Downregulation of DENND2D by promoter hypermethylation is associated with early recurrence of hepatocellular carcinoma

Kanda,Mitsuro; Nomoto,Shuji; Oya,Hisaharu; Takami,Hideki; Hibino,Soki; Hishida,Mitsuhiro; Suenaga,Masaya; Yamada,Suguru; Inokawa,Yoshikuni; Nishikawa,Yoko; Asai,Mikako; Fujii,Tsutomu; Sugimoto,Hiroyuki; Kodera,Yasuhiro

doi:10.3892/ijo.2013.2165

Downregulation of DENND2D by promoter hypermethylation is associated with early recurrence of hepatocellular carcinoma

Authors:
- Mitsuro Kanda
- Shuji Nomoto
- Hisaharu Oya
- Hideki Takami
- Soki Hibino
- Mitsuhiro Hishida
- Masaya Suenaga
- Suguru Yamada
- Yoshikuni Inokawa
- Yoko Nishikawa
- Mikako Asai
- Tsutomu Fujii
- Hiroyuki Sugimoto
- Yasuhiro Kodera
View Affiliations

Affiliations: Department of Gastroenterological Surgery (Surgery II), Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan
Published online on: November 5, 2013 https://doi.org/10.3892/ijo.2013.2165
Pages: 44-52

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

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide and its prognosis is poor. Novel targets for treating recurrence and progression along with associated biomarkers are urgently required. In this study, the expression and regulatory mechanism of DENN/MADD domain containing 2D (DENND2D) were investigated in an attempt to identify a tumor suppressor gene for HCC regulated by silencing through promoter hypermethylation. The levels of DENND2D expression in HCC cell lines and surgical specimens were determined using a quantitative polymerase chain reaction assay and the relationship between the expression levels of DENND2D mRNA and clinicopathological factors was evaluated. The expression and distribution of DENND2D were determined using immunohistochemistry. DNA methylation analysis was performed to determine the regulatory mechanisms of DENND2D expression in HCC. Most HCC cell lines (89%) and surgical specimens (78%) expressed lower levels of DENND2D mRNA compared with normal liver tissue. In contrast, there was no significant difference in the expression levels of DENND2D mRNA between normal tissues of HCC patients with and without cirrhosis. The expression patterns of DENND2D protein and mRNA were consistent. Patients with significantly lower levels of DENND2D mRNA in HCC tissues had remarkably earlier recurrences after hepatectomy and their prognosis worsened. The DENND2D promoter was methylated in eight out of nine HCC cell lines and DNA demethylation reactivated DENND2D mRNA expression. Hypermethylation of DENND2D was frequently detected in HCC tissues (75%) and was significantly associated with downregulation of DENND2D mRNA expression. DENND2D is a candidate tumor suppressor gene that is inactivated by promoter hypermethylation in patients with HCC and may serve as a novel biomarker of early recurrence of HCC.

View Figures

View References

1.	Ferlay J, Shin HR, Bray F, Forman D, Mathers C and Parkin DM: Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 127:2893–2917. 2010. View Article : Google Scholar : PubMed/NCBI
2.	Llovet JM, Burroughs A and Bruix J: Hepatocellular carcinoma. Lancet. 362:1907–1917. 2003. View Article : Google Scholar
3.	Della C and Colombo M: Surveillance for hepatocellular carcinoma. Semin Oncol. 39:384–398. 2012. View Article : Google Scholar
4.	Yang JD and Roberts LR: Hepatocellular carcinoma: a global view. Nat Rev Gastroenterol Hepatol. 7:448–458. 2010. View Article : Google Scholar : PubMed/NCBI
5.	Yu MC and Yuan JM: Environmental factors and risk for hepatocellular carcinoma. Gastroenterology. 127:72–78. 2004. View Article : Google Scholar
6.	Ponder BA: Cancer genetics. Nature. 411:336–341. 2001. View Article : Google Scholar : PubMed/NCBI
7.	Cheng YY, Jin H, Liu X, Siu JM, Wong YP, Ng EK, Yu J, Leung WK, Sung JJ and Chan FK: Fibulin 1 is downregulated through promoter hypermethylation in gastric cancer. Br J Cancer. 99:2083–2087. 2008. View Article : Google Scholar : PubMed/NCBI
8.	Esteller M: Epigenetics in cancer. N Engl J Med. 358:1148–1159. 2008. View Article : Google Scholar
9.	Kanda M, Nomoto S, Okamura Y, Hayashi M, Hishida M, Fujii T, Nishikawa Y, Sugimoto H, Takeda S and Nakao A: Promoter hypermethylation of fibulin 1 gene is associated with tumor progression in hepatocellular carcinoma. Mol Carcinog. 50:571–579. 2011. View Article : Google Scholar : PubMed/NCBI
10.	Kanda M, Nomoto S, Okamura Y, Nishikawa Y, Sugimoto H, Kanazumi N, Takeda S and Nakao A: Detection of metallothionein 1G as a methylated tumor suppressor gene in human hepatocellular carcinoma using a novel method of double combination array analysis. Int J Oncol. 35:477–483. 2009.
11.	Kanda M, Nomoto S, Nishikawa Y, Sugimoto H, Kanazumi N, Takeda S and Nakao A: Correlations of the expression of vascular endothelial growth factor B and its isoforms in hepatocellular carcinoma with clinico-pathological parameters. J Surg Oncol. 98:190–196. 2008. View Article : Google Scholar : PubMed/NCBI
12.	Stenmark H: Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol. 10:513–525. 2009. View Article : Google Scholar : PubMed/NCBI
13.	Pawson T and Nash P: Assembly of cell regulatory systems through protein interaction domains. Science. 300:445–452. 2003. View Article : Google Scholar : PubMed/NCBI
14.	Marat AL and McPherson PS: The connecdenn family, Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery. J Biol Chem. 285:10627–10637. 2010. View Article : Google Scholar : PubMed/NCBI
15.	Sa=to M, Sato K, Liou W, Pant S, Harada A and Grant BD: Regulation of endocytic recycling by C. elegans Rab35 and its regulator RME-4, a coated-pit protein. EMBO J. 27:1183–1196. 2008.PubMed/NCBI
16.	Levivier E, Goud B, Souchet M, Calmels TP, Mornon JP and Callebaut I: uDENN, DENN, and dDENN: indissociable domains in Rab and MAP kinases signaling pathways. Biochem Biophys Res Commun. 287:688–695. 2001. View Article : Google Scholar : PubMed/NCBI
17.	Allaire PD, Marat AL, Dall’Armi C, Di Paolo G, McPherson PS and Ritter B: The Connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes. Mol Cell. 37:370–382. 2010. View Article : Google Scholar : PubMed/NCBI
18.	Knödler A, Feng S, Zhang J, Zhang X, Das A, Peränen J and Guo W: Coordination of Rab8 and Rab11 in primary ciliogenesis. Proc Natl Acad Sci USA. 107:6346–6351. 2010.PubMed/NCBI
19.	Denef N, Chen Y, Weeks SD, Barcelo G and Schüpbach T: Crag regulates epithelial architecture and polarized deposition of basement membrane proteins in Drosophila. Dev Cell. 14:354–364. 2008. View Article : Google Scholar : PubMed/NCBI
20.	Rink J, Ghigo E, Kalaidzidis Y and Zerial M: Rab conversion as a mechanism of progression from early to late endosomes. Cell. 122:735–749. 2005. View Article : Google Scholar : PubMed/NCBI
21.	Girard M, Allaire PD, McPherson PS and Blondeau F: Non-stoichiometric relationship between clathrin heavy and light chains revealed by quantitative comparative proteomics of clathrin-coated vesicles from brain and liver. Mol Cell Proteomics. 4:1145–1154. 2005. View Article : Google Scholar
22.	Majidi M, Hubbs AE and Lichy JH: Activation of extracellular signal-regulated kinase 2 by a novel Abl-binding protein, ST5. J Biol Chem. 273:16608–16614. 1998. View Article : Google Scholar : PubMed/NCBI
23.	Marat AL, Dokainish H and McPherson PS: DENN domain proteins: regulators of Rab GTPases. J Biol Chem. 286:13791–13800. 2011. View Article : Google Scholar : PubMed/NCBI
24.	Postel EH, Weiss VH, Beneken J and Kirtane A: Mutational analysis of NM23-H2/NDP kinase identifies the structural domains critical to recognition of a c-myc regulatory element. Proc Natl Acad Sci USA. 93:6892–6897. 1996. View Article : Google Scholar : PubMed/NCBI
25.	Ling B, Zheng H, Fu G, Yuan J, Shi T, Chen S, Liu Y, Liu Y, Cao Y, Zheng S, Guo S, Han N, Gao Y, Cheng S and Zhang K: Suppression of non-small cell lung cancer proliferation and tumorigenicity by DENND2D. Lung Cancer. 79:104–110. 2013. View Article : Google Scholar : PubMed/NCBI
26.	International Union Against Cancer: TNM Classification of Malignant Tumors. 7th edition. Wiley-Blackwell; New York, NY: 2009
27.	Takai D and Jones PA: Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci USA. 99:3740–3745. 2002. View Article : Google Scholar : PubMed/NCBI
28.	Takai D and Jones PA: The CpG island searcher: a new WWW resource. In Silico Biol. 3:235–240. 2003.PubMed/NCBI
29.	Mann CD, Neal CP, Garcea G, Manson MM, Dennison AR and Berry DP: Prognostic molecular markers in hepatocellular carcinoma: a systematic review. Eur J Cancer. 43:979–992. 2007. View Article : Google Scholar : PubMed/NCBI
30.	Herath NI, Leggett BA and MacDonald GA: Review of genetic and epigenetic alterations in hepatocarcinogenesis. J Gastroenterol Hepatol. 21:15–21. 2006. View Article : Google Scholar : PubMed/NCBI
31.	Tischoff I and Tannapfe A: DNA methylation in hepatocellular carcinoma. World J Gastroenterol. 14:1741–1748. 2008. View Article : Google Scholar : PubMed/NCBI
32.	Bloethner S, Mould A, Stark M and Hayward NK: Identification of ARHGEF17, DENND2D, FGFR3, and RB1 mutations in melanoma by inhibition of nonsense-mediated mRNA decay. Genes Chromosomes Cancer. 47:1076–1085. 2008. View Article : Google Scholar : PubMed/NCBI
33.	Yoshimura S, Gerondopoulos A, Linford A, Rigden DJ and Barr FA: Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors. J Cell Biol. 191:367–381. 2010. View Article : Google Scholar : PubMed/NCBI
34.	Carroll KS, Hanna J, Simon I, Krise J, Barbero P and Pfeffer SR: Role of Rab9 GTPase in facilitating receptor recruitment by TIP47. Science. 292:1373–1376. 2001. View Article : Google Scholar : PubMed/NCBI
35.	Kloer DP, Rojas R, Ivan V, Moriyama K, van Vlijmen T, Murthy N, Ghirlando R, van der Sluijs P, Hurley JH and Bonifacino JS: Assembly of the biogenesis of lysosome-related organelles complex-3 (BLOC-3) and its interaction with Rab9. J Biol Chem. 285:7794–7804. 2010. View Article : Google Scholar : PubMed/NCBI