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Epigenetic inactivation of galanin and GALR1/2 is associated with early recurrence in head and neck cancer

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Abstract

The aim of this study was to investigate the prognostic value of galanin (GAL) and galanin receptor (GALR) promoter hypermethylation in patients with head and neck squamous cell carcinoma (HNSCC). The methylation status of three genes—GAL, GALR1, and GALR2 was examined in HNSCC patient tumors using quantitative methylation-specific PCR (Q-MSP). To determine the prognostic value of GAL, GALR1 and GALR2 methylation status, their associations with various clinical characteristics and patient survival were assessed in HNSCC patient tumors (n = 142). Aberrant methylation of at least one gene was observed in 84 of the 142 (59.2 %) primary tumors analyzed. The methylation index, defined as the ratio between the number of methylated genes and the number of genes examined, was positively correlated with larger tumor size (P = 0.034) and disease recurrence (P < 0.001). In the multivariate logistic-regression analysis, methylation of both GAL and GALR1 exhibited the highest association with poor survival (hazard ratio, 6.83, P = 0.002). Moreover, among patients without lymph node metastasis, a multivariate analysis showed a significant trend for poor survival as the number of hypermethylated genes increased (log-rank test, P = 0.003). CpG hypermethylation is a likely mechanism of GAL and GALR1/2 gene inactivation, indicating that GAL and its receptors play a role in HNSCC tumorigenesis. As such, GAL and GALR1/2 methylation status may serve as an important biomarker for clinical outcome.

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References

  1. Vokes EE, Weichselbaum RR, Lippman SM, Hong WK (1993) Head and neck cancer. N Engl J Med 328(3):184–194. doi:10.1056/NEJM199301213280306

    Article  CAS  PubMed  Google Scholar 

  2. Forastiere A, Koch W, Trotti A, Sidransky D (2001) Head and neck cancer. N Engl J Med 345(26):1890–1900. doi:10.1056/NEJMra001375345/26/1890

    Article  CAS  PubMed  Google Scholar 

  3. Hill SJ (2006) G-protein-coupled receptors: past, present and future. Br J Pharmacol 147(Suppl 1):S27–37. doi:10.1038/sj.bjp.0706455

    PubMed Central  CAS  PubMed  Google Scholar 

  4. Tatemoto K, Rokaeus A, Jornvall H, McDonald TJ, Mutt V (1983) Galanin—a novel biologically active peptide from porcine intestine. FEBS Lett 164(1):124–128. doi:10.1016/0014-5793(83)80033-7

    Article  CAS  PubMed  Google Scholar 

  5. Ottlecz A, Snyder GD, McCann SM (1988) Regulatory role of galanin in control of hypothalamic-anterior pituitary function. Proc Natl Acad Sci USA 85(24):9861–9865

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Vrontakis ME, Torsello A, Friesen HG (1991) Galanin. J Endocrinol Invest 14(9):785–794

    Article  CAS  PubMed  Google Scholar 

  7. Yang Y, Zhang Y, Li XH, Li Y, Qian R, Li J, Xu SL (2015) Involvements of galanin and its receptors in antinociception in nucleus accumbens of rats with inflammatory pain. Neurosci Res 97:20–25. doi:10.1016/j.neures.2015.03.006

    Article  CAS  PubMed  Google Scholar 

  8. Bartfai T, Langel U, Bedecs K, Andell S, Land T, Gregersen S, Ahren B, Girotti P, Consolo S, Corwin R et al (1993) Galanin-receptor ligand M40 peptide distinguishes between putative galanin-receptor subtypes. Proc Natl Acad Sci USA 90(23):11287–11291

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Gutkind JS (1998) Cell growth control by G protein-coupled receptors: from signal transduction to signal integration. Oncogene 17(11 Reviews):1331–1342. doi:10.1038/sj.onc.1202186

  10. Freimann K, Kurrikoff K, Langel U (2015) Galanin receptors as a potential target for neurological disease. Expert Opin Ther Targets 19:1–12. doi:10.1517/14728222.2015.1072513

  11. Henson BS, Neubig RR, Jang I, Ogawa T, Zhang Z, Carey TE, D’Silva NJ (2005) Galanin receptor 1 has anti-proliferative effects in oral squamous cell carcinoma. J Biol Chem 280(24):22564–22571. doi:10.1074/jbc.M414589200

    Article  CAS  PubMed  Google Scholar 

  12. Kanazawa T, Iwashita T, Kommareddi P, Nair T, Misawa K, Misawa Y, Ueda Y, Tono T, Carey TE (2007) Galanin and galanin receptor type 1 suppress proliferation in squamous carcinoma cells: activation of the extracellular signal regulated kinase pathway and induction of cyclin-dependent kinase inhibitors. Oncogene 26(39):5762–5771. doi:10.1038/sj.onc.1210384

    Article  CAS  PubMed  Google Scholar 

  13. Kanazawa T, Kommareddi PK, Iwashita T, Kumar B, Misawa K, Misawa Y, Jang I, Nair TS, Iino Y, Carey TE (2009) Galanin receptor subtype 2 suppresses cell proliferation and induces apoptosis in p53 mutant head and neck cancer cells. Clin Cancer Res 15(7):2222–2230. doi:10.1158/1078-0432.CCR-08-2443

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Kanazawa T, Misawa K, Carey TE (2010) Galanin receptor subtypes 1 and 2 as therapeutic targets in head and neck squamous cell carcinoma. Expert Opin Ther Targ 14(3):289–302. doi:10.1517/14728221003598922

    Article  CAS  Google Scholar 

  15. Misawa K, Ueda Y, Kanazawa T, Misawa Y, Jang I, Brenner JC, Ogawa T, Takebayashi S, Grenman RA, Herman JG, Mineta H, Carey TE (2008) Epigenetic inactivation of galanin receptor 1 in head and neck cancer. Clin Cancer Res 14(23):7604–7613. doi:10.1158/1078-0432.CCR-07-4673

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Misawa K, Kanazawa T, Misawa Y, Uehara T, Imai A, Takahashi G, Takebayashi S, Cole A, Carey TE, Mineta H (2013) Galanin has tumor suppressor activity and is frequently inactivated by aberrant promoter methylation in head and neck cancer. Transl Oncol 6(3):338–346

    Article  PubMed Central  PubMed  Google Scholar 

  17. Misawa Y, Misawa K, Kanazawa T, Uehara T, Endo S, Mochizuki D, Yamatodani T, Carey TE, Mineta H (2014) Tumor suppressor activity and inactivation of galanin receptor type 2 by aberrant promoter methylation in head and neck cancer. Cancer 120(2):205–213. doi:10.1002/cncr.28411

    Article  CAS  PubMed  Google Scholar 

  18. Berger A, Lang R, Moritz K, Santic R, Hermann A, Sperl W, Kofler B (2004) Galanin receptor subtype GalR2 mediates apoptosis in SH-SY5Y neuroblastoma cells. Endocrinology 145(2):500–507. doi:10.1210/en.2003-0649en.2003-0649

    Article  CAS  PubMed  Google Scholar 

  19. Tofighi R, Joseph B, Xia S, Xu ZQ, Hamberger B, Hokfelt T, Ceccatelli S (2008) Galanin decreases proliferation of PC12 cells and induces apoptosis via its subtype 2 receptor (GalR2). Proc Natl Acad Sci USA 105(7):2717–2722. doi:10.1073/pnas.0712300105

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Stewart GD, Van Neste L, Delvenne P, Delree P, Delga A, McNeill SA, O’Donnell M, Clark J, Van Criekinge W, Bigley J, Harrison DJ (2013) Clinical utility of an epigenetic assay to detect occult prostate cancer in histopathologically negative biopsies: results of the MATLOC study. J Urol 189(3):1110–1116. doi:10.1016/j.juro.2012.08.219

    Article  PubMed  Google Scholar 

  21. Brock MV, Hooker CM, Ota-Machida E, Han Y, Guo M, Ames S, Glockner S, Piantadosi S, Gabrielson E, Pridham G, Pelosky K, Belinsky SA, Yang SC, Baylin SB, Herman JG (2008) DNA methylation markers and early recurrence in stage I lung cancer. N Engl J Med 358(11):1118–1128. doi:10.1056/NEJMoa0706550

    Article  CAS  PubMed  Google Scholar 

  22. Misawa K, Kanazawa T, Misawa Y, Imai A, Uehara T, Mochizuki D, Endo S, Takahashi G, Mineta H (2013) Frequent promoter hypermethylation of tachykinin-I and tachykinin receptor type I is a potential biomarker for head and neck cancer. J Cancer Res Clin Oncol. doi:10.1007/s00432-013-1393-5

    PubMed  Google Scholar 

  23. Katz MH (2011) Multivariable analysis: a practical guide for clinicians and public health researchers setting up a multivariable analysis. Cambridge University Press, Cambridge

    Book  Google Scholar 

  24. Herman JG, Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349(21):2042–2054. doi:10.1056/NEJMra023075

    Article  CAS  PubMed  Google Scholar 

  25. Bandholtz S, Wichard J, Kuhne R, Grotzinger C (2012) Molecular evolution of a peptide GPCR ligand driven by artificial neural networks. PLoS ONE 7(5):e36948. doi:10.1371/journal.pone.0036948

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Lappano R, Maggiolini M (2011) G protein-coupled receptors: novel targets for drug discovery in cancer. Nat Rev Drug Discovery 10(1):47–60. doi:10.1038/nrd3320

    Article  CAS  PubMed  Google Scholar 

  27. Jurkowski W, Yazdi S, Elofsson A (2013) Ligand binding properties of human galanin receptors. Mol Membr Biol 30(2):206–216. doi:10.3109/09687688.2012.750384

    Article  PubMed  Google Scholar 

  28. Duan H, Zhang Y, Zhang XM, Xu HH, Shu J, Xu SL (2015) Antinociceptive roles of galanin receptor 1 in nucleus accumbens of rats in a model of neuropathic pain. J Neurosci Res. doi:10.1002/jnr.23611

    PubMed  Google Scholar 

  29. Li L, Yu L, Kong Q (2013) Exogenous galanin attenuates spatial memory impairment and decreases hippocampal beta-amyloid levels in rat model of Alzheimer’s disease. Int J Neurosci 123(11):759–765. doi:10.3109/00207454.2013.800976

    Article  CAS  PubMed  Google Scholar 

  30. Belfer I, Hipp H, Bollettino A, McKnight C, Evans C, Virkkunen M, Albaugh B, Max MB, Goldman D, Enoch MA (2007) Alcoholism is associated with GALR3 but not two other galanin receptor genes. Genes Brain Behav 6(5):473–481. doi:10.1111/j.1601-183X.2006.00275.x

    Article  CAS  PubMed  Google Scholar 

  31. Stevenson L, Allen WL, Turkington R, Jithesh PV, Proutski I, Stewart G, Lenz HJ, Van Schaeybroeck S, Longley DB, Johnston PG (2012) Identification of galanin and its receptor GalR1 as novel determinants of resistance to chemotherapy and potential biomarkers in colorectal cancer. Clin Cancer Res 18(19):5412–5426. doi:10.1158/1078-0432.CCR-12-1780

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Kim KY, Kee MK, Chong SA, Nam MJ (2007) Galanin is up-regulated in colon adenocarcinoma. Cancer Epidemiol Biomark Prev 16(11):2373–2378. doi:10.1158/1055-9965.EPI-06-0740

    Article  CAS  Google Scholar 

  33. Kim JC, Lee HC, Cho DH, Choi EY, Cho YK, Ha YJ, Choi PW, Roh SA, Kim SY, Kim YS (2011) Genome-wide identification of possible methylation markers chemosensitive to targeted regimens in colorectal cancers. J Cancer Res Clin Oncol 137(10):1571–1580. doi:10.1007/s00432-011-1036-7

    Article  CAS  PubMed  Google Scholar 

  34. Doufekas K, Hadwin R, Kandimalla R, Jones A, Mould T, Crowe S, Olaitan A, Macdonald N, Fiegl H, Wik E, Salvesen HB, Widschwendter M (2013) GALR1 methylation in vaginal swabs is highly accurate in identifying women with endometrial cancer. Int J Gynecol Cancer Off J Int Gynecol Cancer Soc 23(6):1050–1055. doi:10.1097/IGC.0b013e3182959103

    Article  Google Scholar 

  35. Rodriguez-Paredes M, Esteller M (2011) Cancer epigenetics reaches mainstream oncology. Nat Med 17(3):330–339. doi:10.1038/nm.2305

    Article  CAS  PubMed  Google Scholar 

  36. Taby R, Issa JP (2010) Cancer epigenetics. CA Cancer J Clin 60(6):376–392. doi:10.3322/caac.20085

    Article  PubMed  Google Scholar 

  37. Jin Z, Olaru A, Yang J, Sato F, Cheng Y, Kan T, Mori Y, Mantzur C, Paun B, Hamilton JP, Ito T, Wang S, David S, Agarwal R, Beer DG, Abraham JM, Meltzer SJ (2007) Hypermethylation of tachykinin-1 is a potential biomarker in human esophageal cancer. Clin Cancer Res 13(21):6293–6300. doi:10.1158/1078-0432.CCR-07-0818

    Article  CAS  PubMed  Google Scholar 

  38. Mori Y, Cai K, Cheng Y, Wang S, Paun B, Hamilton JP, Jin Z, Sato F, Berki AT, Kan T, Ito T, Mantzur C, Abraham JM, Meltzer SJ (2006) A genome-wide search identifies epigenetic silencing of somatostatin, tachykinin-1, and 5 other genes in colon cancer. Gastroenterology 131(3):797–808. doi:10.1053/j.gastro.2006.06.006

    Article  CAS  PubMed  Google Scholar 

  39. Carvalho AL, Henrique R, Jeronimo C, Nayak CS, Reddy AN, Hoque MO, Chang S, Brait M, Jiang WW, Kim MM, Claybourne Q, Goldenberg D, Khan Z, Khan T, Westra WH, Sidransky D, Koch W, Califano JA (2011) Detection of promoter hypermethylation in salivary rinses as a biomarker for head and neck squamous cell carcinoma surveillance. Clin Cancer Res 17(14):4782–4789. doi:10.1158/1078-0432.CCR-11-0324

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Colacino JA, Dolinoy DC, Duffy SA, Sartor MA, Chepeha DB, Bradford CR, McHugh JB, Patel DA, Virani S, Walline HM, Bellile E, Terrell JE, Stoerker JA, Taylor JM, Carey TE, Wolf GT, Rozek LS (2013) Comprehensive analysis of DNA methylation in head and neck squamous cell carcinoma indicates differences by survival and clinicopathologic characteristics. PLoS ONE 8(1):e54742. doi:10.1371/journal.pone.0054742

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Kostareli E, Holzinger D, Bogatyrova O, Hielscher T, Wichmann G, Keck M, Lahrmann B, Grabe N, Flechtenmacher C, Schmidt CR, Seiwert T, Dyckhoff G, Dietz A, Hofler D, Pawlita M, Benner A, Bosch FX, Plinkert P, Plass C, Weichenhan D, Hess J (2013) HPV-related methylation signature predicts survival in oropharyngeal squamous cell carcinomas. J Clin Investig 123(6):2488–2501. doi:10.1172/JCI67010

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors would like to thank Ms. Yuko Mohri for her excellent technical support.

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Authors and Affiliations

  1. Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, 1-20-1 Handayama, Shizuoka, 431-3192, Japan

    Kiyoshi Misawa, Yuki Misawa, Daiki Mochizuki, Atsushi Imai, Shiori Endo & Hiroyuki Mineta

  2. Department of Otolaryngology/Head and Neck Surgery, Jichi Medical University, Tochigi, Japan

    Takeharu Kanazawa

  3. Laboratory of Head and Neck Cancer Biology, Department of Otolaryngology/Head and Neck Surgery, University of Michigan, Ann Arbor, MI, USA

    Thomas E. Carey

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  1. Kiyoshi Misawa
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Correspondence to Kiyoshi Misawa.

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Grant support

A Grant-in-Aid for scientific research (Nos. 24592594, 26462599) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Supplementary material 1 (DOCX 27 kb)

Supplementary Fig. 1

Standard curve plot showing Ct versus initial quantity. Efficiency of primers of target genes (GAL, GALR1 and GALR2) and housekeeping gene (β-actin) checked for methylation quantification using dilutions of universal methylated DNAs control. For each data point three independent analysis were performed. The equation of the linear regression curve as well as the correlation factor is indicated on each graph. (EPS 940 kb)

Supplementary Fig. 2

Comparison of methylation indexes (MI) amongst HPV status in the oropharynx and oral cavity patients. The mean MI for different groups was compared using the Student’s t tests.

Supplementary Fig. 3

Galanin, GALR1, GALR2 and GALR3 mRNA patterns in 55 matched pairs of head and neck tumors and adjacent normal mucosal tissues. The mean MI for different groups was compared using the Paired t tests.

Supplementary Fig. 4

Galanin, GALR1 and GALR2 mRNA patterns for 55 patients according to GAL, GALR1 , and GALR2 methylation status. The changes between cancerous and normal mucosal tissues were considered to be significant, as determined by the Student’s t‑test.

Supplementary Fig. 5

Comparison of Galanin, GALR1 and GALR2 mRNA levels amongst selected clinical parameters. Statistical analyses of the associations between variables were performed by the Student’s t‑test.

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Misawa, K., Misawa, Y., Kanazawa, T. et al. Epigenetic inactivation of galanin and GALR1/2 is associated with early recurrence in head and neck cancer. Clin Exp Metastasis 33, 187–195 (2016). https://doi.org/10.1007/s10585-015-9768-4

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