Epidermal growth factor receptor mutation in combination with expression of MIG6 alters gefitinib sensitivity

BMC Systems Biology

Volumn 5, Issue , 2011, Pages

  Naruo, Yoshimi ^a,b   Nagashima, Takeshi ^a   Ushikoshi Nakayama, Ryoko ^a   Saeki, Yuko ^a   Nakakuki, Takashi ^c   Naka, Takashi ^d   Tanaka, Hiroshi ^b   Tsai, Shih Feng ^e   Okada Hatakeyama, Mariko ^a  

^a RIKEN Center for Integrative Medical Sciences   (Japan)

^b TOKYO MEDICAL AND DENTAL UNIVERSITY   (Japan)

^c KOGAKUIN UNIVERSITY   (Japan)

^d KYUSHU SANGYO UNIVERSITY   (Japan)

^e NATIONAL HEALTH RESEARCH INSTITUTES   (Taiwan)

Author keywords

[No Author keywords available]

Indexed keywords

EPIDERMAL GROWTH FACTOR RECEPTOR; GEFITINIB; MIG 6 PROTEIN, HUMAN; MIG-6 PROTEIN, HUMAN; PRIMER DNA; QUINAZOLINE DERIVATIVE; SIGNAL TRANSDUCING ADAPTOR PROTEIN; TUMOR SUPPRESSOR PROTEIN;

ARTICLE; BIOLOGICAL MODEL; BIOLOGY; DRUG ANTAGONISM; GENETIC TRANSFECTION; GENETICS; HUMAN; METABOLISM; MUTATION; PHOSPHORYLATION; PHYSIOLOGY; POLYACRYLAMIDE GEL ELECTROPHORESIS; SIGNAL TRANSDUCTION; TIME; TUMOR CELL LINE; WESTERN BLOTTING;

ADAPTOR PROTEINS, SIGNAL TRANSDUCING; BLOTTING, WESTERN; CELL LINE, TUMOR; COMPUTATIONAL BIOLOGY; DNA PRIMERS; ELECTROPHORESIS, POLYACRYLAMIDE GEL; HUMANS; MODELS, BIOLOGICAL; MUTATION; PHOSPHORYLATION; QUINAZOLINES; RECEPTOR, EPIDERMAL GROWTH FACTOR; SIGNAL TRANSDUCTION; TIME FACTORS; TRANSFECTION; TUMOR SUPPRESSOR PROTEINS;

EID: 79951563038     PISSN: None     EISSN: 17520509     Source Type: Journal    
DOI: 10.1186/1752-0509-5-29     Document Type: Article

References (79)

1. Olayioye MA, Neve RM, Lane HA, Hynes NE. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 2000, 19:3159-67. 10.1093/emboj/19.13.3159, 313958, 10880430.
2. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001, 2:127-37. 10.1038/35052073, 11252954.
3. Hynes NE, Lane HA. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 2005, 5:341-54. 10.1038/nrc1609, 15864276.
4. Scaltriti M, Baselga J. The epidermal growth factor receptor pathway: a model for targeted therapy. Clin Cancer Res 2006, 12:5268-72. 10.1158/1078-0432.CCR-05-1554, 17000658.
5. Voldborg BR, Damstrup L, Spang-Thomsen M, Poulsen HS. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann Oncol 1997, 8:1197-206. 10.1023/A:1008209720526, 9496384.
6. Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 2003, 284:31-53. 10.1016/S0014-4827(02)00098-8, 12648464.
7. Zandi R, Larsen AB, Andersen P, Stockhausen MT, Poulsen HS. Mechanisms for oncogenic activation of the epidermal growth factor receptor. Cell Signal 2007, 19:2013-23. 10.1016/j.cellsig.2007.06.023, 17681753.
8. Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 1995, 80:179-185. 10.1016/0092-8674(95)90401-8, 7834738.
9. Nicholson RI, Gee JM, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001, 37:S9-15. 10.1016/S0959-8049(01)00231-3, 11597399.
10. Hirsch FR, Varella-Garcia M, Bunn PA, Di Maria MV, Veve R, Bremmes RM, Barón AE, Zeng C, Franklin WA. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 2003, 21:3798-3807. 10.1200/JCO.2003.11.069, 12953099.
11. Sternberg PW, Alberola-Ila J. Conspiracy theory: RAS and RAF do not act alone. Cell 1998, 95:447-50. 10.1016/S0092-8674(00)81612-8, 9827797.
12. Henson ES, Gibson SB. Surviving cell death through epidermal growth factor (EGF) signal transduction pathways: implications for cancer therapy. Cell Signal 2006, 18:2089-97. 10.1016/j.cellsig.2006.05.015, 16815674.
13. Shigematsu H, Gazdar AF. Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers. Int. J. Cancer 2006, 118(2):257-62. 10.1002/ijc.21496, 16231326.
14. Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I, Singh B, Heelan R, Rusch V, Fulton L, Mardis E, Kupfer D, Wilson R, Kris M, Varmus H. EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA 2004, 101:13306-11. 10.1073/pnas.0405220101, 516528, 15329413.
15. Chen YR, Fu YN, Lin CH, Yang ST, Hu SF, Chen YT, Tsai SF, Huang SF. Distinctive activation patterns in constitutively active and gefitinib-sensitive EGFR mutants. Oncogene 2006, 25:1205-15. 10.1038/sj.onc.1209159, 16205628.
16. Yun CH, Boggon TJ, Li Y, Woo MS, Greulich H, Meyerson M, Eck MJ. Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 2007, 11:217-27. 10.1016/j.ccr.2006.12.017, 1939942, 17349580.
17. Fabian MA, Biggs WH, Treiber DK, Atteridge CE, Azimioara MD, Benedetti MG, Carter TA, Ciceri P, Edeen PT, Floyd M, Ford JM, Galvin M, Gerlach JL, Grotzfeld RM, Herrgard S, Insko DE, Insko MA, Lai AG, Lélias JM, Mehta SA, Milanov ZV, Velasco AM, Wodicka LM, Patel HK, Zarrinkar PP, Lockhart DJ. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotechnol 2005, 23:329-36. 10.1038/nbt1068, 15711537.
18. Nagashima T, Ushikoshi-Nakayama R, Suenaga A, Ide K, Yumoto N, Naruo Y, Takahashi K, Saeki Y, Taiji M, Tanaka H, Tsai SF, Hatakeyama M. Mutation of epidermal growth factor receptor is associated with MIG6 expression. FEBS J 2009, 276:5239-51. 10.1111/j.1742-4658.2009.07220.x, 19674104.
19. Zhang X, Pickin KA, Bose R, Jura N, Cole PA, Kuriyan J. Inhibition of the EGF receptor by binding of MIG6 to an activating kinase domain interface. Nature 2007, 450:741-4. 10.1038/nature05998, 18046415.
20. Ferby I, Reschke M, Kudlacek O, Knyazev P, Pantè G, Amann K, Sommergruber W, Kraut N, Ullrich A, Fässler R, Klein R. Mig6 is a negative regulator of EGF receptor-mediated skin morphogenesis and tumor formation. Nat Med 2006, 12:568-73. 10.1038/nm1401, 16648858.
21. Anastasi S, Baietti MF, Frosi Y, Alemà S, Segatto O. The evolutionarily conserved EBR module of RALT/MIG6 mediates suppression of the EGFR catalytic activity. Oncogene 2007, 26:7833-46. 10.1038/sj.onc.1210590, 17599051.
22. Zhang YW, Staal B, Su Y, Swiatek P, Zhao P, Cao B, Resau J, Sigler R, Bronson R, Vande Woude GF. Evidence that MIG-6 is a tumor-suppressor gene. Oncogene 2007, 26:269-76. 10.1038/sj.onc.1209790, 16819504.
23. Amatschek S, Koenig U, Auer H, Steinlein P, Pacher M, Gruenfelder A, Dekan G, Vogl S, Kubista E, Heider KH, Stratowa C, Schreiber M, Sommergruber W. Tissue-wide expression profiling using cDNA subtraction and microarrays to identify tumor-specific genes. Cancer Res 2004, 64:844-56. 10.1158/0008-5472.CAN-03-2361, 14871811.
24. Anastasi S, Sala G, Huiping C, Caprini E, Russo G, Iacovelli S, Lucini F, Ingvarsson S, Segatto O. Loss of RALT/MIG-6 expression in ERBB2-amplified breast carcinomas enhances ErbB-2 oncogenic potency and favors resistance to Herceptin. Oncogene 2005, 24:4540-8. 10.1038/sj.onc.1208658, 15856022.
25. Kholodenko BN, Demin OV, Moehren G, Hoek JB. Quantification of short term signaling by the epidermal growth factor receptor. J Biol Chem 1999, 274:30169-81. 10.1074/jbc.274.42.30169, 10514507.
26. Brightman FA, Fell DA. Differential feedback regulation of the MAPK cascade underlies the quantitative differences in EGF and NGF signalling in PC12 cells. FEBS Lett 2000, 482:169-74. 10.1016/S0014-5793(00)02037-8, 11024454.
27. Schoeberl B, Eichler-Jonsson C, Gilles ED, Müller G. Computational modeling of the dynamics of the MAP kinase cascade activated by surface and internalized EGF receptors. Nat Biotechnol 2002, 20:370-5. 10.1038/nbt0402-370, 11923843.
28. Hatakeyama M, Kimura S, Naka T, Kawasaki T, Yumoto N, Ichikawa M, Kim JH, Saito K, Saeki M, Shirouzu M, Yokoyama S, Konagaya A. A computational model on the modulation of mitogen-activated protein kinase (MAPK) and Akt pathways in heregulin-induced ErbB signalling. Biochem J 2003, 373:451-63. 10.1042/BJ20021824, 1223496, 12691603.
29. Wiley HS, Shvartsman SY, Lauffenburger DA. Computational modeling of the EGF-receptor system: a paradigm for systems biology. Trends Cell Biol 2003, 13:43-50. 10.1016/S0962-8924(02)00009-0, 12480339.
30. Yamada S, Taketomi T, Yoshimura A. Model analysis of difference between EGF pathway and FGF pathway. Biochem Biophys Res Commun 2004, 314:1113-1120. 10.1016/j.bbrc.2004.01.009, 14751248.
31. Sasagawa S, Ozaki Y, Fujita K, Kuroda S. Prediction and validation of the distinct dynamics of transient and sustained ERK activation. Nat Cell Biol 2005, 7:365-73. 10.1038/ncb1233, 15793571.
32. Kiyatkin A, Aksamitiene E, Markevich NI, Borisov NM, Hoek JB, Kholodenko BN. Scaffolding protein Grb2-associated binder 1 sustains epidermal growth factor-induced mitogenic and survival signaling by multiple positive feedback loops. J Biol Chem 2006, 281:19925-38. 10.1074/jbc.M600482200, 2312093, 16687399.
33. Birtwistle MR, Hatakeyama M, Yumoto N, Ogunnaike BA, Hoek JB, Kholodenko BN. Ligand-dependent responses of the ErbB signaling network: experimental and modeling analyses. Mol Syst Biol 2007, 3:144. 10.1038/msb4100188, 2132449, 18004277.
34. Wolf J, Dronov S, Tobin F, Goryanin I. The impact of the regulatory design on the response of epidermal growth factor receptor-mediated signal transduction towards oncogenic mutations. FEBS J 2007, 274:5505-17. 10.1111/j.1742-4658.2007.06066.x, 17916191.
35. Nakakuki T, Yumoto N, Naka T, Shirouzu M, Yokoyama S, Hatakeyama M. Topological analysis of MAPK cascade for kinetic ErbB signaling. PLoS ONE 2008, 3:e1782. 10.1371/journal.pone.0001782, 2262155, 18335053.
36. Ung CY, Li H, Ma XH, Jia J, Li BW, Low BC, Chen YZ. Simulation of the regulation of EGFR endocytosis and EGFR-ERK signaling by endophilin-mediated RhoA-EGFR crosstalk. FEBS Lett 2008, 582:2283-90. 10.1016/j.febslet.2008.05.026, 18505685.
37. Li H, Ung CY, Ma XH, Li BW, Low BC, Cao ZW, Chen YZ. Simulation of crosstalk between small GTPase RhoA and EGFR-ERK signaling pathway via MEKK1. Bioinformatics 2009, 25:358-64. 10.1093/bioinformatics/btn635, 19074159.
38. Kimura S, Konagaya A. High dimensional function optimization using a new genetic local search suitable for parallel computers. Proc of the 2003 Int Conf on Systems, Man, and Cybernetics 2003, 335-342.
39. Hartman JL, Tippery NP. Systematic quantification of gene interactions by phenotypic array analysis. Genome Biol 2004, 5:R49. 10.1186/gb-2004-5-7-r49, 463315, 15239834.
40. Keith CT, Borisy AA, Stockwell BR. Multicomponent therapeutics for networked systems. Nat Rev Drug Disco 2005, 4:71-8.
41. Lemmon MA, Bu Z, Ladbury JE, Zhou M, Pinchasi D, Lax I, Engelman DM, Schlessinger J. Two EGF molecules contribute additively to stabilization of the EGFR dimer. EMBO J 1997, 16:281-94. 10.1093/emboj/16.2.281, 1169635, 9029149.
42. Schlessinger J. Signal transduction by allosteric receptor oligomerization. Trends Biochem Sci 1988, 13:443-7. 10.1016/0968-0004(88)90219-8, 3075366.
43. Schlessinger J. Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 2002, 110:669-72. 10.1016/S0092-8674(02)00966-2, 12297041.
44. Hashimoto N, Zhang WR, Goldstein BJ. Insulin receptor and epidermal growth factor receptor dephosphorylation by three major rat liver protein-tyrosine phosphatases expressed in a recombinant bacterial system. Biochem J 1992, 284:569-76. 1132676, 1599438.
45. Zhang ZY, Walsh AB, Wu L, McNamara DJ, Dobrusin EM, Miller WT. Determinants of substrate recognition in the protein-tyrosine phosphatase, PTP1. J Biol Chem 1996, 271:5386-92. 10.1074/jbc.271.10.5386, 8621392.
46. Levkowitz G, Waterman H, Zamir E, Kam Z, Oved S, Langdon WY, Beguinot L, Geiger B, Yarden Y. c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev 1998, 12:3663-74. 10.1101/gad.12.23.3663, 317257, 9851973.
47. Muthuswamy SK, Gilman M, Brugge JS. Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo- and heterodimers. Mol Cell Biol 1999, 19:6845-57. 84681, 10490623.
48. Buday L, Downward J. Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell 1993, 73:611-20. 10.1016/0092-8674(93)90146-H, 8490966.
49. Batzer AG, Blaikie P, Nelson K, Schlessinger J, Margolis B. The phosphotyrosine interaction domain of Shc binds an LXNPXY motif on the epidermal growth factor receptor. Mol Cell Biol 1995, 15:4403-9. 230680, 7542744.
50. Ravichandran KS. Signaling via Shc family adapter proteins. Oncogene 2001, 20:6322-30. 10.1038/sj.onc.1204776, 11607835.
51. Margolis B, Skolnik EY. Activation of Ras by receptor tyrosine kinases. J Am Soc Nephrol 1994, 5:1288-99.
52. Boriack-Sjodin PA, Margarit SM, Bar-Sagi D, Kuriyan J. The structural basis of the activation of Ras by Sos. Nature 1998, 394:337-43. 10.1038/28548, 9690470.
53. Liu XQ, Pawson T. The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains. Mol Cell Biol 1991, 11:2511-6. 360020, 1850098.
54. Wang Z, Tung PS, Moran MF. Association of p120 ras GAP with endocytic components and colocalization with epidermal growth factor (EGF) receptor in response to EGF stimulation. Cell Growth Differ 1996, 7:123-33.
55. Bernards A, Settleman J. GAP control: regulating the regulators of small GTPases. Trends Cell Biol 2004, 14:377-85. 10.1016/j.tcb.2004.05.003, 15246431.
56. Koide H, Satoh T, Nakafuku M, Kaziro Y. GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells. Proc Natl Acad Sci USA 1993, 90:8683-6. 10.1073/pnas.90.18.8683, 47422, 8378348.
57. Chong H, Vikis HG, Guan KL. Mechanisms of regulating the Raf kinase family. Cell Signal 2003, 5:463-9.
58. Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J 2000, 351:289-305. 10.1042/0264-6021:3510289, 1221363, 11023813.
59. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 2001, 22:153-83. 10.1210/er.22.2.153, 11294822.
60. Keyse SM. Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Curr Opin Cell Biol 2000, 12:186-92. 10.1016/S0955-0674(99)00075-7, 10712927.
61. Zhou B, Wang ZX, Zhao Y, Brautigan DL, Zhang ZY. The specificity of extracellular signal-regulated kinase 2 dephosphorylation by protein phosphatases. J Biol Chem 2002, 277:31818-25. 10.1074/jbc.M203969200, 12082107.
62. Chuang CF, Ng SY. Functional divergence of the MAP kinase pathway. ERK1 and ERK2 activate specific transcription factors. FEBS Lett 1994, 346:229-34. 10.1016/0014-5793(94)00480-3, 8013639.
63. Gille H, Kortenjann M, Thomae O, Moomaw C, Slaughter C, Cobb MH, Shaw PE. ERK phosphorylation potentiates Elk-1-mediated ternary complex formation and transactivation. EMBO J 1995, 14:951-62. 398167, 7889942.
64. Kerkhoff E, Houben R, Löffler S, Troppmair J, Lee JE, Rapp UR. Regulation of c-myc expression by Ras/Raf signalling. Oncogene 1998, 16:211-6. 10.1038/sj.onc.1201520, 9464539.
65. Schaeffer HJ, Weber MJ. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 1999, 19:2435-44. 84036, 10082509.
66. Miyake S, Lupher ML, Druker B, Band H. The tyrosine kinase regulator Cbl enhances the ubiquitination and degradation of the platelet-derived growth factor receptor alpha. Proc Natl Acad Sci USA 1998, 95:7927-32. 10.1073/pnas.95.14.7927, 20906, 9653117.
67. Harari PM. Epidermal growth factor receptor inhibition strategies in oncology. Endocr Relat Cancer 2004, 11:689-708. 10.1677/erc.1.00600, 15613446.
68. Normanno N, De Luca A, Bianco C, Strizzi L, Mancino M, Maiello MR, Carotenuto A, De Feo G, Caponigro F, Salomon DS. Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 2006, 366:2-16. 10.1016/j.gene.2005.10.018, 16377102.
69. Meert AP, Martin B, Delmotte P, Berghmans T, Lafitte JJ, Mascaux C, Paesmans M, Steels E, Verdebout JM, Sculier JP. The role of EGF-R expression on patient survival in lung cancer: a systematic review with meta-analysis. Eur Respir J 2002, 20:975-81. 10.1183/09031936.02.00296502, 12412692.
70. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J, Haber DA. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004, 350:2129-39. 10.1056/NEJMoa040938, 15118073.
71. Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, Naoki K, Sasaki H, Fujii Y, Eck MJ, Sellers WR, Johnson BE, Meyerson M. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004, 304:1497-500. 10.1126/science.1099314, 15118125.
72. Selvaggi G, Novello S, Torri V, Leonardo E, De Giuli P, Borasio P, Mossetti C, Ardissone F, Lausi P, Scagliotti GV. Epidermal growth factor receptor overexpression correlates with a poor prognosis in completely resected non-small-cell lung cancer. Ann Oncol 2004, 15:28-32. 10.1093/annonc/mdh011, 14679115.
73. Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, Kris MG, Varmus H. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2005, 2:e73. 10.1371/journal.pmed.0020073, 549606, 15737014.
74. Citri A, Yarden Y. EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol 2006, 7:505-16. 10.1038/nrm1962, 16829981.
75. Carey KD, Garton AJ, Romero MS, Kahler J, Thomson S, Ross S, Park F, Haley JD, Gibson N, Sliwkowski MX. Kinetic analysis of epidermal growth factor receptor somatic mutant proteins shows increased sensitivity to the epidermal growth factor receptor tyrosine kinase inhibitor, erlotinib. Cancer Res 2006, 66:8163-8171. 10.1158/0008-5472.CAN-06-0453, 16912195.
76. Sakai K, Yokote H, Murakami-Murofushi K, Tamura T, Saijo N, Nishio K. In-frame deletion in the EGF receptor alters kinase inhibition by gefitinib. Biochem J 2006, 397:537-43. 10.1042/BJ20051962, 1533307, 16623663.
77. Mulloy R, Ferrand A, Kim Y, Sordella R, Bell DW, Haber DA, Anderson KS, Settleman J. Epidermal growth factor receptor mutants from human lung cancers exhibit enhanced catalytic activity and increased sensitivity to gefitinib. Cancer Res 2007, 67:2325-30. 10.1158/0008-5472.CAN-06-4293, 17332364.
78. Yun CH, Mengwasser KE, Toms AV, Woo MS, Greulich H, Wong KK, Meyerson M, Eck MJ. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci USA 2008, 105:2070-5. 10.1073/pnas.0709662105, 2538882, 18227510.
79. Guha U, Chaerkady R, Marimuthu A, Patterson AS, Kashyap MK, Harsha HC, Sato M, Bader JS, Lash AE, Minna JD, Pandey A, Varmus HE. Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS. Proc Natl Acad Sci USA 2008, 105:14112-7. 10.1073/pnas.0806158105, 2531065, 18776048.