Hypermethylation of the RSK4 promoter associated with BRAF V600E promotes papillary thyroid carcinoma

Yin,Yanan; Che,Kui; Hu,Jianxia; Hua,Hui; Dong,Anbing; Wang,Jueru; Yu,Jiarui; Zhang,Qing; Zhao,Shihua; Zhao,Yuhang; Wang,Ping; Wang,Fei; Wang,Yangang; Chi,Jingwei; Sun,Wenhai

doi:10.3892/ijo.2020.4999

Hypermethylation of the RSK4 promoter associated with BRAF V600E promotes papillary thyroid carcinoma

Authors:
- Yanan Yin
- Kui Che
- Jianxia Hu
- Hui Hua
- Anbing Dong
- Jueru Wang
- Jiarui Yu
- Qing Zhang
- Shihua Zhao
- Yuhang Zhao
- Ping Wang
- Fei Wang
- Yangang Wang
- Jingwei Chi
- Wenhai Sun
View Affiliations

Affiliations: Department of Endocrinology and Metabolism, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266000, P.R. China, Qingdao Key Laboratory of Thyroid Diseases, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266000, P.R. China, Department of Thyroid Surgery, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266000, P.R. China
Published online on: February 25, 2020 https://doi.org/10.3892/ijo.2020.4999
Pages: 1284-1293

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Ribosomal S6 kinase 4 (RSK4) is a putative tumor suppressor gene which is inactivated by epigenetic events in a number human malignancies; however, its role in papillary thyroid carcinoma (PTC) remains largely unknown. The aim of this study was to explore the methylation status of the RSK4 promoter in PTC, and to determine its potential role in thyroid carcinogenesis. Reverse transcription‑quantitative PCR (RT‑qPCR) and western blot analyses were performed to examine the RSK4 mRNA and protein levels, respectively. Methylation‑specific PCR (MSP) and bisulfite genomic sequencing (BGS) were used to analyze methylation status of the RSK4 gene. Sanger sequencing was further carried out to detect the BRAF V600E mutation. Cell proliferation assay was finally performed to evaluate the role of hypermethylation in the growth of PTC cells. The association between RSK4 methylation and the clinicopathological characteristics of patients with PTC was assessed. The methylation frequency of the RSK4 promoter in PTC tissues was higher than that in paired paracancerous tissues. Coincidentally, the RSK4 mRNA levels were also downregulated in PTC tissues when compared with the paracancerous counterparts. The hypermethylation of RSK4 was associated with tumor size and lymph node metastasis. Furthermore, the BRAF V600E mutation may influence RSK4 expression and methylation. Moreover, RSK4 hypermethylation was observed in thyroid cancer cell lines, which was consistent with a lack of RSK4 expression. Upon the 5‑Aza‑deoxycytidine treatment of thyroid cancer cells, RSK4 expression was significantly upregulated, while cell proliferation was inhibited. On the whole, the findings of the present study demonstrate that the hypermethylation of the RSK4 promoter may be one of the mechanisms responsible for the poor RSK4 expression in PTC. Thus, these data suggest that RSK4 may serve as a molecular target for the early diagnosis and treatment of PTC.

View Figures

View References

1	Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J and Jemal A: Global cancer statistics, 2012. CA Cancer J Clin. 65:87–108. 2015. View Article : Google Scholar : PubMed/NCBI
2	Haugen BR, Alexander EK, Bible KC, Doherty GM, Mandel SJ, Nikiforov YE, Pacini F, Randolph GW, Sawka AM, Schlumberger M, et al: 2015 American thyroid association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: The American thyroid association guidelines task force on thyroid nodules and differentiated thyroid cancer. Thyroid. 26:1–133. 2016. View Article : Google Scholar :
3	Cabanillas ME, McFadden DG and Durante C: Thyroid cancer. Lancet. 388:2783–2795. 2016. View Article : Google Scholar : PubMed/NCBI
4	Tuttle RM, Ball DW, Byrd D, Dilawari RA, Doherty GM, Duh QY, Ehya H, Farrar WB, Haddad RI, Kandeel F, et al: Thyroid carcinoma. J Natl Compr Canc Netw. 8:1228–1274. 2010. View Article : Google Scholar : PubMed/NCBI
5	Riesco-Eizaguirre G and Santisteban P: ENDOCRINE TUMOURS: Advances in the molecular pathogenesis of thyroid cancer: Lessons from the cancer genome. Eur J Endocrinol. 175:R203–R217. 2016. View Article : Google Scholar : PubMed/NCBI
6	López-Vicente L, Pons B, Coch L, Teixidó C, Hernández-Losa J, Armengol G, Ramon Y and Cajal S: RSK4 inhibition results in bypass of stress-induced and oncogene-induced senescence. Carcinogenesis. 32:470–476. 2011. View Article : Google Scholar : PubMed/NCBI
7	Lopez-Vicente L, Armengol G, Pons B, Coch L, Argelaguet E, Lleonart M, Hernandez-Losa J, de Torres I and Ramon y Cajal S: Regulation of replicative and stress-induced senescence by RSK4, which is down-regulated in human tumors. Clin Cancer Res. 15:4546–4553. 2009. View Article : Google Scholar : PubMed/NCBI
8	Arechavaleta-Velasco F, Zeferino-Toquero M, Estrada-Moscoso I, Imani-Razavi FS, Olivares A, Perez-Juarez CE and Diaz-Cueto L: Ribosomal S6 kinase 4 (RSK4) expression in ovarian tumors and its regulation by antineoplastic drugs in ovarian cancer cell lines. Med Oncol. 33:112016. View Article : Google Scholar : PubMed/NCBI
9	Dewdney SB, Rimel BJ, Thaker PH, Thompson DM Jr, Schmidt A, Huettner P, Mutch DG, Gao F and Goodfellow PJ: Aberrant methylation of the X-linked ribosomal S6 kinase RPS6KA6 (RSK4) in endometrial cancers. Clin Cancer Res. 17:2120–2129. 2011. View Article : Google Scholar : PubMed/NCBI
10	Jiang Y, Ye X, Ji Y, Zhou X, Yang H, Wei W and Li Q: Aberrant expression of RSK4 in breast cancer and its role in the regulation of tumorigenicity. Int J Mol Med. 40:883–890. 2017. View Article : Google Scholar : PubMed/NCBI
11	Anjum R and Blenis J: The RSK family of kinases: Emerging roles in cellular signalling. Nat Rev Mol Cell Biol. 9:747–758. 2008. View Article : Google Scholar : PubMed/NCBI
12	Xing M: Molecular pathogenesis and mechanisms of thyroid cancer. Nat Rev Cancer. 13:184–199. 2013. View Article : Google Scholar : PubMed/NCBI
13	Lin JD, Fu SS, Chen JY, Lee CH, Chau WK, Cheng CW, Wang YH, Lin YF, Fang WF and Tang KT: Clinical manifestations and gene expression in patients with conventional papillary thyroid carcinoma carrying the BRAF(V600E) mutation and BRAF pseudogene. Thyroid. 26:691–704. 2016. View Article : Google Scholar : PubMed/NCBI
14	Xing M: Genetic-guided risk assessment and management of thyroid cancer. Endocrinol Metab Clin North Am. 48:109–124. 2019. View Article : Google Scholar : PubMed/NCBI
15	Yang T, Chen C, Pan NF, Sun LY, Jiang XL, Li JN, Tang Y and Jiang Y: BRAF V600E mutation and TERT promoter mutation in papillary thyroid carcinomas and their association with clinicopathological characteristics. Sichuan Da Xue Xue Bao Yi Xue Ban. 50:919–924. 2019.In Chinese. PubMed/NCBI
16	Myers AP, Corson LB, Rossant J and Baker JC: Characterization of mouse Rsk4 as an inhibitor of fibroblast growth factor-RAS-extracellular signal-regulated kinase signaling. Mol Cell Biol. 24:4255–4266. 2004. View Article : Google Scholar : PubMed/NCBI
17	Peixoto P, Grandvallet C, Feugeas JP, Guittaut M and Hervouet E: Epigenetic control of autophagy in cancer cells: A key process for cancer-related phenotypes. Cells. 8:E16562019. View Article : Google Scholar : PubMed/NCBI
18	Skvortsova K, Stirzaker C and Taberlay P: The DNA methylation landscape in cancer. Essays Biochem. 63:797–811. 2019. View Article : Google Scholar : PubMed/NCBI
19	Curtis CD and Goggins M: DNA methylation analysis in human cancer. Methods Mol Med. 103:123–136. 2005.
20	Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonak J, Lind K, Sindelka R, Sjöback R, Sjögreen B, Strömbom L, et al: The real-time polymerase chain reaction. Mol Aspects Med. 27:95–125. 2006. View Article : Google Scholar : PubMed/NCBI
21	Niskakoski A, Kaur S, Staff S, Renkonen-Sinisalo L, Lassus H, Järvinen HJ, Mecklin JP, Bützow R and Peltomäki P: Epigenetic analysis of sporadic and Lynch-associated ovarian cancers reveals histology-specific patterns of DNA methylation. Epigenetics. 9:1577–1587. 2014. View Article : Google Scholar
22	Mao Y and Xing M: Recent incidences and differential trends of thyroid cancer in the USA. Endocr Relat Cancer. 23:313–322. 2016. View Article : Google Scholar : PubMed/NCBI
23	Liu R, Bishop J, Zhu G, Zhang T, Ladenson PW and Xing M: Mortality risk stratification by combining BRAF V600E and TERT promoter mutations in papillary thyroid cancer: Genetic duet of BRAF and TERT promoter mutations in thyroid cancer mortality. JAMA Oncol. 3:202–208. 2017. View Article : Google Scholar
24	Tsumagari K, Abd Elmageed ZY, Sholl AB, Friedlander P, Abdraboh M, Xing M, Boulares AH and Kandil E: Simultaneous suppression of the MAP kinase and NF-kB pathways provides a robust therapeutic potential for thyroid cancer. Cancer Lett. 368:46–53. 2015. View Article : Google Scholar : PubMed/NCBI
25	Liu D, Yang C, Bojdani E, Murugan AK and Xing M: Identification of RASALl as a major tumor suppressor gene in thyroid cancer. J Natl Cancer Inst. 105:1617–1627. 2013. View Article : Google Scholar : PubMed/NCBI
26	Khalaileh A, Dreazen A, Khatib A, Apel R, Swisa A, Kidess-Bassir N, Maitra A, Meyuhas O, Dor Y and Zamir G: Phosphorylation of ribosomal protein S6 attenuates DNA damage and tumor suppression during development of pancreatic cancer. Cancer Res. 73:1811–1820. 2013. View Article : Google Scholar : PubMed/NCBI
27	Li Q, Jiang Y, Wei W, Ji Y, Gao H and Liu J: Frequent epigenetic inactivation of RSK4 by promoter methylation in cancerous and non-cancerous tissues of breast cancer. Med Oncol. 31:7932014. View Article : Google Scholar
28	Rafiee M, Keramati MR, Ayatollahi H, Sadeghian MH, Barzegar M, Asgharzadeh A and Alinejad M: Down-regulation of ribosomal S6 kinase RPS6KA6 in acute myeloid leukemia patients. Cell J. 18:159–164. 2016.PubMed/NCBI
29	LLeonart ME, Vidal F, Gallardo D, Diaz-Fuertes M, Rojo F, Cuatrecasas M, López-Vicente L, Kondoh H, Blanco C, Carnero A, et al: New p53 related genes in human tumors: Significant downregulation in colon and lung carcinomas. Oncol Rep. 16:603–608. 2006.PubMed/NCBI
30	Xiao K, Yu Z, Shi DT, Lei Z, Chen H, Cao J, Tian W, Chen W and Zhang HT: Inactivation of BLU is associated with methylation of Sp1-binding site of BLU promoter in gastric cancer. Int J Oncol. 47:621–631. 2015. View Article : Google Scholar : PubMed/NCBI
31	Foy JP, Pickering CR, Papadimitrakopoulou VA, Jelinek J, Lin SH, William WN Jr, Frederick MJ, Wang J, Lang W, Feng L, et al: New DNA methylation markers and global DNA hypomethylation are associated with oral cancer development. Cancer Prev Res (Phila). 8. pp. 1027–1035. 2015, View Article : Google Scholar
32	Xing M, Alzahrani AS, Carson KA, Shong YK, Kim TY, Viola D, Elisei R, Bendlova B, Yip L, Mian C, et al: Association between BRAF V600E mutation and recurrence of papillary thyroid cancer. J Clin Oncol. 33:42–50. 2015. View Article : Google Scholar :
33	Xing M, Westra WH, Tufano RP, Cohen Y, Rosenbaum E, Rhoden KJ, Carson KA, Vasko V, Larin A, Tallini G, et al: BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab. 90:6373–6379. 2005. View Article : Google Scholar : PubMed/NCBI
34	Wang F, Zhao S, Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, et al: braf V600E confers male sex disease-specific mortality risk in patients with papillary thyroid cancer. J Clin Oncol. 36:2787–2795. 2018. View Article : Google Scholar : PubMed/NCBI
35	Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE and Fagin JA: High prevalence of BRAF mutations in thyroid cancer: Genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res. 63:1454–1457. 2003.PubMed/NCBI