Wnt antagonist gene DKK2 is epigenetically silenced and inhibits renal cancer progression through apoptotic and cell cycle pathways.
OBJECTIVE
Wnt/beta-catenin signaling is involved in renal cancer. DKK2, a Wnt antagonist, is silenced in some cancers, although its function has not been investigated. We hypothesized that DKK2 may be epigenetically silenced and inhibits progression of renal cell carcinoma (RCC).


METHODS
RCC cell lines and a normal kidney cell line were used for methylation and chromatin immunoprecipitation assays. To assess various functions of DKK2, we established stable DKK2-transfected cells and examined them with regard to cell viability, colony formation, apoptosis, cell cycle, and invasive capability. A total of 52 patients with confirmed conventional RCC were enrolled in this study.


RESULTS
RCC cell lines had decreased levels of DKK2, which were significantly increased after treatment with 5-Aza-2'-deoxycytidine alone or 5-Aza-2'-deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the levels of acetyl H3, acetyl H4, and dimethylated H3K4 were decreased, whereas the level of dimethylated H3K9 was increased in RCC cell lines compared with HK2 cells. Increased methylation in RCC tissues was associated with higher grades, pathologic stages, and pathologic tumor in RCC. Functional analysis showed that the numbers of viable A498 cells were significantly decreased in DKK2-transfected cells compared with mock cells. The number of apoptotic cells and S/G(2)-M phase cells was significantly increased and decreased after DKK2 transfection, respectively. Corresponding to these results, Bcl2 and cyclin D1 expression were also decreased in DKK2-overexpressing cells.


CONCLUSIONS
DKK2 is epigenetically silenced by methylation in higher grades and stages of RCC. These results suggest that DKK2 inhibits renal cancer progression through apoptotic and cell cycle pathways.
Hirata
Hiroshi
H
Department of Urology, San Francisco Veterans Affairs Medical Center and University of California at San Francisco, San Francisco, California, USA.
Hinoda
Yuji
Y
Nakajima
Koichi
K
Kawamoto
Ken
K
Kikuno
Nobuyuki
N
Kawakami
Kazumori
K
Yamamura
Soichiro
S
Ueno
Koji
K
Majid
Shahana
S
Saini
Sharanjot
S
Ishii
Nobuhisa
N
Dahiya
Rajvir
R
D000328:Adult; D000368:Aged; D017209:Apoptosis; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle; D047369:Chromatin Immunoprecipitation; D000077209:Decitabine; D018450:Disease Progression; D005260:Female; D020868:Gene Silencing; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D007680:Kidney Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D051153:Wnt Proteins / Q000037:antagonists & inhibitors
C498622:DKK2 protein, human; D006877:Hydroxamic Acids; D036341:Intercellular Signaling Peptides and Proteins; D051153:Wnt Proteins; C012589:trichostatin A; D000077209:Decitabine; D001374:Azacitidine