuentzer J, Kneissl B, Comtesse N, Elnakady YA, et al. GeneTrail—advanced gene set enrichment analysis. Nucleic Acids Research. Oxford University Press; 2007;35: W186–92. 17526521

95. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, et al. Circos: an information aesthetic for comparative genomics. Genome Research. Cold Spring Harbor Lab; 2009;19: 1639–1645. doi: 10.1101/gr.092759.109 19541911