<em>GHSR</em> methylation‑dependent expression of a variant ligand and receptor of the ghrelin system induces thymoma tumorigenesis

Tegshee,Bilguun; Kondo,Kazuya; Soejima,Shiho; Muguruma,Kyoka; Tsuboi,Mitsuhiro; Kajiura,Koichiro; Kawakami,Yukikiyo; Kawakita,Naoya; Toba,Hiroaki; Yoshida,Mitsuteru; Takizawa,Hiromitsu; Tangoku,Akira

doi:10.3892/ol.2021.13054

GHSR methylation‑dependent expression of a variant ligand and receptor of the ghrelin system induces thymoma tumorigenesis

Authors:
- Bilguun Tegshee
- Kazuya Kondo
- Shiho Soejima
- Kyoka Muguruma
- Mitsuhiro Tsuboi
- Koichiro Kajiura
- Yukikiyo Kawakami
- Naoya Kawakita
- Hiroaki Toba
- Mitsuteru Yoshida
- Hiromitsu Takizawa
- Akira Tangoku
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Affiliations: Department of Oncological Medical Services, Graduate School of Biomedical Sciences, Tokushima University, Tokushima, Tokushima 770‑8509, Japan, Department of Thoracic, Endocrine Surgery and Oncology, Graduate School of Biomedical Sciences, Tokushima University, Kuramoto‑cho, Tokushima 770‑8503, Japan
Published online on: September 17, 2021 https://doi.org/10.3892/ol.2021.13054
Article Number: 793
Copyright: © Tegshee et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 4.0].

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Abstract

Our previous study reported that the DNA methylation of growth hormone secretagogue receptor (GHSR) was significantly higher in thymoma or thymic carcinoma (TC) than in normal thymic tissue samples. Thymic epithelial tumors (TETs) with higher GHSR DNA methylation were associated with significantly worse prognosis than those with lower levels of DNA methylation. Diversified components of the ghrelin‑GHSR axis may exert opposing effects in cancer progression, depending on the cancer type in question. However, the precise function of the axis remains unclear. In the present study, the mRNA expression of five key components of the ghrelin system [native ligand ghrelin, variant ligand In‑1 ghrelin, native receptor GHSR1a, variant receptor GHSR1b and acylation enzyme ghrelin O‑acyltransferase (GOAT)] were examined in 58 TET samples by reverse transcription‑quantitative PCR, and protein expression of GHSR1a and GHSR1b was assessed in 20 TETs using immunohistochemistry. The results revealed that In‑1 ghrelin, GHSR1b (variant forms) and GOAT were more strongly expressed in thymoma compared with thymic‑adjacent tissue. By contrast, no significant differences were observed in the expression of ghrelin and GHSR1a (native forms) between thymoma and thymic tissue. The mRNA expression of In‑1 ghrelin and GHSR1b (variant forms) was positively associated with GHSR methylation in thymoma tissue samples. However, a relationship was not found between ghrelin, GHSR1a or GOAT expression (native forms) and GHSR methylation in thymoma. Immunohistochemical analysis revealed that mRNA expression of GHSR1a and GHSR1b generally correlated with expression of the corresponding protein, and that the expression of GHSR1b was increased in advanced‑stage TETs. These results indicate that the DNA methylation of GHSR is associated with a shift from native expression (ghrelin and GHSR1a) to variant expression (In‑1 ghrelin and GHSR1b), which induces the tumorigenesis of thymoma, but not TC.

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1	Tateo V, Manuzzi L, De Giglio A, Parisi C, Lamberti G, Campana D and Pantaleo MA: Immunobiology of thymic epithelial tumors: Implications for immunotherapy with immune checkpoint inhibitors. Int J Mol Sci. 21:90562020. View Article : Google Scholar : PubMed/NCBI
2	Travis WD, Brambilla E, Nicholson AG, Yatabe Y, Austin JH, Beasley MB, Chirieac LR, Dacic S, Duhig E, et al: The 2015 World Health Organization Classification of Lung Tumors: Impact of genetic, clinical and radiologic advances since the 2004 classification. J Thorac Oncol. 10:1243–1260. 2015. View Article : Google Scholar : PubMed/NCBI
3	Detterbeck FC, Nicholson AG, Kondo K, Van Schil P and Moran C: The Masaoka-Koga stage classification for thymic malignancies: clarification and definition of terms. J Thorac Oncol: Cancer. 6:S1710–1716. 2011. View Article : Google Scholar : PubMed/NCBI
4	Lababede O and Meziane MA: The eighth edition of TNM staging of lung cancer: Reference chart and diagrams. Oncologist. 23:844–848. 2018. View Article : Google Scholar : PubMed/NCBI
5	Kondo K: Therapy for thymic epithelial tumors. Gen Thorac Cardiovasc Surg. 62:468–474. 2014. View Article : Google Scholar : PubMed/NCBI
6	Shimosato Y, Mukai K and Matsuno Y: Tumors of the mediastinum. AFIP Atlas of Tumor Pathology, Series 4. Armed Forces Institute of Pathology; Washington, DC: 2010
7	Kondo K and Monden Y: Therapy for thymic epithelial tumors: A clinical study of 1,320 patients from Japan. Ann Thorac Surg. 76:878–885. 2003. View Article : Google Scholar : PubMed/NCBI
8	Berghmans T, Durieux V, Holbrechts S, Jungels C, Lafitte JJ, Meert AP, Moretti L, Ocak S, Roelandts M, et al: Systemic treatments for thymoma and thymic carcinoma: A systematic review. Lung Cancer. 126:25–31. 2018. View Article : Google Scholar : PubMed/NCBI
9	Girard N, Ruffini E, Marx A, Faivre-Finn C and Peters S: Thymic epithelial tumours: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 26:v40–v55. 2015. View Article : Google Scholar : PubMed/NCBI
10	Scorsetti M, Leo F, Trama A, D'Angelillo R, Serpico D, Macerelli M, Zucali P, Gatta G and Garassino MC: Thymoma and thymic carcinomas. Crit Rev Oncol Hematol. 99:332–350. 2016. View Article : Google Scholar : PubMed/NCBI
11	Kelly RJ, Petrini I, Rajan A, Wang Y and Giaccone G: Thymic malignancies: from clinical management to targeted therapies. J Clin Oncol. 29:4820–4827. 2011. View Article : Google Scholar : PubMed/NCBI
12	Suzuki E, Sasaki H, Kawano O, Endo K, Haneda H, Yukiue H, Kobayashi Y, Yano M and Fujii Y: Expression and mutation statuses of epidermal growth factor receptor in thymic epithelial tumors. Jpn J Clin Oncol. 36:351–356. 2006. View Article : Google Scholar : PubMed/NCBI
13	Cimpean AM, Raica M, Encica S, Cornea R and Bocan V: Immunohistochemical expression of vascular endothelial growth factor A (VEGF), and its receptors (VEGFR1, 2) in normal and pathologic conditions of the human thymus. Ann Anat. 190:238–245. 2008. View Article : Google Scholar : PubMed/NCBI
14	Girard N, Teruya-Feldstein J, Payabyab EC, Riely GJ, Rusch VW, Kris MG and Zakowski MF: Insulin-like growth factor-1 receptor expression in thymic malignancies. J Thorac Oncol. 5:1439–1446. 2010. View Article : Google Scholar : PubMed/NCBI
15	Weissferdt A, Fujimoto J, Kalhor N, Rodriguez J, Bassett R, Wistuba II and Moran CA: Expression of PD-1 and PD-L1 in thymic epithelial neoplasms. Mod Pathol. 30:826–833. 2017. View Article : Google Scholar : PubMed/NCBI
16	Zucali PA, De Pas T, Palmieri G, Favaretto A, Chella A, Tiseo M, Caruso M, Simonelli M, Perrino M, et al: Phase II study of everolimus in patients with thymoma and thymic carcinoma previously treated with cisplatin-based chemotherapy. J Clin Oncol. 36:342–349. 2018. View Article : Google Scholar : PubMed/NCBI
17	Thomas A, Rajan A, Berman A, Tomita Y, Brzezniak C, Lee MJ, Lee S, Ling A, Spittler AJ, et al: Sunitinib in patients with chemotherapy-refractory thymoma and thymic carcinoma: an open-label phase 2 trial. Lancet Oncol. 16:177–186. 2015. View Article : Google Scholar : PubMed/NCBI
18	Kishibuchi R, Kondo K, Soejima S, Tsuboi M, Kajiura K, Kawakami Y, Kawakita N, Sawada T, Toba H, et al: DNA methylation of GHSR, GNG4, HOXD9 and SALL3 is a common epigenetic alteration in thymic carcinoma. Int J Oncol. 56:315–326. 2020.PubMed/NCBI
19	Moskalev EA, Jandaghi P, Fallah M, Manoochehri M, Botla SK, Kolychev OV, Nikitin EA, Bubnov VV, von Knebel Doeberitz M, et al: GHSR DNA hypermethylation is a common epigenetic alteration of high diagnostic value in a broad spectrum of cancers. Oncotarget. 6:4418–4427. 2015. View Article : Google Scholar : PubMed/NCBI
20	Kojima M and Kangawa K: Ghrelin: Structure and Function. Physiol Rev. 85:495–522. 2005. View Article : Google Scholar : PubMed/NCBI
21	Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, et al: A receptor in pituitary and hypothalamus that functions in growth hormone release. Science. 273:974–977. 1996. View Article : Google Scholar : PubMed/NCBI
22	Seim I, Herington AC and Chopin LK: New insights into the molecular complexity of the ghrelin gene locus. Cytokine Growth Factor Rev. 20:297–304. 2009. View Article : Google Scholar : PubMed/NCBI
23	Travis WD, Brambilla E, Burke AP, Marx A and Nicholson AG: introduction to the 2015 World Health Organization classification of tumors of the lung, pleura, thymus, and heart. J Thorac Oncol. 10:1240–1242. 2015. View Article : Google Scholar : PubMed/NCBI
24	Luque RM, Sampedro-Nuñez M, Gahete MD, Ramos-Levi A, Ibáñez-Costa A, Rivero-Cortés E, Serrano-Somavilla A, Adrados M, Culler MD, et al: In1-ghrelin, a splice variant of ghrelin gene, is associated with the evolution and aggressiveness of human neuroendocrine tumors: Evidence from clinical, cellular and molecular parameters. Oncotarget. 6:19619–19633. 2015. View Article : Google Scholar : PubMed/NCBI
25	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
26	Lin T-C and Hsiao M: Ghrelin and cancer progression. Biochim Biophys Acta Rev Cancer. 1868:51–57. 2017. View Article : Google Scholar : PubMed/NCBI
27	Volante M, Allia E, Fulcheri E, Cassoni P, Ghigo E, Muccioli G and Papotti M: Ghrelin in fetal thyroid and follicular tumors and cell lines: expression and effects on tumor growth. Am J Pathol. 162:645–654. 2003. View Article : Google Scholar : PubMed/NCBI
28	Cassoni P, Papotti M, Ghè C, Catapano F, Sapino A, Graziani A, Deghenghi R, Reissmann T, Ghigo E, et al: Identification, characterization, and biological activity of specific receptors for natural (ghrelin) and synthetic growth hormone secretagogues and analogs in human breast carcinomas and cell lines. J Clin Endocrinol Metab. 86:1738–1745. 2001. View Article : Google Scholar : PubMed/NCBI
29	Bai RX, Wang WP, Zhao PW and Li CB: Ghrelin attenuates the growth of HO-8910 ovarian cancer cells through the ERK pathway. Braz J Med Biol Res. 49:e50432016. View Article : Google Scholar : PubMed/NCBI
30	Díaz-Lezama N, Hernández-Elvira M, Sandoval A, Monroy A, Felix R and Monjaraz E: Ghrelin inhibits proliferation and increases T-type Ca²⁺ channel expression in PC-3 human prostate carcinoma cells. Biochem Biophys Res Commun. 403:24–29. 2010. View Article : Google Scholar : PubMed/NCBI
31	Cassoni P, Allia E, Marrocco T, Ghè C, Ghigo E, Muccioli G and Papotti M: Ghrelin and cortistatin in lung cancer: Expression of peptides and related receptors in human primary tumors and in vitro effect on the H345 small cell carcinoma cell line. J Endocrinol Invest. 29:781–790. 2006. View Article : Google Scholar : PubMed/NCBI
32	Lin TC, Liu YP, Chan YC, Su CY, Lin YF, Hsu SL, Yang CS and Hsiao M: Ghrelin promotes renal cell carcinoma metastasis via Snail activation and is associated with poor prognosis. J Pathol. 237:50–61. 2015. View Article : Google Scholar : PubMed/NCBI
33	Dixit VD, Weeraratna AT, Yang H, Bertak D, Cooper-Jenkins A, Riggins GJ, Eberhart CG and Taub DD: Ghrelin and the growth hormone secretagogue receptor constitute a novel autocrine pathway in astrocytoma motility. J Biol Chem. 281:16681–16690. 2006. View Article : Google Scholar : PubMed/NCBI
34	Waseem T, Javaid ur R, Ahmad F, Azam M and Qureshi MA: Role of ghrelin axis in colorectal cancer: A novel association. Peptides. 29:1369–1376. 2008. View Article : Google Scholar : PubMed/NCBI
35	Duxbury MS, Waseem T, Ito H, Robinson MK, Zinner MJ, Ashley SW and Whang EE: Ghrelin promotes pancreatic adenocarcinoma cellular proliferation and invasiveness. Biochem Biophys Res Commun. 309:464–468. 2003. View Article : Google Scholar : PubMed/NCBI
36	Ibáñez-Costa A, Gahete MD, Rivero-Cortés E, Rincón-Fernández D, Nelson R, Beltrán M, de la Riva A, Japón MA, Venegas-Moreno E, et al: In1-ghrelin splicing variant is overexpressed in pituitary adenomas and increases their aggressive features. Sci Rep. 5:87142015. View Article : Google Scholar : PubMed/NCBI
37	Hormaechea-Agulla D, Gahete MD, Jiménez-Vacas JM, Gómez-Gómez E, Ibáñez-Costa A, L-López F, Rivero-Cortés E, Sarmento-Cabral A, Valero-Rosa J, et al: The oncogenic role of the In1-ghrelin splicing variant in prostate cancer aggressiveness. Mol Cancer. 16:1462017. View Article : Google Scholar : PubMed/NCBI
38	Gahete MD, Córdoba-Chacón J, Hergueta-Redondo M, Martínez-Fuentes AJ, Kineman RD, Moreno-Bueno G, Luque RM and Castaño JP: A Novel Human Ghrelin Variant (In1-Ghrelin) and Ghrelin-O-Acyltransferase Are Overexpressed in Breast Cancer: Potential Pathophysiological Relevance. PLoS One. 6:e233022011. View Article : Google Scholar : PubMed/NCBI
39	Rincón-Fernández D, Culler MD, Tsomaia N, Moreno-Bueno G, Luque RM, Gahete MD and Castaño JP: In1-ghrelin splicing variant is associated with reduced disease-free survival of breast cancer patients and increases malignancy of breast cancer cells lines. Carcinogenesis. 39:447–457. 2018. View Article : Google Scholar : PubMed/NCBI
40	Jeffery PL, Murray RE, Yeh AH, McNamara JF, Duncan RP, Francis GD, Herington AC and Chopin LK: Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines. Endocr Relat Cancer. 12:839–850. 2005. View Article : Google Scholar : PubMed/NCBI
41	Barzon L, Pacenti M, Masi G, Stefani AL, Fincati K and Palù G: Loss of growth hormone secretagogue receptor 1a and overexpression of type 1b receptor transcripts in human adrenocortical tumors. Oncology. 68:414–421. 2005. View Article : Google Scholar : PubMed/NCBI
42	Herrera-Martínez AD, Gahete MD, Sánchez-Sánchez R, Salas RO, Serrano-Blanch R, Salvatierra Á, Hofland LJ, Luque RM, Gálvez-Moreno MA, et al: The components of somatostatin and ghrelin systems are altered in neuroendocrine lung carcinoids and associated to clinical-histological features. Lung Cancer. The components of somatostatin and ghrelin systems are altered in neuroendocrine lung carcinoids and associated to clinical-histological features. Lung Cancer. 109:128–136. 2017. View Article : Google Scholar : PubMed/NCBI
43	De Vriese C, Grégoire Fo, De Neef P, Robberecht P and Delporte C: Ghrelin is produced by the human erythroleukemic HEL cell line and involved in an autocrine pathway leading to cell proliferation. Endocrinology. 146:1514–1522. 2005. View Article : Google Scholar : PubMed/NCBI
44	Leung PK, Chow KB, Lau PN, Chu KM, Chan CB, Cheng CH and Wise H: The truncated ghrelin receptor polypeptide (GHS-R1b) acts as a dominant-negative mutant of the ghrelin receptor. Cell Signal. 19:1011–1022. 2007. View Article : Google Scholar : PubMed/NCBI
45	Takahashi K, Furukawa C, Takano A, Ishikawa N, Kato T, Hayama S, Suzuki C, Yasui W, Inai K, et al: The neuromedin U-growth hormone secretagogue receptor 1b/neurotensin receptor 1 oncogenic signaling pathway as a therapeutic target for lung cancer. Cancer Res. 66:9408–9419. 2006. View Article : Google Scholar : PubMed/NCBI
46	Gómez-Gómez E, Jiménez-Vacas JM, Carrasco-Valiente J, Herrero-Aguayo V, Blanca-Pedregosa AM, León-González AJ, Valero-Rosa J, Fernández-Rueda JL, González-Serrano T, et al: Plasma ghrelin O-acyltransferase (GOAT) enzyme levels: A novel non-invasive diagnosis tool for patients with significant prostate cancer. J Cell Mol Med. 22:5688–5697. 2018. View Article : Google Scholar : PubMed/NCBI
47	Herrera-Martínez AD, Gahete MD, Sánchez-Sánchez R, Alors-Perez E, Pedraza-Arevalo S, Serrano-Blanch R, Martínez-Fuentes AJ, Gálvez-Moreno MA, Castaño JP, et al: Ghrelin-O-Acyltransferase (GOAT) enzyme as a novel potential biomarker in gastroenteropancreatic neuroendocrine tumors. Clin Transl Gastroenterol. 9:1962018. View Article : Google Scholar : PubMed/NCBI
48	Jones PA and Baylin SB: The fundamental role of epigenetic events in cancer. Nat Rev Genet. 3:415–428. 2002. View Article : Google Scholar : PubMed/NCBI
49	Brena RM, Plass C and Costello JF: Mining methylation for early detection of common cancers. PLoS Med. 3:e4792006. View Article : Google Scholar : PubMed/NCBI
50	Ordway JM, Budiman MA, Korshunova Y, Maloney RK, Bedell JA, Citek RW, Bacher B, Peterson S, Rohlfing T, et al: Identification of Novel High-Frequency DNA Methylation Changes in Breast Cancer. PLoS One. 2:e13142007. View Article : Google Scholar : PubMed/NCBI
51	Daugaard I, Dominguez D, Kjeldsen TE, Kristensen LS, Hager H, Wojdacz TK and Hansen LL: Identification and validation of candidate epigenetic biomarkers in lung adenocarcinoma. Sci Rep. 6:358072016. View Article : Google Scholar : PubMed/NCBI
52	Nakagawa T, Matsusaka K, Misawa K, Ota S, Takane K, Fukuyo M, Rahmutulla B, Shinohara KI, Kunii N, et al: Frequent promoter hypermethylation associated with human papillomavirus infection in pharyngeal cancer. Cancer Lett. 407:21–31. 2017. View Article : Google Scholar : PubMed/NCBI
53	Verlaat W, Snijders PJF, Novianti PW, Wilting SM, De Strooper LM, Trooskens G, Vandersmissen J, Van Criekinge W, Wisman GB, et al: Genome-wide DNA methylation profiling reveals methylation markers associated with 3q gain for detection of cervical precancer and cancer. Clin Cancer Res:. 23:3813–3822. 2017. View Article : Google Scholar : PubMed/NCBI
54	Coppede F, Stoccoro A, Lazzarotti A, Spisni R and Migliore L: Investigation of GHSR and GHRL methylation in colorectal cancer. Epigenomics. 10:1525–1539. 2018. View Article : Google Scholar : PubMed/NCBI
55	Coppedè F, Stoccoro A, Nicolì V, Gallo R, De Rosa A, Guida M, Maestri M, Lucchi M, Ricciardi R, et al: Investigation of GHSR methylation levels in thymomas from patients with Myasthenia Gravis. Gene. 752:1447742020. View Article : Google Scholar : PubMed/NCBI