Aberrant GRK6 promoter methylation is associated with poor prognosis in hypopharyngeal squamous cell carcinoma

Qiu,Xiaoxia; Chen,Jianqiu; Zhang,Zhenxin; You,Yiwen; Wang,Zhiwei

doi:10.3892/or.2015.4469

Aberrant GRK6 promoter methylation is associated with poor prognosis in hypopharyngeal squamous cell carcinoma

Authors:
- Xiaoxia Qiu
- Jianqiu Chen
- Zhenxin Zhang
- Yiwen You
- Zhiwei Wang
View Affiliations

Affiliations: Department of Otorhinolaryngology/Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, P.R. China, Department of Otolaryngology Head and Neck Surgery, General Hospital of Jinan Military Region, Jinan, Shandong 250031, P.R. China, Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, P.R. China
Published online on: December 1, 2015 https://doi.org/10.3892/or.2015.4469
Pages: 1027-1033

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

Hypopharyngeal squamous cell carcinoma (HSCC) is one of the most common head and neck cancers with high invasiveness and poor prognosis. To identify targeted therapeutics against metastasis, a better understanding of the regulation of HSCC cell invasion is needed. In recent years, G protein-coupled receptor kinases (GRKs) have been implicated in cancer metastasis through phosphorylation of the activated form of G protein coupled receptors (GPCRs). However, there is little information regarding GRKs expression in HSCC. In the present study, we examined GRK6 expression in HSCC and also assessed the possible cause of its aberrant expression, as well as its clinical significance. Real-time quantitative PCR (qPCR) and western blotting were performed to analyze the expression of GRK6 in HSCC tissues and corresponding non-malignant tissues. Subsequently, paired HSCC and corresponding non-malignant tissues were evaluated for the methylation status of GRK6 gene promoter using methylation-specific PCR (MSP). Furthermore, we investigated the methylation status and the clinicopathological significance of GRK6 in 45 paired HSCC and corresponding non-malignant tissues. The suppression of GRK6 in hypopharyngeal cell line FaDu by GRK6-shRNA lentivirus transfection was utilized to detect the role of GRK6 in hypopharyngeal cancer progression. Our results showed that the expression of GRK6 mRNA and protein was significantly lower in HSCC than in corresponding adjacent non-tumor tissues, and this downregulation was found to be in accordance with aberrant methylation of the gene. Hypermethylation of the gene was observed in 77.8% (35/45) of the HSCC tissues, while it was found in only 42.2% (19/45) of the corresponding non-malignant tissues. GRK6 methylation was related to depth of tumor invasion and TNM stage. Upon treatment with 5-aza-2'-deoxycytidine, GRK6 expression was upregulated in FaDu cells, and cell invasion was signinficantly inhibited. Furthermore, the suppression of GRK6 by shRNA transfection enhanced FaDu cells invasion. Our results indicate that the aberrant methylation of GRK6 gene promoter may underlie its downregulation in HSCC, and may play an important role in the metastasis of HSCC.

View Figures

View References

1	Hall SF, Groome PA, Irish J and O'Sullivan B: The natural history of patients with squamous cell carcinoma of the hypopharynx. Laryngoscope. 118:1362–1371. 2008. View Article : Google Scholar : PubMed/NCBI
2	Wycliffe ND, Grover RS, Kim PD and Simental A Jr: Hypopharyngeal cancer. Top Magn Reson Imaging. 18:243–258. 2007. View Article : Google Scholar : PubMed/NCBI
3	Pitcher JA, Freedman NJ and Lefkowitz RJ: G protein-coupled receptor kinases. Annu Rev Biochem. 67:653–692. 1998. View Article : Google Scholar : PubMed/NCBI
4	Premont RT and Gainetdinov RR: Physiological roles of G protein-coupled receptor kinases and arrestins. Annu Rev Physiol. 69:511–534. 2007. View Article : Google Scholar : PubMed/NCBI
5	Métayé T, Gibelin H, Perdrisot R and Kraimps JL: Pathophysiological roles of G-protein-coupled receptor kinases. Cell Signal. 17:917–928. 2005. View Article : Google Scholar : PubMed/NCBI
6	Dorn GW II: GRK mythology: G-protein receptor kinases in cardiovascular disease. J Mol Med Berl. 87:455–463. 2009. View Article : Google Scholar : PubMed/NCBI
7	Ahmed MR, Berthet A, Bychkov E, Porras G, Li Q, Bioulac BH, Carl YT, Bloch B, Kook S, Aubert I, et al: Lentiviral overexpression of GRK6 alleviates L-dopa-induced dyskinesia in experimental Parkinson's disease. Sci Transl Med. 2:28ra282010.PubMed/NCBI
8	Eijkelkamp N, Heijnen CJ, Lucas A, Premont RT, Elsenbruch S, Schedlowski M and Kavelaars A: G protein-coupled receptor kinase 6 controls chronicity and severity of dextran sodium sulphate-induced colitis in mice. Gut. 56:847–854. 2007. View Article : Google Scholar : PubMed/NCBI
9	Murga C and Mayor F Jr: GRK6, a gatekeeper of visceral hyperalgesia. Brain Behav Immun. 23:16–17. 2009. View Article : Google Scholar
10	Tiedemann RE, Zhu YX, Schmidt J, Yin H, Shi CX, Que Q, Basu G, Azorsa D, Perkins LM, Braggio E, et al: Kinome-wide RNAi studies in human multiple myeloma identify vulnerable kinase targets, including a lymphoid-restricted kinase, GRK6. Blood. 115:1594–1604. 2010. View Article : Google Scholar :
11	Sutton N, Smith N, Thomas AJ, Raghuwanshi SK and Richardson RM: Abstract 2821: GRK6 deficiency promotes tumor aggressiveness and metastasis in a murine model of human lung cancer. Cancer Res. 73(Suppl 8): 2821. 2013. View Article : Google Scholar
12	Yuan L, Zhang H, Liu J, Rubin JB, Cho YJ, Shu HK, Schniederjan M and MacDonald TJ: Growth factor receptor-Src-mediated suppression of GRK6 dysregulates CXCR4 signaling and promotes medulloblastoma migration. Mol Cancer. 12:182013. View Article : Google Scholar : PubMed/NCBI
13	Hara S, Arawaka S, Sato H, Machiya Y, Cui C, Sasaki A, Koyama S and Kato T: Serine 129 phosphorylation of membrane-associated alpha-synuclein modulates dopamine transporter function in a G protein-coupled receptor kinase-dependent manner. Mol Biol Cell. 24:1649–1660. 2013. View Article : Google Scholar :
14	Benovic JL and Gomez J: Molecular cloning and expression of GRK6. A new member of the G protein-coupled receptor kinase family. J Biol Chem. 268:19521–19527. 1993.PubMed/NCBI
15	Zhou Y, Huang X, Wu H, Xu Y, Tao T, Xu G, Cheng C and Cao S: Decreased expression and role of GRK6 in spinal cord of rats after chronic constriction injury. Neurochem Res. 38:2168–2179. 2013. View Article : Google Scholar : PubMed/NCBI
16	Willets JM, Challiss RA and Nahorski SR: Endogenous G protein-coupled receptor kinase 6 regulates M3 muscarinic acetylcholine receptor phosphorylation and desensitization in human SH-SY5Y neuroblastoma cells. J Biol Chem. 277:15523–15529. 2002. View Article : Google Scholar : PubMed/NCBI
17	Chen Y, Lu B, Yang Q, Fearns C, Yates JR III and Lee JD: Combined integrin phosphoproteomic analyses and small interfering RNA-based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res. 69:3713–3720. 2009. View Article : Google Scholar : PubMed/NCBI
18	Teicher BA and Fricker SP: CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res. 16:2927–2931. 2010. View Article : Google Scholar : PubMed/NCBI
19	McCormick PJ, Segarra M, Gasperini P, Gulino AV and Tosato G: Impaired recruitment of Grk6 and beta-Arrestin 2 causes delayed internalization and desensitization of a WHIM syndrome-associated CXCR4 mutant receptor. PLoS One. 4:e81022009. View Article : Google Scholar : PubMed/NCBI
20	Vroon A, Heijnen CJ, Raatgever R, Touw IP, Ploemacher RE, Premont RT and Kavelaars A: GRK6 deficiency is associated with enhanced CXCR4-mediated neutrophil chemotaxis in vitro and impaired responsiveness to G-CSF in vivo. J Leukoc Biol. 75:698–704. 2004. View Article : Google Scholar : PubMed/NCBI
21	Raghuwanshi SK: GRK6 deficiency promotes angiogenesis, tumor progression and metastasis. J Immunol. 190:8962013. View Article : Google Scholar
22	Premont RT, Claing A, Vitale N, Freeman JL, Pitcher JA, Patton WA, Moss J, Vaughan M and Lefkowitz RJ: beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein. Proc Natl Acad Sci USA. 95:14082–14087. 1998. View Article : Google Scholar : PubMed/NCBI
23	Ribas C, Penela P, Murga C, Salcedo A, García-Hoz C, Jurado-Pueyo M, Aymerich I and Mayor F Jr: The G protein-coupled receptor kinase (GRK) interactome: Role of GRKs in GPCR regulation and signaling. Biochim Biophys Acta. 1768:913–922. 2007. View Article : Google Scholar
24	Pronin AN, Satpaev DK, Slepak VZ and Benovic JL: Regulation of G protein-coupled receptor kinases by calmodulin and localization of the calmodulin binding domain. J Biol Chem. 272:18273–18280. 1997. View Article : Google Scholar : PubMed/NCBI
25	Razin A and Riggs AD: DNA methylation and gene function. Science. 210:604–610. 1980. View Article : Google Scholar : PubMed/NCBI
26	Wei DM, Liu DY, Lei DP, Jin T, Wang J and Pan XL: Aberrant methylation and expression of DAPk1 in human hypopharyngeal squamous cell carcinoma. Acta Otolaryngol. 135:70–78. 2015. View Article : Google Scholar
27	Christman JK: 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: Mechanistic studies and their implications for cancer therapy. Oncogene. 21:5483–5495. 2002. View Article : Google Scholar : PubMed/NCBI